Patent Publication Number: US-2009239293-A1

Title: Grooved High Density Plate

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 11/086,262 filed Mar. 22, 2005, which is a continuation-in-part of U.S. application Ser. No. 10/944,673 filed Sep. 17, 2004, and U.S. application Ser. No. 10/944,672 filed Sep. 17, 2004. U.S. patent application Ser. No. 10/944,673 claims a benefit to U.S. Provisional Application No. 60/504,500 filed on Sep. 19, 2003; U.S. Provisional Application No. 60/504,052 filed on Sep. 19, 2003; U.S. Provisional Application No. 60/589,224 filed Jul. 19, 2004; U.S. Provisional Application No. 60/589,225 filed on Jul. 19, 2004; and U.S. Provisional Application No. 60/601,716 filed on Aug. 13, 2004. U.S. patent application Ser. No. 10/944,672 claims the benefit of U.S. Provisional Application No. 60/589,224 filed Jul. 19, 2004. 
    
    
     All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. 
     INTRODUCTION 
     Currently, genomic analysis, including that of the estimated 30,000 human genes is a major focus of basic and applied biochemical and pharmaceutical research. Such analysis may aid in developing diagnostics, medicines, and therapies for a wide variety of disorders. However, the complexity of the human genome and the interrelated functions of genes often make this task difficult. There is a continuing need for methods and apparatus to aid in such analysis. 
    
    
     
       DRAWINGS 
       The skilled artisan will understand that the drawings, described herein, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. 
         FIG. 1(   a ) is a perspective view illustrating a high-density sequence detection system according to some embodiments of the present teachings; 
         FIG. 1(   b ) is a perspective view illustrating a high-density sequence detection system according to some embodiments of the present teachings; 
         FIG. 1(   c ) is a side view illustrating the high-density sequence detection system of  FIG. 1(   b ); 
         FIG. 2  is a top perspective view illustrating a microplate in accordance with some embodiments; 
         FIG. 3  is a top perspective view illustrating a microplate in accordance with some embodiments; 
         FIG. 4  is an enlarged perspective view illustrating a microplate in accordance with some embodiments comprising a plurality of wells comprising a circular rim portion; 
         FIG. 5  is an enlarged perspective view illustrating a microplate in accordance with some embodiments comprising a plurality of wells comprising a square-shaped rim portion; 
         FIG. 6  is a cross-sectional view illustrating a well comprising a pressure relief bore according to some embodiments; 
         FIG. 7  is a cross-sectional view illustrating the well of  FIG. 6  wherein the pressure relief bore is partially filled; 
         FIG. 8  is a cross-sectional view illustrating a well comprising an offset pressure relief bore according to some embodiments, being filled by a spotting device; 
         FIG. 9  is a cross-sectional view illustrating the well of  FIG. 8  being filled by a micro-piezo dispenser; 
         FIG. 10  is a cross-sectional view illustrating a microplate employing a plurality of apertures, a foil seal, and a sealing cover according to some embodiments; 
         FIG. 11  is a top view illustrating a microplate in accordance with some embodiments comprising one or more grooves; 
         FIG. 12  is an enlarged top view illustrating a corner of the microplate illustrated in  FIG. 11 ; 
         FIG. 13  is a cross-sectional view of the microplate of  FIG. 12  taken along Line  13 - 13 ; 
         FIG. 14  is an enlarged top view illustrating a corner of a microplate according to some embodiments; 
         FIG. 15  is a cross-sectional view of the microplate of  FIG. 14  taken along Line  15 - 15 ; 
         FIG. 16  is a top view illustrating a microplate in accordance with some embodiments comprising at least one thermally isolated portion; 
         FIG. 17  is a side view illustrating the microplate of  FIG. 16 ; 
         FIG. 18  is a bottom view illustrating the microplate of  FIG. 16 ; 
         FIG. 19  is an enlarged cross-sectional view illustrating the microplate of  FIG. 16  taken along Line  19 - 19 ; 
         FIG. 20  is an exploded perspective view illustrating a filling apparatus according to some embodiments; 
         FIG. 21  is a cross-sectional perspective view of the filling apparatus of  FIG. 20 ; 
         FIG. 22  is a cross-sectional perspective view of a filling apparatus according to some embodiments; 
         FIG. 22(   b ) is a cross-sectional view of a portion of a filling apparatus comprising a plurality of staging capillaries, microfluidic channels, and ramp features according to some embodiments; 
         FIG. 23(   a ) is a top schematic view of a filling apparatus according to some embodiments; 
         FIG. 23(   b ) is a top perspective view of a portion of a filling apparatus comprising a plurality of staging capillaries, microfluidic channels, and ramp features according to some embodiments; 
         FIG. 24  is a bottom perspective view of an output layer of a filling apparatus comprising spacer features according to some embodiments; 
         FIGS. 25(   a )-( f ) are top schematic views of a filling apparatus according to some embodiments; 
         FIG. 26  is a cross-sectional view illustrating a well of a microplate according to some embodiments; 
         FIG. 27  is a cross-sectional view illustrating a well of an inverted microplate according to some embodiments; 
         FIG. 28  is a cross-sectional view illustrating a sealing cover according to some embodiments; 
         FIG. 29  is a cross-sectional view illustrating a hot roller apparatus that can be used to seal a sealing cover to a microplate according to some embodiments; 
         FIG. 30  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising an inflatable transparent bag; 
         FIG. 31  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a moveable transparent window; 
         FIG. 32  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising an inverted microplate; 
         FIG. 33  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a plurality of apertures in a microplate; 
         FIG. 34  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a pressure chamber engaging a sealing cover; 
         FIG. 35  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a pressure chamber used together with an inverted microplate; 
         FIG. 36  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a pressure chamber used together with a microplate comprising a plurality of apertures; 
         FIG. 37  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a pressure chamber engaging a thermocycler block; 
         FIG. 38  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a vacuum assist system; 
         FIG. 39  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a pressure chamber engaging a thermocycler block and a microplate; 
         FIG. 40  is a cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a pressure chamber and a relief port; 
         FIG. 41  is an exploded cross-sectional view illustrating a pressure clamp system according to some embodiments comprising a heatable transparent window; 
         FIG. 42  is a top perspective view illustrating an upright configuration, according to some embodiments, of a thermocycler system, an excitation system, a detection system, and a microplate; 
         FIG. 43  is a side view illustrating the upright configuration of the thermocycler system, the excitation system, the detection system, and the microplate of  FIG. 42 ; 
         FIG. 44  is a perspective view illustrating an inverted configuration, according to some embodiments, of a thermocycler system, an excitation system, a detection system, and a microplate; 
         FIG. 45  is an enlarged perspective view illustrating an excitation system according to some embodiments comprising a plurality of LED excitation sources; 
         FIG. 46  is an enlarged perspective view illustrating an excitation system according to some embodiments comprising a plurality of LED excitation sources; 
         FIG. 47  is a side view illustrating the inverted configuration of the thermocycler system, the excitation system, the detection system, and the microplate of  FIG. 44 ; 
         FIG. 48  is a perspective view illustrating an inverted configuration, according to some embodiments, of a thermocycler system, an excitation system comprising individually mirrored excitation sources, a detection system, and a microplate; 
         FIG. 49  is an enlarged perspective view illustrating the excitation system comprising individually mirrored excitation sources of  FIG. 48 ; 
         FIG. 50  is a graph exemplifying vignetting and shadowing relative to excitation source position; 
         FIG. 51  is a graph exemplifying vignetting and shadowing and an illumination profile according to some embodiments; 
         FIG. 52  is a schematic view illustrating an excitation source comprising a lens according to some embodiments; 
         FIG. 53  is a schematic view illustrating an excitation source comprising a concave mirror according to some embodiments; 
         FIG. 54  is a schematic view illustrating an excitation source comprising a concave mirror and a lens according to some embodiments; 
         FIG. 55  is a schematic view illustrating multiple excitation sources focused to a point on a microplate according to some embodiments; 
         FIG. 56  is a schematic view illustrating multiple excitation sources focused to multiple points to achieve a desired irradiance profile according to some embodiments; 
         FIG. 57  is a flow chart illustrating a manufacturing procedure of preloaded microplates according to some embodiments; 
         FIG. 58  is a flow chart illustrating the use of a database system according to some embodiments; 
         FIG. 59  is a top perspective view illustrating a multipiece microplate in accordance with some embodiments; 
         FIG. 60  is an exploded perspective view illustrating the multipiece microplate of  FIG. 59  in accordance with some embodiments; 
         FIG. 61  is a top view illustrating the multipiece microplate in accordance with some embodiments; 
         FIG. 62  is a cross-sectional view of the multipiece microplate of  FIG. 61  taken along Line  62 - 62 ; 
         FIG. 63  is an enlarged cross-sectional view of cap portion and main body portion of the multipiece microplate of  FIG. 62 ; 
         FIG. 64  is a top schematic view illustrating a loading distribution system comprising a conveyer, a plurality of dispensing stations, a plurality of robots, and a plurality of microplate hotels according to some embodiments; 
         FIG. 65  is a perspective view illustrating a loading distribution system according to some embodiments; 
         FIG. 66(   a ) is a side view illustrating a loading distribution system according to some embodiments, comprising a dispensing device, a source plate and wash station, and a carriage; 
         FIG. 66(   b ) is a side view illustrating a loading distribution system according to some embodiments, comprising a dispensing device, a source plate station, a wash station, and a carriage; 
         FIGS. 68(   a )-( c ) are top-plan views illustrating various uses of a source plate and wash pallet; 
         FIG. 69  is a top-plan view illustrating a ceiling mounted plate-handling device adapted to retrieve a microplate from a hotel according to some embodiments; 
         FIG. 70  is a perspective view illustrating a carriage capable of holding a microplate according to some embodiments; 
         FIG. 71  is a perspective view illustrating a table coupled to a carriage utilizing a spring allowing the table to float in X and Y axis with respect to the carriage according to some embodiments; 
         FIG. 72  is a perspective view illustrating an embodiment of a locating ratchet adapted to hold a microplate on the table according to some embodiments; 
         FIG. 73  is a perspective view illustrating a lifting device to allow the table to float in Z axis with respect to the carriage according to some embodiments; 
         FIG. 74  is a perspective view illustrating a pressure source adapted to communicate with a vacuum connection shoe according to some embodiments; 
         FIG. 75  is a perspective view illustrating of a loading distribution system comprising a pair of rails and a guide channel to lift the table off of the carriage according to some embodiments 
         FIG. 76  is a perspective view illustrating an air slide connecting the pair of rails and a guide channel according to some embodiments; 
         FIG. 77  is a perspective view illustrating a loading distribution system comprising the carriage, the table, and an alignment stage according to some embodiments; 
         FIG. 78  is a perspective view illustrating a lifting stage adapted to lift a carriage according to some embodiments; 
         FIGS. 79(   a )-( b ) are perspective views illustrating a visual inspection station including a carriage alignment device according to some embodiments; 
         FIG. 80  is a top-plan view illustrating a table comprising a vacuum trench and a gasket according to some embodiments; 
         FIG. 81  is a perspective view illustrating a dispensing device including a plurality of dispensers according to some embodiments; 
         FIG. 82  is a perspective view illustrating a plate gripper robot according to some embodiments; 
         FIG. 83  is a perspective view illustrating a plate gripper robot, gripping a microplate in a lower jaw according to some embodiments; 
         FIGS. 84-90  are progressive perspective views illustrating a plate gripper robot depositing and picking-up microplates from a table and/or a plate storage unit according to some embodiments; 
         FIG. 91  is a perspective view illustrating a source plate and wash pallet according to some embodiments; 
         FIG. 92  is a perspective view illustrating a source plate and wash station, wherein a source plate and a washing tray each comprise a respective lid thereupon according to some embodiments; 
         FIG. 93  is a perspective view illustrating a source plate and wash station, wherein a de-lidded source plate allowing a dispensing device to access fluids stored in or on the source plate according to some embodiments; 
         FIG. 94  is a perspective view illustrating a source plate and wash station, wherein the source plate stays lidded and the washing tray can be accessed by a dispensing device according to some embodiments; 
         FIG. 95  is a perspective view illustrating a source plate and wash station positioned to enable a robot gripper to access a lidded source plate according to some embodiments; 
         FIG. 96  is a perspective view illustrating a source plate and wash station positioned to a allow a dispensing station to access a source plate according to some embodiments; 
         FIG. 97  is a perspective view illustrating a source plate and wash station positioned to a allow a dispensing station to access the washing tray according to some embodiments; 
         FIG. 98  is a front-plan view illustrating a source plate and wash station in a wait position alongside a dispensing device and a conveyer according to some embodiments; 
         FIG. 99  is a front-plan view illustrating a source plate and wash station in a deployed position alongside a dispensing device and a conveyer according to some embodiments; 
         FIG. 100  is a perspective view illustrating a hotel and a movable entry guide according to some embodiments; 
         FIG. 101  is a process flow diagram illustrating a software command and control architecture for a loading distribution system, according to some embodiments; 
         FIG. 102  is an illustration a sample distribution mapping for an eight dispenser sample filler, according to some embodiments; 
         FIG. 103  is an illustration of using a dead row to prevent cross-contamination in sample loadings from a filler according to some embodiments; 
         FIG. 104  is a top-plan view illustrating a robot accessing microplate hotels, source plate hotels, and a plurality of dispensing devices according to some embodiments; 
         FIG. 105  is a top-plan view illustrating a mapping of fluid locations of a 384-well source plate into a dispensing device comprising 96 dispensers and further into a 6,144-well microplate according to some embodiments; 
         FIG. 106  is an exploded top perspective view illustrating a filling apparatus comprising an intermediate layer according to some embodiments; 
         FIG. 107  is an exploded bottom perspective view illustrating the filling apparatus comprising the intermediate layer according to some embodiments; 
         FIG. 108  is a cross-sectional view illustrating the filling apparatus comprising the intermediate layer according to some embodiments; 
         FIG. 109  is a cross-sectional view illustrating the filling apparatus comprising the intermediate layer and nodules according to some embodiments; 
         FIG. 110  is a top schematic view of the filling apparatus comprising the intermediate layer and nodules according to some embodiments; 
         FIG. 111  is a cross-sectional view illustrating the filling apparatus comprising the intermediate layer, nodules, and sealing feature according to some embodiments; 
         FIG. 112  is a bottom perspective view of the intermediate layer of the filling apparatus according to some embodiments; 
         FIG. 113  is an exploded top perspective view illustrating a clamp system for a filling apparatus according to some embodiments; 
         FIG. 114  is an exploded top perspective view illustrating a filling apparatus comprising a vent layer according to some embodiments; 
         FIG. 115  is an exploded bottom perspective view illustrating the filling apparatus comprising the vent layer according to some embodiments; 
         FIG. 116  is a cross-sectional view illustrating the filling apparatus comprising the vent layer and a vent manifold according to some embodiments; 
         FIG. 117  is a top schematic view of the filling apparatus comprising the vent layer and vent apertures positioned between staging capillaries according to some embodiments; 
         FIG. 118  is a top schematic view of the filling apparatus comprising the vent layer and oblong vent apertures according to some embodiments; 
         FIG. 119  is a cross-sectional view illustrating the filling apparatus comprising the vent layer and pressure bores according to some embodiments; 
         FIG. 120  is a perspective view illustrating a filling apparatus comprising one or more assay input ports positioned on an end of an input layer according to some embodiments; 
         FIG. 121  is a perspective view illustrating a filling apparatus comprising one or more assay input ports positioned on a side of an input layer according to some embodiments; 
         FIG. 122  is a perspective view illustrating a filling apparatus comprising one or more assay input ports positioned on opposing sides of an input layer according to some embodiments; 
         FIG. 123  is a perspective view with portions illustrated in cross-section illustrating an assay input port according to some embodiments; 
         FIG. 124  is a cross-sectional view illustrating the filling apparatus of  FIGS. 120-123  according to some embodiments; 
         FIGS. 125-131  and  133  are cross-sectional views illustrating the progressive filling of a microplate according to some embodiments; 
         FIG. 132  is a top schematic view of the filling apparatus comprising reduced material areas for, at least in part, use in staking according to some embodiments; 
         FIGS. 134-139  are cross-sectional views illustrating the progressive filling of a microplate using a filling apparatus employing fluid overfill reservoirs according to some embodiments; 
         FIG. 140  is a cross-sectional view illustrating a filling apparatus employing fluid overfill reservoirs disposed in an output layer according to some embodiments; 
         FIGS. 141(   a )-( g ) are top schematic views illustrating various possible positions of the staging capillaries relative to corresponding microfluidic channels according to some embodiments; 
         FIGS. 142(   a )-( g ) are cross-sectional views illustrating various possible positions and configurations microfluidic channels and staging capillaries according to some embodiments; 
         FIG. 143  is an exploded perspective view illustrating a filling apparatus comprising a floating insert and cover according to some embodiments; 
         FIG. 144  is a cross-sectional view illustrating the filling apparatus comprising the floating insert according to some embodiments; 
         FIG. 145  is an exploded perspective view illustrating a filling apparatus comprising a floating insert according to some embodiments; 
         FIG. 146  is a cross-sectional view illustrating a floating insert according to some embodiments; 
         FIG. 147  is a cross-sectional view illustrating a floating insert comprising post members according to some embodiments; 
         FIG. 148  is a cross-sectional view illustrating a floating insert comprising tapered members according to some embodiments; 
         FIG. 149  is a cross-sectional view illustrating a floating insert comprising tapered members and a flanged base portion according to some embodiments; 
         FIG. 150  is a cross-sectional view illustrating the floating insert comprising tapered members and the flanged base portion inserted into a corresponding depression according to some embodiments; 
         FIG. 151  is a cross-sectional view illustrating the floating insert comprising tapered members and the flanged base portion inserted into the corresponding depression and assay flow therebetween according to some embodiments; 
         FIG. 152  is a cross-sectional view illustrating the floating insert comprising tapered members and the flanged base portion being forced down onto the corresponding depression according to some embodiments; 
         FIGS. 153-155  are cross-sectional views illustrating the progressive filling and release of assay from the filling apparatus illustrated in  FIG. 145  according to some embodiments; 
         FIGS. 156 and 157  are cross-sectional views illustrating the filling and release of assay from a filling apparatus comprising weight members according to some embodiments; 
         FIG. 158  is a perspective view illustrating a filling apparatus comprising a surface wire assembly and reservoir pockets according to some embodiments; 
         FIG. 159  is a cross-sectional view illustrating the filling apparatus comprising the surface wire assembly according to some embodiments; 
         FIGS. 160-162  are cross-sectional views illustrating the progressive filling of a plurality of staging capillaries according to some embodiments; 
         FIG. 163  is a perspective view illustrating a filling apparatus comprising a surface wire assembly, a reservoir trough, and absorbent member according to some embodiments; 
         FIG. 164  is a perspective view illustrating the filling apparatus comprising the surface wire assembly, the reservoir trough, and absorbent member further comprising a sloping portion according to some embodiments; 
         FIG. 165  is a perspective view illustrating a filling apparatus comprising a surface wire assembly, reservoir pockets, and absorbent members according to some embodiments; 
         FIG. 166  is a perspective view illustrating the filling apparatus comprising the surface wire assembly, reservoir pockets, and absorbent members further comprising a sloping overflow channel portion according to some embodiments; 
         FIG. 167  is a perspective view illustrating a funnel member comprising an assay chamber according to some embodiments; 
         FIG. 168  is a perspective view illustrating a funnel member comprising multiple discrete assay chambers according to some embodiments; 
         FIG. 169  is a perspective view illustrating a funnel member comprising multiple discrete assay chambers according to some embodiments; 
         FIG. 170  is a cross-sectional view illustrating a funnel member comprising a tip portion according to some embodiments; 
         FIG. 171  is a cross-sectional view illustrating a funnel member comprising a tip portion and a wiper member according to some embodiments; 
         FIG. 172  is a cross-sectional view illustrating a funnel member comprising a tip portion and a planar cavity according to some embodiments; 
         FIG. 173  is a cross-sectional view illustrating a funnel member comprising a tip portion and a wiper member spaced apart from the tip portion according to some embodiments; 
         FIG. 174  is a bottom perspective view illustrating a funnel member comprising multiple offset discrete assay chambers according to some embodiments; 
         FIG. 175  is a top plan view illustrating a funnel member comprising multiple offset discrete assay chambers and one or more apertures according to some embodiments; 
         FIG. 176  is a cross-sectional view illustrating a funnel member comprising multiple offset discrete assay chambers and one or more apertures according to some embodiments; 
         FIG. 177  is a top perspective view illustrating a multipiece funnel member comprising multiple offset discrete assay chambers and an internal siphon passage according to some embodiments; 
         FIG. 178  is a cross-sectional view illustrating the multipiece funnel member comprising multiple offset discrete assay chambers and the internal siphon passage according to some embodiments; 
         FIG. 179  is an exploded top perspective view illustrating a multipiece funnel member comprising portions separated generally vertically according to some embodiments; 
         FIG. 180  is an exploded top perspective view illustrating a multipiece funnel member comprising portions separated generally horizontally according to some embodiments; 
         FIG. 181  is a cross-sectional view illustrating a sealing cover according to some embodiments; 
         FIG. 182  is a perspective view illustrating a sealing cover roll according to some embodiments; 
         FIG. 183  is a perspective view illustrating a manual sealing cover applicator according to some embodiments; 
         FIG. 184  is a perspective view illustrating a fixture for use with a manual sealing cover applicator according to some embodiments; 
         FIG. 185  is a perspective view, with portions illustrated in cross-section, illustrating the manual sealing cover applicator according to some embodiments; 
         FIG. 186  is a side view, with portions illustrated in cross-section, illustrating the manual sealing cover applicator in a closed position according to some embodiments; 
         FIG. 187  is a side view, with portions illustrated in cross-section, illustrating the manual sealing cover applicator in an opened position according to some embodiments; 
         FIG. 188  is a perspective view illustrating an automated sealing cover applicator employing a sealing cover roll according to some embodiments; 
         FIG. 189  is a perspective view, with portions removed for clarity, illustrating the automated sealing cover applicator employing the sealing cover roll according to some embodiments; 
         FIG. 190  is a cross-sectional view illustrating the automated sealing cover applicator employing the sealing cover roll according to some embodiments; 
         FIG. 191  is a perspective view illustrating a sealing cover roll cartridge according to some embodiments; 
         FIG. 192  is a cross-sectional view illustrating the sealing cover roll cartridge according to some embodiments; 
         FIG. 193  is a perspective view, with portions removed for clarity, illustrating the automated sealing cover applicator employing a single sheet cartridge according to some embodiments; 
         FIG. 194  is a perspective view, with portions removed for clarity, illustrating a single sheet applicator assembly according to some embodiments; 
         FIG. 195  is a perspective view, with portions removed for clarity, illustrating a single cover cartridge according to some embodiments; 
         FIG. 196  is an enlarged cross-sectional view illustrating the single cover cartridge according to some embodiments; 
         FIG. 197  is an exploded perspective view illustrating the single cover cartridge according to some embodiments; 
         FIGS. 198-201  are cross-sectional views illustrating progressive steps of applying a single sealing cover to a microplate according to some embodiments; 
         FIG. 202  is an exploded view illustrating an inverted configuration of a pressure chamber according to some embodiments; 
         FIG. 203  is a cross-sectional view illustrating section A-A of the pressure chamber of  FIG. 202  in combination with a thermocycler system according to some embodiments; 
         FIG. 204  is a side view illustrating a clamp mechanism in a locked condition according to some embodiments; 
         FIG. 205  is a side view illustrating a clamp mechanism in an unlocked condition according to some embodiments; 
         FIG. 206  is a bottom perspective view illustrating a clamp mechanism in a locked condition according to some embodiments; 
         FIG. 207  is a pneumatic diagram illustrating a pneumatic system for a pressure chamber and a clamp mechanism according to some embodiments; 
         FIG. 208  is a perspective view illustrating the pneumatic system of  FIG. 207  according to some embodiments; 
         FIG. 209  is a flow diagram illustrating a method of clamping a chamber to a thermocycler system according to some embodiments; 
         FIG. 210  is a flow diagram illustrating a method of performing a leak test on a chamber according to some embodiments; 
         FIG. 211  is a flow diagram illustrating a method of unclamping a chamber from a thermocycler system according to some embodiments; 
         FIG. 212  is a cross-sectional view illustrating an adjustable lens and camera mount according to some embodiments; and 
         FIG. 213  is a flowchart illustrating a process for determining bias. 
     
    
    
     DESCRIPTION OF VARIOUS EMBODIMENTS 
     The following description of various embodiments is merely exemplary in nature and is in no way intended to limit the present teachings, applications, or uses. Although the present teachings will be discussed in some embodiments as relating to polynucleotide amplification, such as PCR, such discussion should not be regarded as limiting the present teaching to only such applications. 
     The section headings and sub-headings used herein are for general organizational purposes only and are not to be construed as limiting the subject matter described in any way. 
     High-Density Sequence Detection System 
     In some embodiments, a high density sequence detection system comprises one or more components useful in an analytical method or chemical reaction, such as the analysis of biological and other materials containing polynucleotides. Such systems are, in some embodiments, useful in the analysis of assays, as further described below. High density sequence detection systems, in some embodiments, comprise an excitation system and a detection system which can be useful for analytical methods involving the generation and/or detection of electromagnetic radiation (e.g., visible, ultraviolet or infrared light) generated during analytical procedures. In some embodiments, such procedures include those comprising the use of fluorescent or other materials that absorb and/or emit light or other radiation under conditions that allow quantitative and/or qualitative analysis of a material (e.g., assays among those described herein). In some embodiments useful for polynucleotide amplification and/or detection, a high density sequence detection system can further comprise a thermocycler. In some embodiments, a high density sequence system can further comprise microplate and components for, e.g., filling and handling the microplate, such as a pressure clamp system. It will be understood that, although high density sequence detection systems are described herein with respect to specific microplates, assays and other embodiments, such systems and components thereof are useful with a variety of analytical platforms, equipment, and procedures. 
     Referring to  FIG. 1 , a high-density sequence detection system  10  is illustrated in accordance with some embodiments of the present teachings. In some embodiments, high-density sequence detection system  10  comprises a microplate  20  containing an assay  1000  (see  FIGS. 26 and 27 ), a thermocycler system  100 , a pressure clamp system  110 , an excitation system  200 , and a detection system  300  disposed in a housing  1008 . 
     In some embodiments, assay  1000  can comprise any material that is useful in, the subject of, a precursor to, or a product of, an analytical method or chemical reaction. In some embodiments for amplification and/or detection of polynucleotides, assay  1000  comprises one or more reagents (such as PCR master mix, as described further herein); an analyte (such as a biological sample comprising DNA, a DNA fragment, cDNA, RNA, or any other nucleic acid sequence), one or more primers, one or more primer sets, one or more detection probes; components thereof; and combinations thereof. In some embodiments, assay  1000  comprises a homogenous solution of a DNA sample, at least one primer set, at least one detection probe, a polymerase, and a buffer, as used in a homogenous assay (described further herein). In some embodiments, assay  1000  can comprise an aqueous solution of at least one analyte, at least one primer set, at least one detection probe, and a polymerase. In some embodiments, assay  1000  can be an aqueous homogenous solution. In some embodiments, assay  1000  can comprise at least one of a plurality of different detection probes and/or primer sets to perform multiplex PCR, which can be useful, for example, when analyzing a whole genome (e.g., 20,000 to 30,000 genes, or more) or other large numbers of genes or sets of genes. 
     Microplate 
     In some embodiments, a microplate comprises a substrate useful in the performance of an analytical method or chemical reaction. In some embodiments, a microplate can comprise one or more material retention regions, configured to hold or support a material (e.g., an assay, as discussed below, or other solid or liquid) at one or more locations on or in the microplate. In some embodiments, such material retention regions can be wells, through-holes, hydrophilic spots or pads, and the like. In some embodiments, such as shown in  FIG. 2-19 , material retention regions comprise wells, as at  26 . In some embodiments, such wells can comprise a feature on or in the surface of the microplate wherein assay  1000  is contained at least in part by physical separation from adjacent features. Such well features can include, in some embodiments, depressions, indentations, ridges, and combinations thereof, in regular or irregular shapes. In some embodiments a microplate is single-use, wherein it is filled or otherwise used with a single assay for a single experiment or set of experiments, and is thereafter discarded. In some embodiments, a microplate is multiple-use, wherein it can be operable for use in a plurality of experiments or sets of experiments. 
     Referring now to  FIGS. 2-19 , in some embodiments, microplate  20  comprises a substantially planar construction having a first surface  22  and an opposing second surface  24  (see  FIG. 12-19 ). First surface  22  comprises a plurality of wells  26  disposed therein or thereon. The overall positioning of the plurality of wells  26  can be referred to as a well array. Each of the plurality of wells  26  is sized to receive assay  1000  ( FIGS. 26 and 27 ). As illustrated in  FIGS. 26 and 27 , assay  1000  is disposed in at least one of the plurality of wells  26  and sealing cover  80  ( FIG. 26 ) is disposed thereon (as will be discussed herein). In some embodiments, one or more of the plurality of wells  26  may not be completely filled with assay  1000 , thereby defining a headspace  1006  ( FIG. 26 ), which can define an air gap or other gas gap. 
     In some embodiments, the material retention regions of microplate  20  can comprise a plurality of reaction spots on the surface of the microplate. In such embodiments, a reaction spot can be an area on the substrate which localizes, at least in part by non-physical means, assay  1000 . In such embodiments, assay  1000  can be localized in sufficient quantity, and isolation from adjacent areas on the microplate, so as to facilitate an analytical or chemical reaction (e.g., amplification of one or more target DNA) in the material retention region. Such localization can be accomplished by physical and chemical modalities, including, for example, physical containment of reagents in one dimension and chemical containment in one or more other dimensions. 
     In some embodiments, the surface of the microplate  20  comprises an enhanced surface which can comprise a physical or chemical modality on or in the surface of the microplate so as to enhance support of, or filling of, assay  1000  in a material retention region (e.g., a well or a reaction spot). Such modifications can include chemical treatment of the surface, or coating the surface. In some embodiments, such chemical treatment can comprise chemical treatment or modification of the surface of the microplate so as to form relatively hydrophilic and hydrophobic areas. In some embodiments, a surface tension array can be formed comprising a pattern of hydrophilic sites forming reaction spots on a hydrophobic matrix, such that the hydrophilic sites can be spatially segregated by hydrophobic regions. Reagents delivered to the array can be constrained by surface tension difference between hydrophilic and hydrophobic sites. 
     In some embodiments, the chemical modality can comprise chemical treatment or modification of the surface or other material of microplate  20  so as to affix one or more components of assay  1000  to the microplate. In such embodiments, assay  1000  can be affixed to microplate  20 , directly or indirectly, so that assay  1000  is operable for analysis or reaction, but is not removed or otherwise displaced from the microplate prior to the analysis or reaction during routine handling of the microplate. In some embodiments, assay  1000  can be affixed to the surface so as form a patterned array (immobilized reagent array) of reaction spots. In some embodiments, an immobilization reagent array can comprise a hydrogel affixed to the microplate. Such hydrogels can include, for example, cellulose gels, such as agarose and derivatized agarose (e.g., low melting agarose, monoclonal anti-biotin agarose, and streptavidin derivatized agarose); xanthan gels; synthetic hydrophilic polymers, such as crosslinked polyethylene glycol, polydimethyl acrylamide, polyacrylamide, polyacrylic acid (e.g., cross-linked with dysfunctional monomers or radiation cross-linking), and micellar networks; and combinations thereof. 
     In some embodiments, one or more components of assay  1000  can be affixed to microplate  20  by covalent or non-covalent bonding to the surface of the microplate. In certain embodiments, assay  1000  an be bonded, anchored or tethered to a second moiety (immobilization moiety) which, in turn, can be anchored to the surface of the microplate. In some embodiments, such anchoring is through a chemically releasable or cleavable moeity, such that assay  1000  can be released or made available for analysis or reaction after reacting with a cleaving reagent prior to, during, or after the microplate assembly. Such release methods can include a variety of enzymatic, or non-enzymatic means, such as chemical, thermal, or photolytic treatment. In some embodiments, chemical moieties for immobilization moieties can include those comprising carbamate, ester, amide, thiolester, (N)-functionalized thiourea, functionalized maleimide, amino, disulfide, amide, hydrazone, streptavidin, avidin/biotin, and gold-sulfide groups. 
     Microplate Footprint With reference to  FIGS. 2-19 , microplate  20  generally comprises a main body or substrate  28 . In some embodiments, main body  28  is substantially planar. In some embodiments, microplate  20  comprises an optional skirt or flange portion  30  disposed about a periphery of main body  28  (see  FIG. 2 ). Skirt portion  30  can form a lip around main body  28  and can vary in height. Skirt portion  30  can facilitate alignment of microplate  20  on thermocycler block  102 . Additionally, skirt portion  30  can provide additional rigidity to microplate  20  such that during handling, filling, testing, and the like, microplate  20  remains rigid, thereby ensuring assay  1000 , or any other components, disposed in each of the plurality of wells  26  does not contaminate adjacent wells. However, in some embodiments, microplate  20  can employ a skirtless design (see  FIGS. 3-5 ) depending upon user preference. 
     In order to facilitate use with existing equipment, robotic implements, and instrumentation, the footprint dimensions of main body  28  and/or skirt portion  30  of microplate  20 , in some embodiments, can conform to standards specified by the Society of Biomolecular Screening (SBS) and the American National Standards Institute (ANSI), published January 2004 (ANSI/SBS 3-2004). In some embodiments, the footprint dimensions of main body  28  and/or skirt portion  30  of microplate  20  are about 127.76 mm (5.0299 inches) in length and about 85.48 mm (3.3654 inches) in width. In some embodiments, the outside corners of microplate  20  comprise a corner radius of about 3.18 mm (0.1252 inches). In some embodiments, microplate  20  comprises a thickness of about 0.5 mm to about 3.0 mm. In some embodiments, microplate  20  comprises a thickness of about 1.25 mm. In some embodiments, microplate  20  comprises a thickness of about 2.25 mm. One skilled in the art will recognize that microplate  20  and skirt portion  30  can be formed in dimensions other than those specified herein. 
     Plurality of Wells 
     In order to increase throughput of genotyping, gene expression, and other assays, in some embodiments, microplate  20  comprises an increased quantity of the plurality of wells  26  beyond that employed in prior conventional microplates. In some embodiments, microplate  20  comprises 6,144 wells. According to the present teachings, microplate  20  can comprise, but is not limited to, any of the array configurations of wells described in Table 1. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Total Number of Wells 
                 Rows × Columns 
                 Approximate Well Area 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 96 
                  8 × 12 
                 9 × 9 
                 mm 
               
               
                 384 
                 16 × 24 
                 4.5 × 4.5 
                 mm 
               
               
                 1536 
                 32 × 48 
                 2.25 × 2.25 
                 mm 
               
               
                 3456 
                 48 × 72 
                 1.5 × 1.5 
                 mm 
               
               
                 6144 
                 64 × 96 
                 1.125 × 1.125 
                 mm 
               
               
                 13824 
                  96 × 144 
                 0.75 × .075 
                 mm 
               
               
                 24576 
                 128 × 192 
                 0.5625 × 6.5625 
                 mm 
               
               
                 55296 
                 192 × 288 
                 0.375 × 0.375 
                 mm 
               
               
                 768 
                 24 × 32 
                 3 × 3 
                 mm 
               
               
                 1024 
                 32 × 32 
                 2.25 × 3 
                 mm 
               
               
                 1600 
                 40 × 40 
                 1.8 × 2.7 
                 mm 
               
               
                 1280 
                 32 × 40 
                 2.25 × 2.7 
                 mm 
               
               
                 1792 
                 32 × 56 
                 2.25 × 1.714 
                 mm 
               
               
                 2240 
                 40 × 56 
                 1.8 × 1.714 
                 mm 
               
               
                 864 
                 24 × 36 
                 3 × 3 
                 mm 
               
               
                 4704 
                 56 × 84 
                 1.257 × 1.257 
                 mm 
               
               
                 7776 
                  72 × 108 
                 1 × 1 
                 mm 
               
               
                 9600 
                  80 × 120 
                 0.9 × .09 
                 mm 
               
               
                 11616 
                  88 × 132 
                 0.818 × 0.818 
                 mm 
               
               
                 16224 
                 104 × 156 
                 0.692 × 0.692 
                 mm 
               
               
                 18816 
                 112 × 168 
                 0.643 × 0.643 
                 mm 
               
               
                 21600 
                 120 × 180 
                 0.6 × 0.6 
                 mm 
               
               
                 27744 
                 136 × 204 
                 0.529 × 0.529 
                 mm 
               
               
                 31104 
                 144 × 216 
                 0.5 × 0.5 
                 mm 
               
               
                 34656 
                 152 × 228 
                 0.474 × 0.474 
                 mm 
               
               
                 38400 
                 160 × 240 
                 0.45 × 0.45 
                 mm 
               
               
                 42336 
                 168 × 252 
                 0.429 × 0.429 
                 mm 
               
               
                 46464 
                 176 × 264 
                 0.409 × 0.409 
                 mm 
               
               
                 50784 
                 184 × 256 
                 0.391 × 0.391 
                 mm 
               
               
                   
               
            
           
         
       
     
     Well Shape 
     According to some embodiments, as illustrated in  FIGS. 4 and 5 , each of the plurality of wells  26  can be substantially equivalent in size. The plurality of wells  26  can have any cross-sectional shape. In some embodiments, as illustrated in  FIGS. 4 ,  26 , and  27 , each of the plurality of wells  26  comprises a generally circular rim portion  32  ( FIG. 4 ) with a downwardly-extending, generally-continuous sidewall  34  that terminate at a bottom wall  36  interconnected to sidewall  34  with a radius. A draft angle of sidewall  34  can be used in some embodiments. In some embodiments, the draft angle provides benefits including increased ease of manufacturing and minimizing shadowing (as discussed herein). The particular draft angle is determined, at least in part, by the manufacturing method and the size of each of the plurality of wells  26 . In some embodiments, circular rim portion  32  can be about 1.0 mm in diameter, the depth of each of the plurality of wells  26  can be about 0.9 mm, the draft angle of sidewall  34  can be about 1° to 5° or greater and each of the plurality of wells  26  can have a center-to-center distance of about 1.125 mm. In some embodiments, the volume of each of the plurality of wells  26  can be about 500 nanoliters. 
     According to some embodiments, as illustrated in  FIG. 5 , each of the plurality of wells  26  comprises a generally square-shaped rim portion  38  with downwardly-extending sidewalls  40  that terminate at a bottom wall  42 . A draft angle of sidewalls  40  can be used. Again, the particular draft angle is determined, at least in part, by the manufacturing method and the size of each of the plurality of wells  26 . In some embodiments of wells  26  of  FIG. 5 , generally square-shaped rim portion  38  can have a side dimension of about 1.0 mm in length, a depth of about 0.9 mm, a draft angle of about 1° to 5° or greater, and a center-to-center distance of about 1.125 mm, generally indicated at A (see  FIG. 27 ). In some embodiments, the volume of each of the plurality of wells  26  of  FIG. 5  can be about 500 nanoliters. In some embodiments, the spacing between adjacent wells  26 , as measured at the top of a wall dividing the wells, is less than about 0.5 m. In some embodiments, this spacing between adjacent wells  26  is about 0.25 mm. 
     In some embodiments, and in some configurations, the plurality of wells  26  comprising a generally circular rim portion  32  can provide advantages over the plurality of wells  26  comprising a generally square-shaped rim portion  38 . In some embodiments, during heating, it has been found that assay  1000  can migrate through capillary action upward along edges of sidewalls  40 . This can draw assay  1000  from the center of each of the plurality of wells  26 , thereby causing variation in the depth of assay  1000 . Variations in the depth of assay  1000  can influence the emission output of assay  1000  during analysis. Additionally, during manufacture of microplate  20 , in some cases cylindrically shaped mold pins used to form the plurality of wells  26  comprising generally circular rim portion  32  can permit unencumbered flow of molten polymer thereabout. This unencumbered flow of molten polymer results in less deleterious polymer molecule orientation. In some embodiments, generally circular rim portion  32  provides more surface area along microplate  20  for improved sealing with sealing cover  80 , as is discussed herein. 
     Pressure Relief Bores 
     Referring now to  FIGS. 6-9 , in some embodiments, each of the plurality of wells  26  of microplate  20  can comprise a pressure relief bore  44 . In some embodiments, pressure relief bore  44  is sized such that it does not initially fill with assay  1000  due to surface tension. However, when assay  1000  is heated during thermocycling, assay  1000  expands, thereby increasing an internal fluid pressure in each of the plurality of wells  26 . This increased internal fluid pressure is sufficient to permit assay  1000  to flow into pressure relief bore  44  as illustrated in  FIG. 7 , thereby minimizing the pressure exerted on sealing cover  80 . In some embodiments, each of the plurality of wells  26  can have one or a plurality of pressure relief bores  44 . 
     In some embodiments, as illustrated in  FIGS. 8 and 9 , pressure relief bore  44  can be offset within each of the plurality of wells  26  so that each of the plurality of wells  26  can be filled with assay  1000  or other material  1004  via a spotting device  700  ( FIG. 8 ) or a micro-piezo dispenser  702  ( FIG. 9 ). In some embodiments, a top edge  46  of pressure relief bore  44  can be generally square and have minimal or no radius. This arrangement can reduce the likelihood that assay  1000  or other material  1004  will enter pressure relief bore  44  prior to thermocycling. 
     Through-Hole Wells 
     Turning now to  FIGS. 10 ,  33 , and  36 , in some embodiments, each of the plurality of wells  26  of microplate  20  comprises a plurality of apertures  48  being sealed at least on one end by sealing cover  80 . In some embodiments, each of the plurality of apertures  48  is sealed on an opposing end with a foil seal  50 , which can have a clear or opaque adhesive. In these embodiments, foil seal  50  can be placed against thermocycler block  102  to aid in thermal conductivity and distribution. 
     In some embodiments, a layer of mineral oil can be placed at the top of each of the plurality of apertures  48  before, or as an alternative to, placement of sealing cover  80  on microplate  20 . In several of such embodiments, the mineral oil can fill a portion of each of the plurality of apertures  48  and provide an optical interface and can control evaporation of assay  1000 . 
     Grooves 
     Referring to  FIGS. 11-15 , in some embodiments, microplate  20  can comprise grooves  52  and grooves  54  disposed about a periphery of the plurality of wells  26 . In some embodiments, grooves  52  can have depth and width dimensions generally similar to the depth and width dimensions of the plurality of wells  26  ( FIGS. 12 and 13 ). In some embodiments, grooves  54  can have depth and width dimensions less than the depth and width dimensions of the plurality of wells  26  ( FIGS. 14 and 15 ). In some embodiments, as illustrated in  FIG. 12 , additional grooves  56  can be disposed at opposing sides of microplate  20 . In some embodiments, grooves  52 ,  54 , and  56  can improve thermal uniformity among the plurality of wells  26  in microplate  20 . In some embodiments, grooves  52 ,  54 , and  56  can improve the sealing interface formed by sealing cover  80  and microplate  20 . Grooves  52 ,  54 , and  56  can also assist in simplifying the injection molding process of microplate  20 . In some embodiments, a liquid solution similar to assay  1000  can be disposed in grooves  52 ,  54 , and  56  to, in part, improve thermal uniformity during thermocycling. 
     Alignment Features 
     In some embodiments, as illustrated in  FIGS. 2 ,  3 ,  11 , and  14 , microplate  20  comprises an alignment feature  58 , such as a corner chamfer, a pin, a slot, a cut corner, an indentation, a graphic, or other unique feature that is capable of interfacing with a corresponding feature formed in a fixture, reagent dispensing equipment, and/or thermocycler. In some embodiments, alignment feature  58  comprises a nub or protrusion  60  as illustrated in  FIG. 14 . Additionally, in some embodiments, alignment features  58  are placed such that they do not interfere with sealing cover  80  or at least one of the plurality of wells  26 . However, locating alignment features  58  near at least one of the plurality of wells  26  can provide improved alignment with dispensing equipment and/or thermocycler block  102 . 
     Thermally Isolated Portion 
     In some embodiments, as illustrated in  FIGS. 16-19 , microplate  20  comprises a thermally isolated portion  62 . Thermally isolated portion  62  can be disposed along at least one edge of main body  28 . Thermally isolated portion  62  can be generally free of wells  26  and can be sized to receive a marking indicia  64  (discussed in detail herein) thereon. Thermally isolated portion  62  can further be sized to facilitate the handling of microplate  20  by providing an area that can be easily gripped by a user or mechanical device without disrupting the plurality of wells  26 . 
     Still referring to  FIGS. 16-19 , in some embodiments, microplate  20  comprises a first groove  66  formed along first surface  22  and a second groove  68  formed along an opposing second surface  24  of microplate  20 . First groove  66  and second groove  68  can be aligned with respect to each other to extend generally across microplate  20  from a first side  70  to a second side  72 . First groove  66  and second groove  68  can be further aligned upon first surface  22  and second surface  24  to define a reduced cross-section  74  between thermally isolated portion  62  and the plurality of wells  26 . This reduced cross-section  74  can provide a thermal isolation barrier to reduce any heat sink effect introduced by thermally isolated portion  62 , which might otherwise reduce the temperature cycle of some of the plurality of wells  26 . 
     Marking Indicia 
     In some embodiments, as illustrated in  FIGS. 2 ,  16  and  17 , microplate  20  comprises marking indicia  64 , such as graphics, printing, lithograph, pictorial representations, symbols, bar codes, handwritings or any other type of writing, drawings, etchings, indentations, embossments or raised marks, machine readable codes (i.e. bar codes, etc.), text, logos, colors, and the like. In some embodiments, marking indicia  64  is permanent. 
     In some embodiments, marking indicia  64  can be printed upon microplate  20  using any known printing system, such as inkjet printing, pad printing, hot stamping, and the like. In some embodiments, such as those using a light-colored microplate  20 , a dark ink can be used to create marking indicia  64  or vice versa. 
     In some embodiments, microplate  20  can be made of polypropylene and have a surface treatment applied thereto to facilitate applying marking indicia  64 . In some embodiments, such surface treatment comprises flame treatment, corona treatment, treating with a surface primer, or acid washing. However, in some embodiments, a UV-curable ink can be used for printing on polypropylene microplates. 
     Still further, in some embodiments, marking indicia  64  can be printed upon microplate  20  using a CO 2  laser marking system. Laser marking systems evaporate material from a surface of microplate  20 . Because CO 2  laser etching can produce reduced color changes of marking indicia  64  relative to the remaining portions of microplate  20 , in some embodiments, a YAG laser system can be used to provide improved contrast and reduced material deformation. 
     In some embodiments, a laser activated pigment can be added to the material used to form microplate  20  to obtain improved contrast between marking indicia  64  and main body  28 . In some embodiments, an antimony-doped tin oxide pigment can be used, which is easily dispersed in polymers and has marking speeds as high as 190 inches per second. Antimony-doped tin oxide pigments can absorb laser light and can convert laser energy to thermal energy in embodiments where indicia are created using a YAG laser. 
     In some embodiments, marking indicia  64  can identify microplates  20  to facilitate identification during processing. Furthermore, in some embodiments, marking indicia  64  can facilitate data collection so that microplates  20  can be positively identified to properly correlate acquired data with the corresponding assay. Such marking indicia  64  can be employed as part of Good Laboratory Practices (GLP) and Good Manufacturing Practices (GMP), and can further, in some circumstances, reduce labor associated with manually applying adhesive labels, manually tracking microplates, and correlating data associated with a particular microplate. 
     In some embodiments, marking indicia  64  can assist in alignment by placing a symbol or other machine-readable graphic on microplate  20 . An optical sensor or optical eye  1491  ( FIG. 204 ) can detect marking indicia  64  and can determine a location of microplate  20 . In some embodiments, such location of microplate  20  can then be adjusted to achieve a predetermined position using, for example, a drive system of high-density sequence detection system  10 , sealing cover applicator  1100 , or other corresponding systems. 
     In some embodiments, the type (physical properties, characteristics, etc.) of marking indicia employed on a microplate can be selected so as to reduce thermal and/or chemical interference during thermocycling relative to what might otherwise occur with other types of marking indicia (e.g., common prior indicia designs, such as adhesive labels). For example, adhesive labels can, in some circumstances, interfere (e.g., chemically interact) with one or more reagents (e.g., dyes) being used. 
     Referring to  FIG. 2 , in some embodiments, a radio frequency identification (RFID) tag  76  can be used to electronically identify microplate  20 . RFID tag  76  can be attached or molded within microplate  20 . An RFID reader (not illustrated) can be integrated into high-density sequence detection system  10  to automatically read a unique identification and/or data handling parameters of microplate  20 . Further, RFID tag  76  does not require line-of-sight for readability. It should be appreciated that RFID tag  76  can be variously configured and used according to various techniques, such as those described in commonly-assigned U.S. Patent Application No. ______, entitled “SAMPLE CARRIER DEVICE INCORPORATING RADIO FREQUENCY IDENTIFICATION, AND METHOD” filed herewith (Attorney Docket No. 5010-193). 
     Multi-Piece Construction 
     In some embodiments, such as illustrated in  FIGS. 59-63 , microplate  20  can comprise a multi-piece construction. In some embodiments, microplate  20  can comprise main body  28  and a separate cap portion  95  that can be connected with main body  28 . In some embodiments, cap portion  95  can be sized and/or shaped to mate with main body  28  such that the combination thereof results in a footprint that conforms to the above-described SBS and/or ANSI standards. Alternatively, main body  28  and/or cap portion  95  can comprise non-standard dimensions, as desired. 
     Cap portion  95  can be coupled with main body  28  in a variety of ways. In some embodiments, cap portion  95  comprises a cavity  96  ( FIG. 63 ), such as a mortis, sized and/or shaped to receive a support member  97 , such as a tenon, extending from main body  28  to couple cap portion  95  with main body  28 . In some embodiments, cavity  96  of cap portion  95  and support member  97  of main body  28  can comprise an interference fit or other locking feature, such as a hook member, to at least temporarily join main body  28  and cap portion  95  during assembly. In some embodiments, support member  97  of main body  28  can comprise a cap alignment feature  98  that can interface with a corresponding feature  99  on cap portion  95  to properly align cap portion  95  relative to main body  28 . In some embodiments, cap portion  95  can comprise alignment feature  58  for use in later alignment of microplate  20  as described herein. In some embodiments, alignment feature  58  can be disposed on main body  28  to reduce tolerance buildup caused by the interface of cap portion  95  and main body  28 . 
     In some embodiments, cap portion  95  can be formed directly on main body  28 , such as through over-molding. In such embodiments, main body  28  can be placed within a mold cavity that generally closely conforms to main body  28  and defines a cap portion cavity generally surrounding support member  97  of main body  28 . Over-molding material can then be introduced about support member  97  within cap portion cavity to form cap portion  95  thereon. 
     In some embodiments, cap portion  95  comprises marking indicia  64  on any surface(s) thereon (e.g. top surface, bottom surface, side surface). In some embodiments, cap portion  95  can comprise an enlarged print area thereon relative to embodiments employing first groove  66  ( FIG. 16-19 ). In some embodiments, cap portion  95  can be made of a material different from main body  28 . In some embodiments, cap portion  95  can be made of a material that is particularly conducive to a desired form of printing or marking, such as through laser marking. In some embodiments, a laser-activated pigment can be added to the material used to form cap portion  95  to obtain improved contrast between marking indicia  64  and cap portion  95 . In some embodiments, an antimony-doped tin oxide pigment can be used. In some embodiments, cap portion  95  can be color-coded to aid in identifying a particular microplate relative to others. 
     In some embodiments, cap portion  95  can serve to provide a thermal isolation barrier through the interface of cavity member  96  and support member  97  to reduce any heat sink effect of cap portion  95  relative to main body  28  to maintain a generally consistent temperature cycle of the plurality of wells  26 . Cap portion  95  can be made, for example, of a non-thermally conductive material, such as one or more of those set forth herein, to, at least in part, help to thermally isolate cap portion  95  from main body  28 . 
     In some embodiments, cap portion  95  can serve to conceal any injection molding gates coupled to support member  97  during molding. During manufacturing, as such gates are removed from any product, aesthetic variations can result. Any such aesthetic variations in main body  28  can be concealed in some embodiments using cap portion  95 . In some case, injection-molding gates can lead to a localized increase in flourescence. In some embodiments, such localized increase in flourescence can be reduced using cap portion  95 . 
     Microplate Material 
     In some embodiments, microplate  20  can comprise, at least in part, a thermally conductive material. In some embodiments, a microplate, in accordance with the present teachings, can be molded, at least in part, of a thermally conductive material to define a cross-plane thermal conductivity of at least about 0.30 W/mK or, in some embodiments, at least about 0.58 W/mK. Such thermally conductive materials can provide a variety of benefits, such as, in some cases, improved heat distribution throughout microplate  20 , so as to afford reliable and consistent heating and/or cooling of assay  1000 . In some embodiments, this thermally conductive material comprises a plastic formulated for increased thermal conductivity. Such thermally conductive materials can comprise, for example and without limitation, at least one of polypropylene, polystyrene, polyethylene, polyethyleneterephthalate, styrene, acrylonitrile, cyclic polyolefin, syndiotactic polystyrene, polycarbonate, liquid crystal polymer, conductive fillers or plastic materials; and mixtures or combinations thereof. In some embodiments, such thermally conductive materials include those known to those skilled in the art with a melting point greater than about 130° C. For example, microplate  20  can be made of commercially available materials such as RTP199×104849, COOLPOLY E1201, or, in some embodiments, a mixture of about 80% RTP199X104849 and 20% polypropylene. 
     In some embodiments, microplate  20  can comprise at least one carbon filler, such as carbon, graphite, impervious graphite, and mixtures or combinations thereof. In some cases, graphite has an advantage of being readily and cheaply available in a variety of shapes and sizes. One skilled in the art will recognize that impervious graphite can be non-porous and solvent-resistant. Progressively refined grades of graphite or impervious graphite can provide, in some cases, a more consistent thermal conductivity. 
     In some embodiments, one or more thermally conductive ceramic fillers can be used, at least in part, to form microplate  20 . In some embodiments, the thermally conductive ceramic fillers can comprise boron nitrate, boron nitride, boron carbide, silicon nitride, aluminum nitride, and mixtures or combinations thereof. 
     In some embodiments, microplate  20  can comprise an inert thermally conductive coating. In some embodiments, such coatings can include metals or metal oxides, such as copper, nickel, steel, silver, platinum, gold, copper, iron, titanium, alumina, magnesium oxide, zinc oxide, titanium oxide, and mixtures thereof. 
     In some embodiments, microplate  20  comprises a mixture of a thermally conductive material and other materials, such as non-thermally conductive materials or insulators. In some embodiments, the non-thermally conductive material comprises glass, ceramic, silicon, standard plastic, or a plastic compound, such as a resin or polymer, and mixtures thereof to define a cross-plane thermal conductivity of below about 0.30 W/mK. In some embodiments, the thermally conductive material can be mixed with liquid crystal polymers (LCP), such as wholly aromatic polyesters, aromatic-aliphatic polyesters, wholly aromatic poly(ester-amides), aromatic-aliphatic poly(ester-amides), aromatic polyazomethines, aromatic polyester-carbonates, and mixtures thereof. In some embodiments, the composition of microplate  20  can comprise from about 30% to about 60%, or from about 38% to about 48% by weight, of the thermally conductive material. 
     The thermally conductive material and/or non-thermally conductive material can be in the form of, for example, powder particles, granular powder, whiskers, flakes, fibers, nanotubes, plates, rice, strands, hexagonal or spherical-like shapes, or any combination thereof. In some embodiments, the microplate comprises thermally conductive additives having different shapes to contribute to an overall thermal conductivity that is higher than any one of the individual additives alone. 
     In some embodiments, the thermally conductive material comprises a powder. In some embodiments, the particle size used herein can be between 0.10 micron and 300 microns. When mixed homogeneously with a resin in some embodiments, powders provide uniform (i.e. isotropic) thermal conductivity in all directions throughout the composition of the microplate. 
     As discussed above, in some embodiments, the thermally conductive material can be in the form of flakes. In some such embodiments, the flakes can be irregularly shaped particles produced by, for example, rough grinding to a desired mesh size or the size of mesh through which the flakes can pass. In some embodiments, the flake size can be between 1 micron and 200 microns. Homogenous compositions containing flakes can, in some cases, provide uniform thermal conductivity in all directions. 
     In some embodiments, the thermally conductive material can be in the form of fibers, also known as rods. Fibers can be described, among other ways, by their lengths and diameters. In some embodiments, the length of the fibers can be, for example, between 2 mm and 15 mm. The diameter of the fibers can be, for example, between 1 mm and 5 mm. Formulations that include fibers in the composition can, in some cases, have the benefit of reinforcing the resin for improved material strength. 
     In some embodiments, microplate  20  can comprise a material comprising additives to promote other desirable properties. In some embodiments, these additives can comprise flame-retardants, antioxidants, plasticizers, dispersing aids, marking additives, and mold-releasing agents. In some embodiments, such additives are biologically and/or chemically inert. 
     In some embodiments, microplate  20  comprises, at least in part, an electrically conductive material, which can improve reagent dispensing alignment. In this regard, electrically conductive material can reduce static build-up on microplate  20  so that the reagent droplets will not go astray during dispensing. In some embodiments, a voltage can be applied to microplate  20  to pull the reagent droplets into a predetermined position, particularly with a co-molded part where the bottom section can be electrically conductive and the sides of the plurality of wells  26  may not be electrically conductive. In some embodiments, a voltage field applied to the electrically conductive material under the well or wells of interest can pull assay  1000  into the appropriate wells. 
     In some embodiments, microplate  20  can be made, at least in part, of non-electrically conductive materials. In some embodiments, non-electrically conductive materials can at least in part comprise one or more of crystalline silica (3.0 W/mK), aluminum oxide (42 W/mK), diamond (2000 W/mK), aluminum nitride (150-220 W/mK), crystalline boron nitride (1300 W/mK), and silicon carbide (85 W/mK). 
     Microplate Molding 
     In some embodiments, microplate  20  can be molded by first extruding a melt blend comprising a mixture of a polymer and one or more thermally conductive materials and/or additives. In some embodiments, the polymer and thermally conductive additives can be fed into a twin-screw extruder using a gravimetric feeder to create a well-dispersed melt blend. In some embodiments, the extruded melt blend can be transferred through a water bath to cool the melt blend before being pelletized and dried. The pelletized melt blend can then be heated above its melting point by an injection molding machine and then injected into a mold cavity. The mold cavity can generally conform to a desired shape of microplate  20 . In some embodiments, the injection-molding machine can cool the injected melt blend to create microplate  20 . Finally, microplate  20  can be removed from the injection-molding machine. 
     In some embodiments, two or more material types of pellets can be mixed together and the combination then placed in the injection molding machine to be melt blended during the injection molding process. In some embodiments, microplate  20  can be molded by first receiving pellet material from a resin supplier; drying the pellet material in a resin dryer; transferring the dried pellet material with a vacuum system into a hopper of a mold press; molding microplate  20 ; trimming any resultant gates or flash; and packaging microplate  20 . In some embodiments, the mold cavity can be centrally gated along the second surface  24  of microplate  20 . In some embodiments, the mold cavity can be gated along a perimeter of main body  28  and/or skirt portion  30  of microplate  20 . 
     Microplate Spotting, Filling, and Sealing 
     In some embodiments, one or more devices can be used to facilitate the placement of one or more components of assay  1000  within at least some of the plurality of wells  26  of microplate  20 . 
     Microplate Spotting 
     In some embodiments, as illustrated in  FIG. 57 , microplate  20  can be preloaded with at least some component materials of assay  1000 , such as reagents. In some embodiments, as described further herein, such reagents can comprise at least one primer and at least one detection probe. In some embodiments, such reagents can comprise elements facilitating analysis of a whole genome or a portion of a genome. Still further, in some embodiments, such reagents can comprise buffers and/or additives useful for coating, stability, enhanced rehydration, preservation, and/or enhanced dispensing of reagents. 
     In some embodiments, such reagents can be delivered (e.g. spotted) into at least one of the plurality of wells  26  of microplate  20  in very small, e.g. nanoliter, increments using a spotting device  700  ( FIG. 8 ). In some embodiments, spotting device  700  employs one or more piezoelectric pumps, acoustic dispersion, liquid printers, micropiezo dispensers, or the like to deliver such reagents to each of the plurality of wells. In some embodiments, spotting device  700  employs an apparatus and method like or similar to that described in commonly assigned U.S. Pat. Nos. 6,296,702, 6,440,217, 6,579,367, and 6,849,127, issued to Vann et al. 
     According to some embodiments, in operation, as schematically illustrated in  FIG. 57 , reagents, e.g. in an aqueous form or bead form, can be stored on one or more storage plates  704  in a high-humidity storage unit  706 . In some embodiments, high-humidity storage unit  706  can comprise a relative humidity in the range of about 70-100%. However, in some embodiments, high-humidity storage unit  706  can comprise a relative humidity in the range of about 70-85%. The bead form can be like or similar to that described in commonly assigned U.S. Pat. No. 6,432,719 to Vann et al. Some of the plurality of storage plates  704  can be moved out of high-humidity storage unit  706 , as indicated by  708 , and can be placed onto spotting device  700 , as indicated by  710 . A separate unspotted microplate  712  can then be moved out of a low-humidity storage unit  714 , as indicated by  716 . In some embodiments, low-humidity storage unit  714  can comprise a relative humidity in the range of about 0-30%. Unspotted microplate  712  can then be placed on spotting device  700 , as indicated by  718 . Reagents from storage plate  704  can then be spotted onto at least some of the plurality of wells  26  on unspotted microplate  712 . Once at least some of the plurality of wells  26  are spotted, the spotted microplate  720  can then be moved from spotting device  700 , as indicated by  722 . Spotted microplate  720  can then be moved to an optional quality-control station  724 , as indicated by  726 . After quality-control station  724 , spotted microplate  720  can then be moved back to low-humidity storage unit  714 , as indicated by  728 . This procedure of spotting microplates  20  can continue until a desired number (e.g. all) of microplates in storage unit  714  have been spotted with reagents from storage plate  704 . It should be noted that unspotted microplate  712  and spotted microplate  720  are each similar to microplate  20 , however different numerals are used for simplicity in the above description. 
     In some embodiments, the spots of reagents on spotted microplate  720  can be partially or fully dried down, as desired, in the low-humidity of storage unit  714 . In some embodiments, storage unit  714  can also be heated to facilitate this drying. Once the microplates from storage unit  714  have been spotted with reagents from storage plate  704 , storage plate  704  can be removed and designated as a used storage plate  730 . Used storage plate  730  can be removed from spotting device  700  as indicated by  732 . Used storage plate  730  can be returned to high-humidity storage unit  706  as indicated by  734 . The process can continue as the next storage plate  704  is moved out of high-humidity storage unit  706  and into spotting device  700 . In some embodiments, this next storage plate  704  can contain a different set of reagents. The aforementioned process can then be repeated, as desired. This process can continue until all of the plurality of wells  26  on spotted microplate  720  have been spotted or, in some cases, a portion of the plurality of wells  26  have been spotted, while leaving the remaining wells  26  empty. 
     It should be appreciated that this preloading process can vary as desired to accommodate user needs. For instance, in some embodiments, the reagents spotted in each of the plurality of wells  26  can be encapsulated with a material. Such encapsulation can prevent or reduce moisture at room temperature from interacting with the reagents. In some embodiments, each of the plurality of wells  26  can be spotted several times with reagents, such as for multiplex PCR. In some embodiments, these multiple spotted reagents can form layers. In some embodiments of this preloading process, primer sets and detection probes for a whole genome can be spotted from storage plates  704  onto spotted microplate  720 . In other embodiments, a portion of a genome, or subsets of selected genes, can be spotted from source plates  704  onto spotted microplate  720 . 
     In some embodiments, spotted microplate  720  can be sealed with a protective cover, stored, and/or shipped to another location. In some embodiments, the protective cover is releasable from spotted microplate  720  in one piece without leaving adhesive residue on spotted microplate  720 . In some embodiments, the protective cover is visibly different (e.g., a different color) from sealing cover  80  to aid in visual identification and for ease of handling. 
     In some embodiments, the protective cover can be made of a material chosen to reduce static charge generation upon release from spotted microplate  720 . When it is time for spotted microplate  720  to be used, the package seal can be broken and the protective cover can be removed from spotted microplate  720 . In some embodiments, the protective cover can be a pierceable film, a slitted film, or a duckbilled closure to, at least in part, reduce contamination and/or evaporation. An analyte (such a biological sample comprising DNA) can then be added to spotted microplate  720 , along with other materials such as PCR master mix, to form assay  1000  in at least some of the plurality of wells  26 . Spotted microplate  720  can then be sealed with sealing cover  80  as described above. High-density sequence detection system  10  can then be actuated to collect and analyze data. 
     In some embodiments, the filling apparatus comprises a device for depositing (e.g., spotting or spraying) of assay  1000  to specific wells, wherein one or more of the plurality of wells  26  of microplate  20  contains a different assay material than other wells  26  of microplate  20 . In some embodiments, the device can include piezoelectric pumps, acoustic dispersion, liquid printers, or the like. According to some embodiments, a pin spotter can be employed, such as described in PCT Publication No. WO 2004/018104. In some embodiments, a fiber and/or fiber-array spotter can be employed, such as described in U.S. Pat. No. 6,849,127. 
     In some embodiments, the filling apparatus comprises a device for depositing assay  1000  to a plurality of wells, wherein two or more wells contain the same assay material. In some embodiments, microplate  20  comprises two more groups of wells  26 . Each of the groups of wells  26  can comprise a different assay material than at least one other group of wells  26  on microplate  20 . 
     Loading Distribution System 
     Referring to  FIG. 64 , a loading distribution system  800  comprising a conveyer or a track  802  can be used to set up an expandable and flexible microplate loading distribution system. For example,  FIG. 64  depicts four dispensing devices  814 ,  816 ,  818 , and  820 , disposed adjacent a corresponding source plate and wash station  814   a ,  816   a ,  818   a , and  820   a , respectively. Dispensing devices  814 ,  816 ,  818 , and  820  can each comprise a plurality of dispensers, for example, 24-dispensers, 48-dispensers, 96-dispensers, 384-dispensers.  FIG. 81  is a perspective view illustrating dispensing device  814  including a plurality of dispensers  868 , for example, in a SBS standard micro-titer format. One or more of dispensing devices  814 ,  816 ,  818 , and  820  can comprise, for example, the Aurora Scout MPD (MultiTip Piezo Dispenser) available from Aurora Discovery as, for example, a 96-tip dispensing device and/or a 384-tip dispensing device. In some embodiments, the dispensing device can comprise at least 96 dispensing tips in loading distribution system  800 . The dispensing device can comprise, for example, at least 96 dispensing tips, at least 384 dispensing tips, at least 768 dispensing tips, at least 1536 dispensing tips, or more. The dispensing device can comprise a plurality of dispensers and each dispenser can comprise a piezo-electric dispenser. The dispensing device in loading distribution system  800  can comprise a plurality of dispensers and a respective plurality of storage reservoirs. Each dispenser can be designed to dispense a first volume of fluid per dispensing action, and each reservoir can be adapted to store many times the first volume, for example, at least 15 times the first volume, at least 25 times the first volume, at least 50 times the first volume, or at least 100 times the first volume. 
     In some embodiments, each of the plurality of dispensers can be adapted to dispense about 100 nanoliters of liquid or fluid, per dispensing action. The dispensing device can comprise a plurality of spotting devices. The dispensing devices can comprise, for example, piezo-electric devices, acoustic devices, ink-jet devices, pump-action devices, pin spotters, or the like, or a combination thereof. 
     In some embodiments, the number of dispensing devices  814 ,  816 ,  818 , and  820  disposed around a conveyer  802  can be increased or decreased so as to address a desired throughput target. In some embodiments, conveyer  802  can expand (be lengthened) in an X-direction. This can allow more dispensing devices to be disposed around conveyer  802 . Conveyer  802  can comprise a track, for example, SuperTrak™ available from ATS Automation Tooling Systems Inc. However, it should be understood that other tracks can be used. 
     In some embodiments, loading distribution system  800  can comprise a load position  806  on conveyer  802 . Loading distribution system  800  can comprise an unload position  808  on conveyer  802 . Load position  806  and unload position  808  can, according to some embodiments, be a same position along conveyer  802 . 
     The plurality of stations can also include, for example, one or more of an inspection station, a plurality of inspection stations, a tracking station, an identifying tag reader station, or the like, as further described herein. According to some embodiments and as further described below, the table described herein can comprise a plurality of tables, with the number of tables, and corresponding carriages if used, being greater than or equal to the number of processing stations. In some embodiments, the plurality of processing stations in loading distribution system  800  can comprise an inspection station adapted to check an alignment of a microplate on the table. The inspection station can comprise, for example, one or more of a camera, a CCD, a laser, a pattern analyzer, an edge analyzer, and a combination thereof. The plurality of processing stations can comprise, for example, an inspection station adapted to perform a quality control analysis of a spot disposed on the microplate, wherein the inspection station can comprise, for example, one or more of a camera, a CCD, a laser, a pattern analyzer, an edge analyzer, and a combination thereof. In some embodiments, loading distribution system  800  can further comprise, for example, a tracking device adapted to track dispensation of fluid from the dispensing device. The tracking device can track a microplate and be adapted to determine whether and which locations of a microplate have been processed, spotted, or otherwise prepared. The tracking device can, in some embodiments, be adapted to track the use of components of an assay. The tracking device can be adapted, for example, to communicate with an identifying tag reader or with an identifying tag to track the progress of a preparation procedure, for example, to track loading and/or spotting operations at each of many loading and/or spotting sites. The tracking device can be adapted to communicate with machine indicia reader  804  and inspection station  810  illustrated in  FIG. 64 . In some embodiments, a dispensing device can comprise a plurality of dispensing devices and the tracking device can be adapted to track dispensation of fluids from each of the dispensing devices to a microplate. Methods of tracking are further discussed in more detail below. 
     In some embodiments, the plurality of processing stations can comprise a tracking station, for example, an identifying tag reader station adapted to read marking indicia  64  disposed on or in microplate  20 . The identifying tag can be a bar code, a two-dimensional barcode, or other marking indicia reader station adapted to read the identifying tag. The reader station can comprise a reader device or apparatus appropriate to the type of marking indicia employed, e.g., a bar code reader. The identifying tag can, in some embodiments, be a radio frequency identification (RFID) tag and the reader station can comprise a RFID reader. In some embodiments, a marking indicia reader station in loading distribution system  800  can comprise one or more of a bar code reader, a one-dimensional bar code reader, a two-dimensional bar code reader, and an RFID reader. In some embodiments, a marking indicia reader station in loading distribution system  800  can be adapted to read marking indicia on the same surface of the microplate that can engage the table when the microplate is on the table. 
     In some embodiments, loading distribution system  800  can comprise a machine indicia reader  804  disposed along conveyer  802 . Machine indicia reader  804  can, according to some embodiments, comprise a plurality of machine indicia readers, one each disposed prior to every dispensing device along conveyer  802 . In some embodiments, machine indicia reader  804  can be disposed past load position  806  along conveyer  802 . 
     In some embodiments, a method of tracking a microplate is provided. The method can comprise, for example, a first dispensing operation that comprises spotting components of an assay to one or more locations or material retention regions of a microplate, for example, one or more wells of a multiwell microplate, to form a partially loaded microplate. Each well can be spotted with a different set of components of a different respective assay. The method can comprise storing information about the at least partially loaded microplate by writing information into a memory using a value of the machine-readable identifier as an index. The method can comprise storing information about the at least partially loaded microplate by writing information into a memory that is addressable by a value associated with the machine-readable identifier. The stored information can comprise information pertaining to the wells and which wells have been spotted and with what respective components of an assay. By tracking such information, subsequent dispensing operations can be directed to wells that have not been spotted and assay components that have not yet been spotted into respective wells. 
     In some embodiments, the method of tracking can comprise subjecting a microplate to two or more, for example, five or more, dispensing operations and to two or more, for example, five or more, information reading steps with at least one information reading step being conducted prior to or subsequent to each dispensing operation. According to some embodiments, the method of tracking can comprise a reading step followed by a plurality of dispensing operations at a respective plurality of dispensing stations. The method can comprise storing information about the at least partially loaded microplate by writing information to the radio frequency identification tag. The method can comprise: reading information from a machine-readable identifier on a microplate; subjecting the microplate to a first dispensing operation by a first multi-tip dispenser to at least partially load one or more material retention regions of the microplate and form an at least partially loaded microplate; storing information about the at least partially loaded microplate; reading the information stored about the at least partially loaded microplate; and determining, based on the information read about the at least partially loaded microplate, whether to subject the microplate to a subsequent dispensing operation by second multi-tip dispenser that differs from the first multi-tip dispenser. The determining can comprise determining that the at least partially loaded microplate should be subjected to a subsequent dispensing operation, and the method can then further comprise subjecting the microplate to an additional dispensing operation by the second multi-tip dispenser, to further load the microplate. 
     The method of tracking can be used in connection with a system comprising a first multi-tip dispenser located at a first station, a second multi-tip dispenser located at a second station, and a conveyer device connecting the two stations. The method can comprise conveying the microplate from the first station to the second station, along, on, or with, the conveyer device. The conveyer device can comprise, for example, a track and/or a belt or chain. The conveyer device illustrated in  FIGS. 64 and 65  comprises a track along which a carriage and table can ride or traverse. 
     The method of tracking can comprise, for example, reading the information stored about the at least partially loaded microplate by reading the information at a third station. The third station can be located between the first station and the second station, along the conveyer device, or it can be located upstream or downstream of both the first and second stations. The first station and the second station can be located adjacent each other along a track and the method can comprise disposing the microplate on a carriage and conveying the carriage along the track from the first station to the second station. 
     In some embodiments, and as described further below, a system controller  982  ( FIG. 101 ) can manage and track microplates at various locations. Locations for a microplate can comprise, for example, in one or more plate storage units, in or on one or more tables, or in one or more jaws of one or more plate handling devices. In some embodiments, system controller  982  ( FIG. 101 ) can, for example, manage and track microplates at various locations in loading distribution system  800  ( FIGS. 64 and 65 ). Locations for a microplate can comprise, for example, in one or more plate storage units, in or on one or more tables, or in one or more jaws of one or more plate handling devices. In some embodiments, system controller  982  ( FIG. 101 ) can, for example, manage and track source plates at various locations in loading distribution system  800  ( FIGS. 64 and 65 ). Locations for a source plate can comprise, for example, in a source plate storage unit like an incubator, in one or more source plate holders, or in one or more grippers of one or more source plate handling devices. System controller  982  described below with reference to  FIG. 101  can also, for example, track and trace the contents of one or more dispensers, each disposed in one or more respective dispensing devices. For example, system controller  982  can track and trace the contents of one or more dispensers, each disposed in one or more respective dispensing devices. 
     With reference to the perspective views of  FIGS. 64 and 65 , a number of the above-described features of the present teachings can be seen embodied in a high-throughput system for fabricating a microplate. Generally, conveyer  802  transports, in serial fashion, empty microplates from a hotel or storage unit  828  to a position adjacent a load position  806 . Handling device  830  places the microplate on a table and carriage assembly for movement along conveyer  802 . The microplate is then moved by the table and carriage assembly along conveyer  802  to machine indicia reader  804 . The method of tracking can comprise scanning indicia on the bottom of the microplate. This operation can serve, for example, to ensure that the card has been properly placed on the table and to read identifying information into a control computer (not illustrated). Next, the table translates the microplate to dispensing stations  820 ,  818 ,  816 ,  814 , serially, for spotting operations. 
     Having received components of an assay from the dispensing stations, the microplate can then be advanced to a position below an inspection station  810  that inspects each well of the microplate for the presence of spotted components of an assay. If the inspection operations indicate that the microplate has been properly loaded with components of an assay, the microplate is then moved along conveyer  802  to an unload position  808  where the microplate can be unloaded, for example, by handling device  830 , and moved back to the storage unit  828 . If a failure is indicated, on the other hand, unloading at unload position  808  can comprise depositing the microplate in a reject bin. 
     In a subsequent operation, for example, after a new set of respective assay components has been aspirated or loaded in dispensing heads of dispensing stations  820 ,  818 ,  816 , and  814 , a partially loaded microplate can again be moved by handling device  830  onto a table of a carriage on conveyer  802 , and then conveyed again to machine indicia reader  804 . The method of tracking can then comprise reading information stored about the microplate as a result of previous quality control inspection at inspection station  810  and indexed by marking indicia on the microplate. If further spotting of assay components is required, the microplate can then be conveyed to dispensing stations  820 ,  818 ,  816 ,  814  for further dispensing operations, this time with the newly-loaded assay components. After the further dispensing operations, the procedure can be repeated, starting, for example, with another quality control inspection at inspection station  810 . Stored information corresponding to a marking indicia can be compared to predetermined values to determine whether additional spotting is needed or whether the microplate has been completely spotted with all desired assay components. 
     According to some embodiments, the method of tracking can use a control computer (not illustrated) that can integrate the operation of the various assemblies, for example through a program written in an event driven language such as LABVIEW® or LABWINDOWS® (National Instruments Corp., Austin, Tex.). In particular, the LABVIEW software provides a high level graphical programming environment for controlling instruments. U.S. Pat. Nos. 4,901,221; 4,914,568; 5,291,587; 5,301,301; 5,301,336; and 5,481,741 (each expressly incorporated herein in its entirety by reference) disclose various aspects of the LABVIEW graphical programming and development system. The graphical programming environment disclosed in these patents allows a user to define programs or routines by block diagrams, or “virtual instruments.” As this is done, machine language instructions are automatically constructed which characterize an execution procedure corresponding to the displayed procedure. Interface cards for communicating the computer with the motor controllers are also available commercially, for example, from National Instruments Corp. 
     In some embodiments, loading distribution system  800  can comprise an inspection station  810  disposed along conveyer  802 . Inspection station  810  can comprise, according to some embodiments, a plurality of inspection stations, one disposed after each dispensing device along conveyer  802 . In some embodiments, a single inspection station  810  can be disposed after all the dispensing devices along conveyer  802 . 
     In some embodiments, loading distribution system  800  can comprise a plate-handling device  830  disposed on a plate-handling device pathway  832  to access a storage unit  828  adapted to store microplates. Storage unit  828  can also be called a hotel. Loading distribution system  800  can comprise a source plate-handling device  822 . Source plate-handling device  822  can be disposed on a source plate-handling device pathway  824  to access a source plate storage unit  826  housing a plurality of source plates (not illustrated). Source plate storage unit  826  can comprise an incubator, for example, Kendro Cytomat 6001 available from Kendro Laboratory Products. Storage unit  828  can comprise a hotel, for example, one or more 120 Nest Landscape Carousels. Plate-handling device  830  and source plate-handling device  822  can each comprise a Select Compliant Articulated Robot Arm (SCARA) robot, respectively, available, for example, from IAI America, Inc. The SCARA robots can be movable in 4-axis or 5-axis. However, it should be understood that other robot mechanisms can be used. 
     In some embodiments, loading distribution system  800  can comprise a storage unit  828 . Storage unit  828  can comprise a hotel, a carousel, or another rack adapted to hold a plurality of microplates. In some embodiments, storage unit  828  can be accessible by the plate-handling device so that the plate-handling device can retrieve microplates, for example, one at a time, or store microplates therein, for example, one at a time. Loading distribution system  800  can further comprise a plurality of microplates arranged in the storage unit. 
     As illustrated in  FIG. 65 , in some embodiments, dispensing devices  814 ,  816 ,  818 , and  820  can be disposed along conveyer  802  using a respective dispensing device mount  814   c ,  816   c ,  818   c , and  820   c . Each dispensing device  814 ,  816 ,  818 , and  820  can be disposed, for example, adjacent a respective alignment station  814   b ,  816   b ,  818   b , and  820   b . Alignment stations  814   b ,  816   b ,  818   b , and  820   b  can be adapted to move a table (not illustrated) in a Y-direction. 
     In some embodiments, when an alignment station is not provided to move a table in the Y-direction, a dispensing device can be moved in the Y-direction to align a microplate disposed on the table with the dispensing device. 
     As illustrated in  FIG. 66 , in some embodiments, dispensing device  814  can comprise a plurality of dispensers  868 . A carriage  874  can be disposed on conveyer  802 . Carriage  874  can be positioned under dispensers  868 , when dispensing of a fluid in or on microplate  20  is desired. Microplate  20  can be disposed on a table  872 . Table  872  can comprise a vacuum chuck; see  FIG. 80 , adapted to hold microplate  20 . Table  872  can move to align microplate with dispensers  868 . Conveyer  802  can translate carriage  874  away from the dispensing position. Carriage  874  can move along conveyer  802 . 
     In some embodiments, table  872  can be adapted to move along the Y-axis and the alignment stage can be adapted to align the microplate with the dispensing device. Table  872  can be adapted to be rotatable about the Y-axis direction. As described herein, table  872  can comprise a vacuum chuck adapted to apply a vacuum to a surface of a microplate when a microplate is disposed on the table. Loading distribution system  800  can comprise a vacuum source in fluid communication with the vacuum chuck. A vacuum retainment valve can be disposed in fluid communication with the vacuum chuck and can be adapted to maintain a vacuum between the vacuum chuck and the surface of a microplate when a microplate is disposed on the table, for example, when the vacuum chuck is not in fluid communication with the vacuum source. Loading distribution system  800  can comprise a vacuum detector adapted to verify the formation of a vacuum between the surface of a microplate disposed on the table, and the vacuum chuck. 
     In some embodiments, loading distribution system  800  can further comprise an accessory carriage configured to engage a source plate comprising a source of fluids to be loaded into the spotting or other dispensing station. The accessory carriage can be adapted to move the source plate to the dispensing station for aspiration of the fluids from the source plate into the dispensing device. Loading distribution system  800  can further comprise an incubator adapted to store the source plate, for example, to keep it in a cooler and more humid environment relative to the immediately surrounding atmosphere. Loading distribution system  800  can comprise a source plate-handling device adapted to translate a source plate from the incubator to the dispensing station. The incubator can comprise a de-lidder adapted to remove a lid from a source plate in loading distribution system  800 . The de-lidder in loading distribution system  800  can further be adapted to place a lid on a source plate. 
     In some embodiments, when carriage  874  is not positioned beneath dispensing device  814 , a source plate and wash pallet  864  can be positioned under dispensing device  814 . As illustrated in  FIG. 91 , source plate and wash pallet  864  can comprise a washing tray  861  and a source plate holder  863 . Source plate-handling device  822  can pick-up and deposit a source plate from source plate holder  863  using a gripper  823 . Source plate  862  can be covered using a lid  860 . Lid  860  can be placed on source plate  862  by a de-lidder  858 . De-lidder  858  can comprise a lifting device  856  adapted to lift and hold lid  860 . Source plate and wash pallet  864  can be disposed on an elevator mechanism (not illustrated) to move source plate and wash pallet  864  within range of dispensers  868 . Source plate and wash pallet  864  can be in a rest position or a washing position. While in a rest position, washing tray  861  can be covered using a dust cover  866 . Dust cover  866  can be hinged. In some embodiments, loading distribution system  800  can further comprise a plurality of source plates in the incubator, wherein the dispensing device comprises a plurality of multi-tip dispensing heads, and the source plate handling device can be adapted to translate one or more of the plurality of source plates from the incubator to each of the plurality of multi-tip dispensing heads. 
     In  FIG. 66(   b ), a washing tray can be disposed on a washing tray pallet  865 ′ adapted to elevate the washing tray under dispensers  868 ′ of a dispensing device  814 ′. A source plate  862 ′ can be disposed on a source plate pallet  864 ′ that can be positioned under dispensing device  814 ′. Source plate-handling device  822 ′ can comprise dual end effectors to pick-up and deposit a source plate  862 ′ on source plate pallet  864 ′. 
     As illustrated in  FIGS. 68(   a )-( c ), source plate and wash pallet  864  can comprise washing tray  861  and holding source plate  862 . As illustrated in  FIGS. 68(   a )-( c ) a dispensing device can comprise 96-fixed dispensers.  FIG. 68(   a ) illustrates an internal dispenser wash. Dispensers  868  can be immersed in a fluid disposed in internal wash slots  878 .  FIG. 68(   b ) illustrates an external dispenser wash. Dispensers  868  can be immersed in a fluid disposed in external wash slots  876 .  FIG. 68(   c ) illustrates aspiration by dispensers  868 . The illustration depicts 96-dipsensers into a 384-well source plate. Each respective dispenser can be illustrated disposed in every other well along every row and every column. In some embodiments, each dispensing device can be adapted to be loaded by aspirating fluid from a fluid source. The fluid source can be disposed in loading distribution system  800 , for example, in the storage unit or in a separate, second storage unit. Each storage unit can comprise an incubator. 
     As illustrated in  FIG. 69 , a ceiling mounted plate-handling device  830  can be adapted to retrieve microplate  20  from a plate storage unit  828 . Plate-handling device  830  can pick-up and remove microplate  20  from a table  872 . Table  872  can be moved along a conveyer  802 . The ceiling mount configuration can provide for an unobstructed range of motion by plate-handling device  830 . The ceiling mount configuration can provide clearance for an arm of plate-handling device  830 . Plate storage unit  828  can be adapted to translate racks of microplates allowing plate-handling device  830  to access microplates  20  stacked in each rack of plate storage unit  828 . Plate storage unit  828  can provide environmental control. Plate storage unit  828  can be designed for mobility. Plate storage unit  828  can be designed for off-line operator loading and unloading. Microplates  20  can be stored in plate storage unit  828  in a landscape orientation with respect to conveyer  802 . Microplates  20  can be stored in plate storage unit  828  in a portrait orientation with respect to conveyer  802 . 
     In some embodiments, an interval required to unload and reload a microplate from loading distribution system  800  can be a rate-limiting factor when determining throughput of loading distribution system  800 . A plate gripper, automated and robotic, in combination with a carriage adapted to allow simultaneous or substantially simultaneous, unloading and reloading of microplates on the carriage, in a minimum amount of time, can be provided. 
     Referring now to  FIG. 70 , a carriage  874  comprising a table  872  is illustrated. Microplate  20  can be disposed on table  872 . Carriage  874  can comprise locating pins  882   a ,  882   b , and  882   c  disposed on table  872 . A ratchet  888  can be disposed on table  872 . As illustrated in  FIG. 72 , ratchet  888  can be spring-loaded by a spring  910 . When microplate  20  is disposed on table  872 , spring  910  can secure microplate  20  against locating pins  882   a ,  882   b , and  882   c . Spring  910  can be automated. Spring  910  can be actuated and/or released by a manufacturing control system. Spring  910  can be used to position microplate  20  on table  872 , allowing stations disposed along conveyer  902  to be correctly oriented. A self-conveyance device  909  can propel carriage  874  around conveyer  802  (not illustrated). In some embodiments, loading distribution system  800  can further comprise a conveyer on which or with which the table and/or the alignment stage can be moved or translated. Loading distribution system  800  can comprise a carriage, for example, that can ride on, along, and/or with the conveyer. The carriage can be adapted to be translated to one or more of the plurality of processing stations. The carriage can be adapted to translate the table along the conveyer to one or more of the plurality of processing stations. 
     According to some embodiments, table  872  can comprise a plurality of tables and the carriage can comprise a plurality of carriages each respectively adapted to translate one or more of the plurality of tables. Each carriage can comprise a self-conveyance device, for example, a translation motor or servomotor, and the plurality of carriages can be disposed on or along a conveyer. In some embodiments, each of the plurality of carriages can comprise a plurality of automated actuators and a self-conveyance device, for example, wherein the self-conveyance device can comprise a conduit for transferring control signals to the plurality of automated actuators. The conveyer can comprise a track, for example, in the form of a circle, oval, or other loop. The loop can be endless. 
     In some embodiments, loading distribution system  800  can be adapted to convey the table along the X-axis direction. The conveyance can be repeatably positionable to within about 100 micrometers of a predefined location. A conveyer can be used that serially translates one or more of a plurality of tables, for example, with each table being disposed on a respective carriage. The plurality of tables can be translated, for example, consecutively translated, to each of the plurality of processing stations. 
     In some embodiments, a vacuum line supply  890  can provide communication from table  872  to a bellows  896 . Bellows  896  can communicate with a vacuum connection shoe  907 . 
     In some embodiments, carriage  874  can comprise a mechanism to lift or raise a first microplate, allowing a second microplate to be placed under the first microplate. Carriage  874  that transports microplate  20  between stations of loading distribution system  800  can comprise a set of grippers comprising a first cam  884  and a second cam  886 , which can hold up microplate  20  without microplate  20  resting on table  872  of carriage  874 . First cam  884  and second cam  886  can be pivotally attached to self-conveyance device  909 . Table  872  of carriage  874  can move up and down vertically. The normal resting position of table  872  can be at a midpoint of travel for table  872 , rather than a bottom point of travel for table  872 . Table  872  normally rests on a spring plunger  902  via a pin  898 . Table  872  can be lifted off spring plunger  902  for an upward motion. Table  872  can be forced down, in a downward motion, and depress pin  892  into spring plunger  902 . The downward motion can allow first cam  884  and second cam  886  to grab microplate  20  on table  872  and lift microplate  20  up off a surface of table  872 . 
     In some embodiments, rollers  894  and  892  can be attached to first cam  884  and second cam  886 , respectively. A tripod  901  can be disposed in a linear bearing  904 . Linear bearing  904  can be disposed vertically. A travel of tripod  901  can raise and/or lower table  872 . A roller  906  can be attached to tripod  901 . 
       FIG. 71  illustrates a spring  908  that holds table  872  of carriage  874  against one corner. 
       FIG. 73  illustrates a sectioned view of spring plunger  902  that holds table  872  (not illustrated) at an intermediate position in the Z-axis. Table  872  can be lifted off pin  898  to raise table  872  for dispensing or spring  912  can be overpowered to depress table  872  for microplate swapping operation as described herein. 
       FIG. 74  is a perspective view illustrating an embodiment of a pressure source  918  adapted to communicate with vacuum connection shoe  907 . Vacuum connection shoe  907  can comprise a port  920  on the opposite side that can engage with a vacuum supply port  916  disposed in a frame  914  attached to conveyer  902 . Bellows  896 , or other means known in the art, can allow a flexible connection between vacuum connection shoe  907  and table  872  that can move up and down, and shift sideways. 
     In  FIG. 74 , vacuum connection shoe  907  can be disposed next to vacuum port  916  on frame  914 . When a carriage is at a station, for example, a loading station, or a dispensing device station, a valve (not illustrated) opens where vacuum port  916  is disposed on frame  914 . A vacuum retainment valve (not illustrated) can be disposed on carriage  874  along bellow  896  or vacuum line supply  890 . 
     In some embodiments, vacuum connection shoe  907  can be elongated so that a vacuum connection is established before table  872  can reach the stop position at a station. This elongated vacuum connection shoe can make a significant difference in cycle time, as a final deceleration prior to stopping a carriage at a station can be a large part of total transit time for a carriage. 
       FIGS. 75 and 76  illustrate cam rails  922 ,  924  and a slotted rail  926  comprising a slot  930  for vertical motion of first cam  884  and second cam  886  and tripod  901 , respectively. Cam rails  922 ,  924  can be attached to conveyer  802 . Cam rails  922 ,  924  can control the timing of first cam  884  and second cam  886  when performing a grip operation. Slotted rail  926  can control a drop operation of table  872 . The two operations can occur automatically during the motion of carriage  874 . The two operations can occur simultaneously or substantially simultaneously. Carriage  874  transfer speed can take into consideration a use of cam rails  922 ,  924  and slotted rail  926 . First cam  884  and second cam  886  can be fixed to carriage  874 . When a station, for example, a dispensing device station, needs a final registration of microplate  20 , table  872  can float relative to carriage  874 . Table  872  need not float relative to carriage  874  at some stations, for example, a load station or an unload station. 
     Slotted rail  926  that controls the Z-axis movement of table  872  can be fixed to conveyer  802 . Cam rails  922 ,  924  can be mounted to an air-operated slide  921 . Air-operated slide  921  can be attached to slotted rail  926 . When carriage  874  approaches cam rails  922 ,  924 , table  872  can be floating at a midpoint, and first cam  884  and second cam  886  can be open. Cam rails  922 ,  924  can be elevated when carriage  874  approaches a station. Cam rails  922 ,  924  can be rising up, for example, by activating air-operated glide  921 , to meet carriage  874  as it enters a station as long as cam rails  922 ,  924  are in position when roller  906 , a Z-axis control roller, engages with slotted rail  926 . When roller  906  enters slot  930 , tripod  901  can drop. As table  872  rests on tripod  901 , table  872  can drop down with tripod  901 . Prior to dropping tripod  901 , rollers  894  and  892  can engage cam rails  922 ,  924 . As rollers  894  and  892  rise on a ramp of cam rails  922 ,  924 , first cam  884  and second cam  886  attached to rollers  894  and  892 , respectively, close and grip microplate  20 . As a ramp of cam rails  922 ,  924  continues to rise, first cam  884  and second cam  886  can lift microplate  20  off table  872 . When a release of a gripped microplate is desired, first cam  884  and second cam  886  can be dropped, by lowering air-operated slide  921  that in turn lowers cam rails  922 ,  924 . The lowering of cam rails  922 ,  924  can disengage rollers  894  and  892  from cam rails  922 ,  924 , which in turn can open first cam  884  and second cam  886  releasing a gripped microplate  20 . The release can performed when, for example, a plate gripper robot  784  is ready to remove a microplate. Plate gripper robot  784  is illustrated in  FIGS. 82-90  described below. 
       FIG. 77  is a perspective view illustrating an embodiment of a loading distribution system comprising carriage  874 , table  872 , and an alignment stage  932 . Alignment stage  932  can be disposed under a dispensing device mount  931 . A dispensing device (not illustrated) can be attached to dispensing device mount  930 . Table  872  of carriage  874  can engage with alignment stage  932  when carriage  874  lifts. A set of actuators  934 ,  936  engages with three points on table  872  after carriage  874  enters a dispensing station and table  872  has been raised. Alignment stage  932  can comprise a long stroke actuator  935  for the X-axis since microplate  20  disposed on table  872  can index over a substantial distance for some kinds of dispensing, for example, dispensing of fluids for Focused Genome dispensing. The X-axis carries two short stroke Y-axis actuators  934 ,  936 . The Y-axis actuators  934 ,  936  can operate independently from each other to compensate for skew. 
     In some embodiments, loading distribution system  800  can comprise the table, the alignment stage, and a plurality of processing stations. The table can be configured to engage at least one of a plurality of microplates and be movable at least in an X-axis direction. The table can be moved together with a carriage that in-turn can be adapted to move in the X-axis direction. The an alignment stage can be configured to move the table and/or carriage at least in a Y-axis direction that differs from the X-axis direction, for example, that can be perpendicular or at least substantially perpendicular, to the X-axis direction. In some embodiments, substantially perpendicular can mean within about 15 degrees of being perpendicular. The plurality of processing stations can comprise at least one or more dispensing stations and a plate-handling station. Each of the one or more dispensing stations can comprise a dispensing device adapted to dispense fluid into or onto one or more of a plurality of microplates. The plate-handling station can comprise a plate-handling device. The plate-handling device can be adapted to selectively pick up and deposit on the table individual microplates from a plurality of microplates, at least one at a time. In an exemplary embodiment, loading distribution system  800  can further comprise a microplate disposed on the table, wherein the dispensing device comprises at least 24 or more dispensers, and the microplate comprises 768 or more wells, for example, 96 or 384 dispensers and 6,144 wells. 
     In some embodiments, alignment stage  932  works in cooperation with locating pins  882   a ,  882   b , and  882   c . A location of microplate  20  can be offset in varying degrees from the center of dispensing device  814  to satisfy a need to interleave subsets of dot patterns or dispensing locations, and to form stripe pattern offsets for Focused Genome dispensing. A system requiring operator intervention to mechanically align dispensing device  814  with the independent axes of motion, for example, X, Y, and Z-axis, can be very difficult to maintain. In some embodiments, loading distribution system  800  can work without a need for precision alignment by an operator after maintenance on loading distribution system  800  has been performed. Alignment stage  932  can be enhanced with a vision system based adaptive alignment system. A camera (not illustrated) can form an image of microplate  20 . The image can be processed to derive X, Y, and/or Z movement specifications for alignment stage  932 . Table  872  can comprise reference markings (not illustrated) to determine offsets needed to compute the movement specifications. 
       FIG. 78  is a perspective view illustrating an embodiment of a lifting stage  940  adapted to lift carriage  874  in the Z-axis. A motorized slide  938  moves a block  941  with a slot in block  941 , lifting carriage  874  up and down. Roller  906  that controls the Z-axis engages with a slot in block  941  to move table  872  of carriage  874  up for dispensing. Lifting stage  940  can be disposed in a position underneath a dispensing device to allow a Z-direction movement of carriage  874 . 
       FIG. 79(   a ) and  FIG. 79(   b ) are perspective views illustrating two visual inspection station, according to some embodiments. The visual inspection stations can provide an ability to compensate for a large number of potential errors, assist in quality control, and alignment of microplates. 
       FIG. 79(   a ) illustrates a full scan vision station disposed on conveyer  802 . The full scan vision station can perform a full scan of microplate  20  disposed of table  872 . A camera mount  941  can extend from conveyer  802  to position a camera  947  over microplate  20  as it moves around conveyer  802 . A carriage alignment device  945  can engage and properly align table  872  with camera  947 . Carriage alignment device  945  can be a mechanical device to push table  872  into a fixed position by contacting three points on a perimeter of table  872 . This can eliminate servo errors to provide a consistent reference measurement. Carriage alignment device  945  can retract from above conveyer  802 , thus disengaging table  872  from the full scan vision station. Carriage  874  can be docked at a station where camera  947  takes a picture of a fluid pattern deposited on microplate  20 . The full scan vision station can provide quality control. The full scan vision station can be used to provide measurements to alignment system  932 . The full scan vision station can be downstream of the dispensing devices for quality control of microplate  20 . 
     A periphery scan vision system or plate check vision system can be disposed upstream of a dispensing device to check the position and accuracy of microplate  20 , prior to a dispensing by a dispensing device. The periphery scan vision system can utilize a camera mount  941  to hold two cameras  946 ,  948 . Cameras  946 ,  948  can be narrow focus cameras. Cameras  946 ,  948  can check the location of two or three dispensing locations. The periphery scan vision system can comprise a carriage alignment  944  similar in functionality to carriage alignment device  945  described above. The periphery scan vision system can comprise a marker indicia reader station. 
     In some embodiments, a reference microplate can be disposed on table  932 . The reference microplate can comprise an accurately machined microplate mimicking a microplate. The reference microplate can comprise a pattern of etched dots or location that matches the desired pattern on microplates to be manufactured. 
     In some embodiments, a test target microplate can be disposed on table  932 . Flat blank plates can be used for making test patterns of dots. The test target microplate can comprise, for example, a plastic material or a cardboard material. The test target microplate does not need to comprise wells. The test target microplate can comprise a surface providing good contrast with the dot pattern. The surface can comprise a coating that can change color when liquid contacts the coating. 
     In some embodiments, the following sequence of operations can be used adjust loading distribution system  800 . The reference microplate can be placed on a first carriage and the first carriage can be moved to the full scan vision system. The dot pattern on the reference microplate can teach the camera of the full scan vision station, the desired dot locations. Next, a test target microplate can be placed on a second carriage. The second carriage can be moved under a dispensing device. The alignment stage can move the table of the second carriage to the position that the alignment stage guesses to be the correct position. The guess can be based on previous runs. A single test target microplate can be used for one or more of the dispensing devices since the patterns from the individual dispensing stations can be disposed far enough apart so that they do not overlap. Lastly, the second carriage with the test target microplate can be moved to the full scan vision system and the dot pattern of the test target microplate can be compared to the stored memory of the desired pattern. Offsets can be computed to adjust the position of the alignment stages for the next cycle. 
     The above process can be repeated by running another test target microplate through loading distribution system  800  to verify the results of the previous run, until achieving a desired or satisfactory run. The above process need not be repeated. When it is determined that the dot pattern from a particular dispensing device does not or cannot fitted to a desired pattern by adjusting the X, Y and rotary axes, then aiming of dispensers of the dispensing device can be checked and adjusted, if desired. Loading distribution system  800  can alert an operator or it can devise another offset for the off-target dispenser or a subset of the off-target dispensers. The alignment stage can move the table to one position and fire one set of dispensers. The alignment stage can then make a slight adjustment of the alignment of the table and the dispensing device, and fire another dispenser or set of dispensers. The alignment can be dynamic while loading distribution system  800  can be dispensing fluids to the microplates. The slight penalty of a microplate that fails quality control and/or a slight increase in the overall cycle time can be preferable to stopping loading distribution system  800  for maintenance. This process can be useful for expediting, for example, small orders of custom microplates. 
     In some embodiments, once loading distribution system  800  adjusts for a production operation, a microplate can be loaded onto a carriage. The carriage can be moved to the periphery scan vision system. The location of two or more wells can be checked and a new offset for this carriage and microplate set can be added to loading distribution system  800  offsets. This new offset can adjust for variations in carriages, variations in how a microplate is placed on a carriage, and molding variations in the microplates. If the dispensing locations wells are too far or too close to each other or to the edge of the microplate, the microplate can be rejected and the microplate need not be spotted. If the well spacing is within limits but substantially off from the ideal, the error can tend to be cumulative rather than random. This means that each dispensing location can be almost perfectly spaced relative to adjacent dispensing locations, but that this spacing can be always slightly larger or smaller than specification. This can imply that the farthest dispensing locations on the microplate can be out of specification in relation to each other. Loading distribution system  800  can divide the microplate into halves or quadrants, compute an offset for each quadrant, and then dispense to each quadrant with a respective offset. 
     According to some embodiments, a fluid distribution system can comprise: a table configured to engage at least one of a plurality of microplates and movable at least in an X-axis direction and in a Y-axis direction that differs from the X-axis direction; a dispensing device adapted to dispense fluid into or onto one or more of a plurality of microplates; a plate-handling station comprising a plate-handling device adapted to selectively pick-up microplates from and deposit microplates on the table; an inspection station adapted to image a microplate when a microplate is disposed on the table; a calculating device adapted to compute offsets that can comprise at least an X-axis direction offset and a Y-axis direction offset, based on an image provided by the inspection station; and a control device adapted to control an adjustment of a relative position of the table based on offsets computed by the calculating device. 
     According to some embodiments, the calculating device can be adapted to compute positions of at least two dispensing locations on a microplate from an image of the microplate. The calculating device can reject a microplate if the computed positions are not within a predetermined specification. The calculating device can be adapted to divide the image into portions and compute positions of at least two dispensing locations in each image portion. The calculating device can reject a microplate if respective computed positions of an image portion are not within at least one predetermined specification. The control device can be adapted to control movement of the table with the respective offset for each image portion being dispensed to by the dispensing station. The microplate can comprise a reference target plate. 
     According to some embodiments, the system can comprise a marking indicia reader such as marking indicia reader  804  adapted to read a marking indicia disposed on a microplate when a microplate is disposed on the table. The system can comprise a memory or storage device capable of storing offsets indexed by the marking indicia for one or more of a plurality of microplates. The system can comprise an alignment stage configured to move the table in the X-axis direction and in the Y-axis direction. 
     According to some embodiments, the calculating device can compute offsets. Either retrieving from the storage device offsets indexed to a respective marking indicia, or computing and saving into the storage device offsets indexed by the respective marking indicia, for one or more of a plurality of microplates. 
     According to some embodiments, the table can comprise a plurality of tables and each table can comprise a respective table identifier. The storage device can store offsets by the table identifier and marking indicia pair. The computing device can retrieve offsets by the table identifier and marking indicia pair. 
     According to some embodiments, the system can comprise a quality control inspection device adapted to inspect an image of two or more dispensings onto a microplate. The quality control inspection device can be adapted to reject a microplate if an image of two or more dispensings is not within at least one predetermined specification. The quality control inspection device can be adapted to compute dispensing station offsets that can comprise at least an X-axis direction offset and a Y-axis direction offset, based on the image. 
     According to some embodiments, the quality control inspection device can be adapted to inspect an image of a microplate. The quality control inspection device can be adapted to divide the image into portions. The quality control inspection device can be adapted to compute positions of two or more dispensings in each image portion. The quality control inspection device can be adapted to reject a microplate if positions for each image portion of the microplate are not within at least one predetermined specification. The quality control inspection device can be adapted to adjust a dispenser of a dispensing device if positions and volumes for each image portion of the microplate are not within at least one predetermined specification. The microplate can comprise a test target microplate. 
     In some embodiments, loading distribution system  800  can be used dispense dry beads. Loading distribution system  800  can use dry beads rather than fluids to deposit probes. The dry dispensing can face the same issues of how to align a series of interleaved dispensing devices. Dropping dry beads on a test microplate does not provide a useful test pattern. The individual dispensing devices can comprise ink jet heads or sharp pins that can be machined in a fixed pattern relative to the bead outlet points. A test microplate can be run through loading distribution system  800  and the jets or pins can be activated to create a visible dot pattern that can be checked by a vision system. 
       FIG. 80  is a top-plan view illustrating table  872  comprising a vacuum trench  954  and a gasket  956 . When a microplate is disposed on table  872 , a pressure source (not illustrated) can be connected to a vacuum inlet  952 , to form a vacuum between a surface of microplate  20  and table  972 .  FIG. 74  illustrates an embodiment of a pressure source communicating with table  872 . 
       FIG. 80  illustrates an embodiment of table  872  comprising four locating pins and no ratchet, in contrast to table  872  of  FIG. 70 . 
     In some embodiments, a table can provide for initial microplate registration to a carriage at a load station. Vacuum formed between a microplate surface and a table can be used to flatten a microplate. The vacuum can also hold a microplate in place for a dispensing operation. Loading distribution system  800  can operate under a tight tolerance window. A dispensing device and a microplate can be aligned by various devices described to be within, for example, about 100 μm, about 40 μm, or within about 10 μm. These tolerances can allow dispensing into microplates, for example, high-density microplates. The alignment devices can be supplemented with vision and/or laser based active alignment systems, for additional accuracy if desired. Alignment to the tight tolerances can compensate for potential molding errors, head alignment errors, track variability, and table on carriage errors. 
       FIG. 81  is a perspective view illustrating a dispensing device  814  including a plurality of dispensers  868 . 
       FIGS. 82-84  are perspective views illustrating plate gripper robot  784 . Plate gripper robot  784  can comprise a pair of jaws—a lower jaw  786  and an upper jaw  788 . Upper jaw  788  can be mounted above lower jaw  786 . Plate gripper robot  784  can include actuators  784  and  790  to pivotally move an upper jaw-clamping portion  788   a  and a lower jaw-clamping portion  786   a , respectively. 
     In some embodiments, as illustrated in  FIG. 85  lower jaw  786  can bring a first microplate  20   d  to table  872  and can place first microplate  20   d  on table  872  under a second microplate  20   c  that carriage  874  can be holding above table  872  using first cam  884  and second cam  886 . As illustrated in  FIG. 86 , plate gripper robot  784  can release first microplate  20   d  from lower jaw  786 , placing first microplate  20   d  on table  872 . As illustrated in  FIG. 87 , first cam  884  and second cam  886  can release, and upper jaw  788  can grab second microplate  20   c . First cam  884  and second cam  886  can release second microplate  20   c  as described in  FIG. 75  and  FIG. 76 . 
       FIG. 88  illustrates plate gripper robot  784  removing second microplate  20   c  from table  872 . As illustrated in  FIG. 89 , plate gripper robot  784  can transfer second microplate  20   c  to plate storage unit  828 . At plate storage unit  828 , plate gripper robot  784  can place second microplate  20   c  on an empty shelf. The next lower shelf in plate storage unit  828  can be empty to provide clearance for lower jaw  786 . 
     As seen in  FIG. 90 , lower jaw  786  grasps a third microplate  20   e  on from plate storage unit  828  without plate gripper robot  784  needing to shift to another position. Third microplate  20   e  can now be treated as first microplate  20   c  of  FIG. 85  and the process can be repeated again. 
     In some embodiments, after a stack in plate storage unit  828  has been processed, plate gripper robot  784  can shift two microplates from the top of the stack to the bottom of the stack. This can provide empty spaces for the process, and can allow the process to repeat during a next pass. In some embodiments, the table can comprise a plate gripper. The plate gripper can be adapted to grip and/or, lift to an elevated position, a first microplate. Starting with a first microplate disposed on the table, the plate-handling device can be adapted to lift the first microplate and deposit a second microplate underneath the first microplate while the first microplate is in the elevated position. Loading distribution system  800  can comprise a plate gripper release device that can be adapted to release the plate gripper from gripping the first microplate. The plate gripper release device can enable the removal of a first microplate from the plate gripper. 
     Even further details regarding various other uses and configurations of the plate gripper and systems using the same can be found in U.S. patent application entitled “Dual Nest Microplate Spotter” to Lehto (Attorney Docket Number 5010-202), filed the same day as the present application. 
     In some embodiments, a plate gripper robot can approach a table with a new microplate. The plate gripper robot can dispose the new microplate on the table. The plate gripper robot can grip the top microplate. The plate gripper robot can then release the new or bottom microplate. The plate gripper robot can then remove the top microplate. At the plate storage unit, the plate gripper can place the microplate in its top jaws on an empty shelf. There can be two empty adjacent shelves in a hotel, for example, the top empty shelf can receive a microplate, and the next empty shelf can be unused, for example, for gripper clearance. The shelf below the two empty shelves can hold a next microplate. The lower jaws of the plate gripper robot can than grab a microplate from the shelf holding the next microplate without needing to shift to another position along the plate storage unit. The cycle can then be repeated to (1) place a microplate gripped by the lower jaws on the table, (2) grip and remove a microplate raised above the table using the upper jaws, (3) return the microplate in the upper jaws to the plate storage unit, and (4) grab a microplate in the lower jaw from the next shelf holding a microplate. In some embodiments, the plate-handling device in loading distribution system  800  can comprise a two-jaw plate gripper device. The two jaws can be positioned one over the other. Each jaw can be adapted to grip a microplate. The plate-handling device can be adapted to grip and remove a first microplate from the table and substantially simultaneously deposit a second microplate on the table. 
     In some embodiments, a carriage or pallet can move microplates along a conveyer in a portrait orientation. It can be desirable to include as many of the carriage functions as possible off board of the carriage for design simplicity. In some embodiments, a register plate function can be off carriage. A vacuum pallet function applied to chuck can be on carriage. A Z-motion can be off carriage. A Y-motion can be off carriage. A vacuum sensor can be off carriage. A register sensor can be off carriage. A bar code reader can be off carriage. A Docking, Command and Data Acquisition (CDA), signal, and power function can be provided on a carriage. In some embodiments, loading distribution system  800  can comprise a lift. The lift can be configured to move the table in a Z-axis direction. The Z-axis direction can be different from both the X-axis direction and the Y-axis direction. The Z-axis direction can be, for example, perpendicular or substantially perpendicular, to both the X-axis direction and the Y-axis direction. In some embodiments, substantially perpendicular can mean within about 15 degrees of being perpendicular. 
     In some embodiments, the microplate can be pushed at a corner while on a load station of the conveyer. A vacuum chuck can be onboard every carriage. A Z-motion actuator can be disposed beneath the carriage. This can provide clearance and can move the vacuum chuck up to meet a dispensing device. A Y-motion actuator can reside outside of the carriage. The actuator can utilize a ram to drive a table to a reference location. A vacuum sensor can be disposed on the vacuum line supply proximate a carriage docking mechanism. A register sensor-can determine correct microplate placement, for example, by checking a pressure on the vacuum line supply. A machine indicia reader, for example, a bar code reader, can be used with a mirror to reflect a bar code on a microplate to separate reader assembly. In some embodiments, 50-micron repeatability can be desired for X, Y, and Z direction movements at a dispensing station. The carriage can be driven on a conveyer or track by a linear stepper motor. The dispensing device and dispensers therein can be held stationary. Various components, for example, the conveyer, of loading distribution system  800  can be provided with EMI shielding. 
       FIG. 91  is a perspective view illustrating source plate and wash pallet  864  comprising washing tray  861  and source plate holder  863 . A source plate  862  can be disposed in source plate holder  863 . Washing tray  861  can comprise internal wash slots  878  and external wash slots  876 . Washing tray  861  can be available from Aurora Discovery, Inc. 
     Source plate-handling device  822  can pick-up and deposit a source plate  862  from source plate holder  863  using a gripper  823 . Source plate  862  can be covered using a lid  860 . Lid  860  can be placed on source plate  862  by a de-lidding device  868 . De-lidding device  868  can comprise a lifting device  856  adapted to lift and hold lid  860 . Source plate and wash pallet  864  can be disposed on an elevator mechanism (not illustrated) to move source plate and wash pallet  864  within range of dispensers  868 . Source plate and wash station  814   a  can be in a rest position or a washing position, when an elevator mechanism is used. While in a rest position, washing tray  861  can be covered using a dust cover  866 . Dust cover  866  can be hinged. 
       FIG. 92  is a perspective view illustrating a source plate and wash station  814   a  comprising at least one source plate and wash pallet  864 . This embodiment of source plate and wash station  814   a  can service two dispensing stations simultaneously or substantially simultaneously. Washing tray  861  and source plate holder  863  can be placed next to each other on a platform or source plate and wash pallet  864 . Source plate and wash pallet  864  can be disposed on a first slide  867 . Vacuum cups  856  can grab and hold lid  860 , a standard plate cover. Dust cover  866  can cover washing tray  861 . A support  858  can be used to hold vacuum cups  856 . Source plate and wash pallet  864  can normally wait in a position that presses washing tray  861  and source plate  862  up against their respective lids. Washing tray  861  can be covered by dust cover  866  that can be permanently attached to a frame.  FIG. 98  is a side-plan view of source plate and wash station  814   a  in a wait position with respect to conveyer  802  and dispensing device  814 . 
     As illustrated in  FIG. 93 , if source plate and wash station  814   a  can be extended to aspirate a dispensing device from source plate  862 , then source plate and wash pallet  864  drops and vacuum cups  856  retain lid  860 . 
     As illustrated in  FIG. 94 , if source plate and wash station  814   a  is going to extend to wash dispensers of a dispensing stations, vacuum cups  856  do not turn on and lid  860  stays with source plate  872 .  FIG. 99  is a side-plan view of source plate and wash station  814   a  in the wash position with respect to conveyer  802  and dispensing device  814 . 
     As illustrated in  FIG. 95 , to swap source plate  872  out with a fresh source plate from source plate storage unit  826 , a second slide  869  stays retracted. First slide  867  slides crossways, and shifts to one-side so that source plate  872  is not under lid  860  holding mechanism and an external SCARA or 5-axis robot, like store plate-handling unit  822  can load and unload the source plate  872 . 
     As illustrated in  FIG. 96 , source plate and wash station  814   a  can extend on second slide  869  to position source plate  862  for aspiration by a dispensing device. 
     As illustrated in  FIG. 97 , source plate and wash station  814   a  can extend on first slide  867  and second slide  869  to position washing tray  861  to wash dispensers. 
     In some embodiments, for a wash operation carriages can be stopped along the conveyer at locations away from the dispensing devices to allow clearance of a washing tray moving mechanism. The moving mechanism can travel along a fixed linear track that can bring the washing tray to the conveyer. Initially, the washing tray can be located beneath a fixed cover plate that can include an embedded seal surface that the edges of the washing tray can seal against when the bath is in the up or wait position under the fixed cover. The washing tray can be lowered slightly in the Z-direction to unseal the washing tray. The washing tray can then move along a linear track towards the conveyer. When the washing tray is clear of the fixed cover, the washing tray can be raised to present the washing tray to the dispensers of a dispensing station. The washing tray can move down and can index in the Y-direction to accomplish both internal and external tip washing operations. When a wash cycle is complete, the tray can move down and back towards the rest position along the linear track. 
     In some embodiments, for an aspirate operation, a robot arm can remove a correct source plate from an incubator and place it onto a source plate location. The source plate can be moved to a de-lidder that can be mounted under a dust cover. The lid of the source plate can be removed using the de-lidder. 
       FIG. 100  is a perspective view illustrating a hotel and a movable entry guide. In some embodiments, reliable insertion of microplates into shelves can be facilitated by adding an entry guide  974  that captures a leading edge of a microplate. The vertical position of the edge can vary from microplate warping and/or variation in how a microplate can be gripped by a jaw of a plate gripper robot. A shelf  970  can provide support for plate storage unit  828 . Entry guide  974  can be indexed using a linear motor  972 . 
       FIG. 101  is a process flow diagram illustrating a software command and control architecture for a loading distribution system, according to some embodiments. A system controller  982  can networked to an enterprise resource planning (ERP) system  983 , using an inter or intra network  985 . ERP system  983  can provide work order requests to system controller. 
     In some embodiments, system controller  982  ( FIG. 101 ) can manage and track source plates and microplates at various locations in loading distribution system  800  ( FIGS. 64 and 65 ). Locations for a source plate can comprise, for example, in a source plate storage unit like an incubator, in one or more source plate holders, or in one or more grippers of one or more source plate handling devices. Locations for a microplate can comprise, for example, in one or more plate storage units, in or on one or more tables, or in one or more jaws of one or more plate handling devices. System controller  982  can be adapted to track and trace the contents of one or more dispensers, each disposed in one or more respective dispensing devices. 
     When processing a work order or manufacturing microplates, system controller  982  provides control, control, and communication for wash station assemblies module  984 , a tip firing controller  986 , a dispensing assemblies module  988 , an incubator controller  990  also known as a source storage unit controller, an incubator robot controller  992  also known as a storage plate handling device controller, a fluidics controller  994 , a hotel module  996  also known as a storage unit controller, a hotel robot controller  998  also known as a plate handling device controller, a bar code controller  976  also known as a marking indicia reader controller, a XYZ motion controller  978 , and a quality control controller  929 . Wash station assemblies module  984 , tip firing controller  986 , dispensing assemblies module  988 , incubator controller  990 , incubator robot controller  992 , fluidics controller  994 , hotel module  996 , hotel robot controller  998 , and bar code controller  976  can be provided as part of one or more Original Equipment Manufacturer (OEM) packages including Application Protocol Interfaces (API) for all subassemblies. System controller  982  and XYZ motion controller  978  can be provided using real-time manufacturing protocols, for example, Supervisory Control And Data Acquisition (SCADA), a computer system for gathering and analyzing real time data. Quality control controller  929  can comprise a decision maker. QC controller  929  can gather data and status from various systems comprising a loading distribution system, to render a decision for each microplate processed by loading distribution system. 
     In some embodiments, the array of dispensers can be aligned to a microplate, in order to accomplish parallel dispensing of different reagents into different locations at the same time. Dispensers can dispense spots of an assay reagent into one or more locations of a microplate by, for example, aspirating a volume of assay reagent sufficient for multiple spots. The aspirated volume can subsequently be dispersed as spots into multiple locations, where each location receives substantially the same mass of assay reagent. 
     A dilution problem can be observed using arrayed dispensers. Dilution can occur because a dispenser system fluid can dilute an assay reagent, as it is dispensed. Because a dispenser can dispense a volume of the reagent and system fluid, a reduced mass of assay reagent can be deposited into each location from dispensing action to dispensing action. 
     In some embodiments, a dispenser can be programmed to compensate for the dilution affect. The aspirate and dispense arrayed liquid handling technologies, can dispense different amounts of assay reagents for each nozzle for each dispense action. The level of dilution can be measured, and the measured curves can be used to calibrate the effect of dilution. In some embodiments, a method for calibrating the observed diffusion on a tip-by-tip basis, and compensating for the loss of dispensed assay reagent per nozzle from dilution by programming dispensing to dispense more solution per spot, is provided. A required increase in spot volumes can be calculated by mathematically integrating an area under a fluorescence-dispense calibration curve. In some embodiments, dynamic programming of the dispense volumes can provide microplate to microplate reproducibility of dispensed mass of assay reagents (spots), and can reduce assay reagent waste by allowing the use of highly diluted assay reagents from the dispensing device. 
     In some embodiments, methods of spotting assay reagents based on dispenser arrays, into microplates, consistent with the banded format of filling devices, and the production of source plates for spotting, are provided. 
     In some embodiments, assay  1000  can be distributed on microplate  20  using a filling apparatus, such as filling apparatus  400 , a robotic filler, or a manual filler to distribute one or more components of assay  1000  across microplate  20  in columns or bands, for example, as illustrated in  FIG. 102 . For microplates that accommodate more than one sample, the sample distribution can map to this columnar or banded format. 
       FIG. 102  illustrates sample distribution in a banded format using a robotic or manual filler head. The head comprises tips  746 ,  748 ,  750 ,  752 ,  754 ,  756 ,  758 , and  760 , respectively. Tips  746 ,  748 ,  750 ,  752 ,  754 ,  756 ,  758 , and  760  can aspirate fluids from source plate  862 . Source plate  862  can comprise, for example, a 96 or a 384-location plate, including, for example, biological reagents or pre-amplified samples. Tips  746 ,  748 ,  750 ,  752 ,  754 ,  756 ,  758 , and  760  can distribute the aspirated samples across microplate  20  to form bands or columns across microplate  20 , for example, bands about 9 mm wide, bands about 4.5 mm wide, bands about 2.25 mm wide, or bands about 1.125 mm wide. The microplate can include, for example, 6,144 wells. Tips  746 ,  748 ,  750 ,  752 ,  754 ,  756 ,  758 , and  760  can dispense individual samples in bands across a plurality of rows of microplate  20 . As illustrated in  FIG. 102 , tip  746  can correspond to band  746 ′, tip  748  can correspond to band  748 ′, tip  750  can correspond to band  750 ′, tip  752  can correspond to band  752 ′, tip  754  can correspond to band  754 ′, tip  756  can correspond to band  756 ′, tip  758  can correspond to band  758 ′, tip  760  can correspond to band  760 ′, and tip  762  can correspond to band  762 ′. In an exemplary embodiment, tip  746  can load an eight-row column that is a total of 9 mm wide, from one end to the other end of the card, to include band  746 ′ illustrated in  FIG. 102 . With a number of sweeps along the card, back-and-forth, a band of sample can be loaded into the microplate, and with an 8-tip dispenser, the entire 6144 wells of a 6144 well microplate can be loaded with eight motions of the filler to achieve loading one respective well at a time, for each dispenser tip. 
       FIG. 31  illustrates the use of a dead row between sample-loaded wells that can be used to avoid cross-contamination of two rows to be tested, taking advantage of a banded format.  FIG. 103  illustrates a microplate  764 . In the following discussion, rows run from left to right. Microplate  764  includes three rows, illustrated from left to right in the figure, including a first row into which a first sample is loaded and including sample wells  766 . A second row into which a second sample is loaded includes sample wells  770 . The row containing sample wells  768 , located in between the rows respectively containing sample wells  766  and sample wells  770 , can be used as a dead row and can be skipped during a sample loading process. If any of the first or second samples might stray from its intended row, it can be captured in the dead row. That is, if a sample deposited in well or location  766  or well or location  770  of microplate  764 , carries over to an adjacent location  768 , no problem arises because the results of any assays in wells  768  would not be analyzed. For example, when using a robotic or manual filler, any possible cross-contamination between samples can be prevented by leaving approximately one unused row (a “dead row”) between each band of loaded samples in the microplate. The dead row can comprise one or more rows. 
     In some embodiments, a method of avoiding cross-contamination of a plurality of samples disposed in locations of a microplate can be provided. The method can include loading a filling device that can include a plurality of dispensers, each dispenser can include a fluid; translating the filling device along a translation path traversing a microplate that can include rows of locations; and dispensing a band of a respective fluid from each of the dispensers along a portion of the translation path to load rows of the locations, where the bands do not contact one another and the rows include loaded rows and a dead row between otherwise adjacent loaded rows. 
     Bands can contain the same set of samples or assay reagents across the microplate. One row can be eliminated from each band on the microplate. Where one band or one sample is provided on the microplate, there can be no need for a dead row to prevent sample cross-contamination. 
     In some embodiments, the dead rows of a microplate can be left empty or can be spotted with one or more components of assay. A buffer, for example, a TaqMan buffer, comprising no templates in common with the assay reagents in the bands, can be used to fill locations in a dead row. In some embodiments, each microplate can comprise an m×n configuration. Dead rows do not have to comprise wells or fluid locations. Dead rows can comprise other markings or features, for example, mold ejector pins can be disposed in the dead rows to improve a release of the microplate from a mold. Dead row wells or locations can be loaded with a calibrating dye or other marker or control substance useful in calibrating, for example, with respect to fluorescence or background noise. Dead row wells or locations can be loaded with a dye or other marker useful in providing identifiable locations on the microplate. 
       FIG. 104  illustrates a system according to some embodiments for manufacturing source plates and spotted microplates. Loading distribution system  800  can include: a plate-handling station  774  for moving at least one microplate; a first dispensing station  780  and a second dispensing station  782 ; a source incubator  776 ; and a microplate incubator  778 . Each dispense station can dispense fluid, for example, into or onto a microplate. Each dispense station can aspirate fluid from one or more source plate. Plate-handling station  774  can move source plates (not illustrated) in and out of source incubators  778 . Plate-handling station  774  can move and microplates in and out of dispensing stations  780 ,  782 . The source plates can be stored in incubators when not in use. 
     In some embodiments, source plates can be stored, optionally lidded, in source incubator  776  that can circulate, for example, high humidity filtered air around the source plates. This can, for example, prevent evaporation of the assay reagents. There can be a delay between when source plates are prepared and when they are used for spotting destination microplates. The delay can be problematic because evaporation can adversely change the concentration of the reagents. 
     In some embodiments, the spotted assay reagents can be dried and the microplates can be protected from dust during production. Drying of microplates can take place in microplate incubator  778 . The destination microplates can be stored, optionally lidded, in microplate incubator  778  that can circulate low humidity filtered air around the microplates. Because the spotted assay reagents can be dried within microplate incubator  778 , a post-batch drying step for the microplates can be eliminated. In some embodiments, loading distribution system  800  can be housed in an enclosure such that the housing can enclose loading distribution system  800 . The housing can comprise a class  1000  or cleaner clean room. 
     Plate-handling station  774  can be adapted to selectively pick up and deposit in dispensing station  780 ,  782 , individual microplates, at least one at a time. The plate-handling station  774  can include, for example, a robotic arm. The plate-handling station  774  can be adapted to simultaneously remove a first microplate from an incubator and deposit a second microplate an incubator. Dispensing stations  780  and  782  can include at least 96 dispensing tips, or at least 384 dispensing tips. Each dispensing station can include a plurality (two or more) of dispensers. Dispensing stations  780  and  782  can further include a plurality of (two or more) storage reservoirs. The source incubator  776  can store a source plate. The microplate incubator  778  can store a microplate that is unspotted, partially spotted, or fully spotted. The source incubator  776  can include circulated high humidity filtered air in order to prevent evaporation of the source assay reagents from the stored source plate. Microplate incubator  778  can include circulated low humidity filtered air to dry the spotted assay reagents. Microplate incubator  778  can maintain the spotted dried assay reagents in a dried state on the spotted microplate. Microplate incubator  778  can prevent a post-batch drying step. 
     The plate-handling station  774  can be adapted to selectively pick up and deposit individual source plates from the source hotel  776 , microplates from the microplate hotel  778 , or microplates and/or source plates from dispensing station  780 ,  782 . The plate-handling station can transfer source plates from the dispensing station  780  and  782  to the appropriate source incubator  776 . The plate-handling station can transfer microplates from the dispensing station  780  and  782  to the appropriate microplate incubator  778 . The source plates and/or the microplates can optionally be lidded. The incubators can include a device for lidding and de-lidding a source plate. 
     In some embodiments, methods and systems are provided that improve the manufacturing of microplates by: increasing microplate to microplate reducibility and reducing assay reagent waste; preventing sample cross-contamination from the use of robotic and manual fillers; reducing evaporation loss of assay reagents from source plates; assisting in the drying of spotted assay reagents on microplates, and avoiding a post-batch step of drying the microplates; and reducing dust contamination of both source and microplates. 
       FIG. 105  is a top-plan view illustrating a mapping of fluid locations of a 384-location source plate into a dispensing device comprising 96 dispensers, further into a 6,144-microplate. Microplate  20  can comprise a plurality of grids, for example, 96-grids. A grid  854  can comprise 64 locations. Each of the locations in a grip of microplate  20  can be dispensed into or onto by a respective dispenser  868  of dispensing device  814 , when dispensing device  814  comprises 96-dispensers. A quarter of a grid  852 , 16 locations, illustrates a location map pattern. The locations in quarter of a grid  852  can be addresses as 1, 2, 3, and 4 for a first row; 7, 8, 9, and 10 for a second row; 17, 18, 19, and 20 for a third row; and 25, 26, 27, and 28 for a fourth row. Loading distribution system  800  can dispense into a location number 1 during a first pass over microplate  20 , location number 2 during a second pass over microplate  20 , and so on so forth. To accomplish this, loading distribution system  800  can control the X and Y placement of microplate  20  using X-Y alignment, for example, as provided by alignment stage  932  as described above when dispensing device  814  is fixed or stationary with relative to microplate  20 , or by offsetting each dispenser  868  of dispensing device  814 . 
     In some embodiments, source plate  862  can be divided into 96-grids, each grid  848  comprising 4-locations for fluid aspiration. Loading distribution system  800  can aspirate from a location number 1 during a first pass over source plate  862 , location number 2 during a second pass over source plate  862 , and so on so forth. To accomplish this, loading distribution system  800  can control the X and Y placement of source plate  862  using X-Y alignment, for example, as provided by source plate and wash station  814   a  as described above when dispensing device  814  is fixed or stationary with relative to microplate  20 , or by offsetting each dispenser  868  of dispensing device  814  while holding source plate  862  in fixed position. 
     In some embodiments, a system and method for manufacturing a microplate comprising a plurality of fluid samples, for example, about 768 or more samples, about 1536 or more fluids, about 3072 or more fluids, about 6,144 or more fluids, about 12,288 or more fluids, are described. In some embodiments the plurality of fluids can all be the same fluid and in some embodiments each fluid can be different from all the other fluids. The plurality of fluids can reside in or on a microplate. 
     In some embodiments, fluids to loading distribution system  800  can be provided using a source plate, for example, a multiwell source plate. The source plate can comprise 24 or more wells, for example, 48 or more wells, 96 or more wells, 192 or more wells, 384 or more wells, or 768 or more wells. 
     In some embodiments, a dispensing device comprising a plurality of dispensers can be used in the present teachings. The dispensers can number 24 or more tips, for example, 48 or more tips, 96 or more tips, 192 or more tips, 384 or more tips. The dispensers can be, for example, piezo-electric spotting tips. The dispensers can be disposed in an SBS microtiter footprint, for example, the footprint and pitch distribution of a standard 96 well microtiter plate, a 192 well microtiter footprint pitch, a 384 well microtiter footprint, etc. In some embodiments, the dispensers can be fixed in position. In some embodiments, the dispensers can be moveable within a subportion of the dispensing device. 
     According to some embodiments, a system utilizing a 384-well source plate using a 96-dispenser device can be used to manufacture a microplate comprising, for example, 6,144 wells. Loading distribution system  800  can utilize, for example, 16, 384 well source plates, to access 6,144 unique fluids from the 36 times 384 or 6,144 wells. A 96-dispenser device can access a 384-source plate four times, each time drawing 96 unique fluids into corresponding 96-dispensers. Thus, the dispensing device can aspirate from a 384 well source plate  4  times. Sixteen source plates and 64 aspirations can be utilized to aspirate 6,144 unique fluids. A dispenser can be positioned over a target microplate comprising 6,144 wells, 64 times. For a 96 tip dispenser spotting a 6144 well microplate, each of the 64 dispensations per dispenser tip can be offset from the other dispensations so that each dispenser tip dispenses to 64 different combinations of X and Y coordinates, for example, so each tip spots 64 different wells. 
     In some embodiments, a method of dispensing can comprise: (a) loading a dispensing device comprising n fixed dispensers with a first plurality of fluids from a first source plate, wherein the source plate comprises m fluids, wherein n is an integer greater than or equal to two, and m is a positive whole number multiple of n; (b) moving a first microplate into a receiving position with respect to the fixed dispensers; (c) dispensing n fluids from the dispensers onto or into a first set of n locations on or in the first microplate, (d) moving at least one additional microplate into receiving position with respect to the dispensers; (e) dispensing n fluids from the dispensers onto or into a first set of n locations on or in the at least one additional microplate; (f) loading the n dispensers with a second plurality of fluids from a second source plate, wherein the second source plate comprises m fluids; (g) moving the first microplate into a receiving position with respect to the fixed dispensers; (h) dispensing n fluids from the dispensers onto or into a second set of n locations on or in the first microplate; (i) moving the at least one additional microplate into receiving position with respect to the dispensers; and (j) dispensing n fluids from the dispensers onto or into a second set of n locations on or in the at least one additional microplate. The first source plate can be the same as the second source plate, or they can be different source plates. 
     The method of dispensing can further involve loading from a plurality of source plates, for example, four, eight, 16, 32, 64, 96, 384, or more. In some embodiments, the first and second source plates can be the same and the first plurality of fluids can be a different plurality of fluids than the second plurality of fluids. In some embodiments, the first plurality of fluids can be the same plurality of fluids as the second plurality of fluids. In some embodiments, the first plurality of fluids can comprise a first plurality of mixtures, and each mixture can comprise two or more reagents for a nucleic acid sequence reaction. The method can comprise spotting a microplate that comprises, for example, 6,144 or more wells. 
     In some embodiments, a method of dispensing fluids is provided that comprises: (a) aspirating a first fluid volume into a dispenser adapted to dispense fluid volumes of one microliter or less; (b) dispensing a desired amount of the fluid volume, to form a dispensed portion, (c) calculating the volume of the dispensed portion, and (d) calculating an adjusted desired volume that compensates for a difference between the desired volume and the volume of the dispensed portion. The method can further comprise: (e) dispensing an adjusted desired volume of the fluid volume, to form a second dispensed portion, (f) calculating the volume of the second dispensed portion, and (g) calculating an adjusted desired volume that compensates for a difference between the adjusted desired volume and the volume of the dispensed portion. The method can comprise repeating the dispensing and two calculating steps for each dispensation of the dispenser. The method can be used on a piezo-electric dispenser, on an acoustic dispenser, or the like. 
     The method of dispensing a fluid can comprise calculating the volume by remembering a count of the number of dispensings per aspiration, and looking up in a table a level of dilution determined by the count. As fluid can be dispensed from the dispenser, the loss of volume can comprise an effect on the dispensed amount and the method can improve dispensing accuracy. A computer control unit and a memory can be used to track the dispensing and determine adjustments to be made if compensation is needed for a loss of volume per dispensation. The dispenser can comprise a plurality of dispensers and the calculating can comprise calculating a level of dilution of the dispensed volume for each dispenser of the plurality of dispensers. The dispenser can comprise a plurality of dispensers and the adjusting can comprise adjusting the dispensed volume of each dispenser of the plurality of dispensers. 
     In some embodiments, a method of loading a microplate is provided that comprises: translating a filling device comprising a plurality of dispensers, each dispenser comprising a fluid, along a translation path traversing a microplate comprising rows of wells, wherein the wells can comprise an average minimum dimension equal to a first dimension; and dispensing a band of a respective fluid from each of the dispensers along a portion of the translation path to load rows of the wells, wherein the bands do not contact one another and the rows include at least two adjacent loaded rows of wells which can be spaced apart from one another by a dimension that is about the same as or greater than the first dimension. The at least two adjacent loaded rows of wells can be separated from one another by at least one dead row of wells, that is, at least one row of wells that has not purposefully been loaded, but rather, that may receive some overspray or overshoot of fluids intended to be dispensed into the loaded wells. In place of a dead row of wells, the method can comprise dispensing to a microplate that includes a thickened sidewall between the two adjacent loaded rows, wherein the sidewall can be at least as wide as the average width of each of the well. The sidewall can be as high as all of the other sidewalls between adjacent wells of the microplate. 
     The method of loading a microplate can comprise the dispensation of, for example, one or more biological sample. The method can comprise the dispensation of, for example, a biological reagent, an assay, a probe, a primer, an oligonucleotide, and a combination thereof. The plurality of the wells of the microplate can each be preloaded with components for a same kind of assay or for respective different kinds of assays. Each well in each row of wells loaded by one of the bands can comprise components for a same kind of assay. In some embodiments, the method can comprise dispensing a marker fluid in the at least one dead row of wells, for example, a control liquid, dye, or optical marker. The marker fluid can be used to calibrate fluorescence signals and/or to provide for location identification like a milepost or landmarker. 
     In some embodiments, loading distribution system  800  can be used to transfer assay components such as oligonucleotides from source plates, for example, 384-well source plates, to microplates  20 . Loading distribution system  800  can produce a plurality of microplates  20  simultaneously in batches. Batches can comprise a plurality of source plates, for example, 2, 4, 8, 16, 32, or more source plates. Batches can comprise a plurality of target microplates, for example, about 5 or more, about 10 or more, about 100 or more, or about 200 or more, microplates per batch. Loading distribution system  800  can be integrated into a manufacturing system. The manufacturing system can provide, for example, work orders, a manufacturing historian, or logger. The manufacturing system can comprise an enterprise resource planning (ERP) system. Loading distribution system  800  can maintain queues for source and target microplates. Loading distribution system  800  can provide different temperature and humidity control environments for the source and the target microplates. A cache of source and target microplates can be disposed in appropriate stations of loading distribution system  800 . This can allow for the unattended operation of loading distribution system  800 . 
     In some embodiments, control software and/or a dispensing device can be utilized that is configurable for a list of variables. Exemplary variables can be found herein in the EXAMPLE section. Loading distribution system  800  can utilize, for example, a 96-dispenser dispensing device, or a 384-dispenser dispensing device. Loading distribution system  800  can utilize, for example, 1, 2, 4, 8, 16, or more than 16 dispensing devices. Loading distribution system  800  can be designed to mitigate a throughput bottleneck at a dispensing device. 
     In some embodiments, Incoming Quality Control (IQC) requirements for microplate  20  can be used for a Whole Genome Array (WGA), a Focused Gene Set(s) (FGS) system, or a custom gene-set(s) system. The IQC can require, for example, a 100% inspection of a microplate in from about 1 second to about 60 seconds, from about 1 second to about 10 seconds, or from about 3 seconds to about 6 seconds. The inspection can comprise tests for, for example, an absence or presence of spots, spot metrics, and/or volume and concentration measurements (CPM). The IQC system can comprise hardware and/or software. In some embodiments, the IQC station can comprise a fluorescence detection system using, for example, infrared dye spiking or blue LED excitation of spots. The IQC station can be a data logger. The IQC can be a decision maker. 
     In some embodiments, a dispensing device can be configured to disable rows of dispensers. For example, a 96 dispenser-dispensing device can mimic 12, 24, and 48 dispenser configurations. In some embodiments, the unused dispensers can be disabled, for example, using software. In some embodiments, the unused dispensers can be physically removed from a dispense position. A manifold in the dispensing device can be reconfigured to gang disabled tips. A common valve disposed on the manifold can shut-off unused dispensers to prevent them from aspirating air. The different dispensing devices can be swapped manually or robotically. 
     An exemplary loading distribution system can provide many different combinations of variables as exemplified in the table below: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Counts 
                 Unit 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Variable 
                   
                   
               
               
                 number of tips per head 
                 96 
               
               
                 number of spotting heads 
                 4 
               
               
                 number of replicates per tip per source 
                 1 
               
               
                 plate well 
               
               
                 moving time between 2 stations 
                 1 
                 sec 
               
               
                 move time between replicates on microplate 
                 0.5 
                 sec 
               
               
                 tip firing cycle time for each spotting 
                 1 
                 sec 
               
               
                 number of stations for other functions 
                 4 
               
               
                 number of dispenses per tip per source plate 
                 1 
               
               
                 number of high-density microplates per batch 
                 150 
               
               
                 number of source plates per batch 
                 16 
               
               
                 number of passes for each microplate 
                 16 
               
               
                 volume in tip per aspirate 
                 3 
                 μl 
               
               
                 volume per dispense 
                 0.03 
                 μl 
               
               
                 percent of volume dispensed per aspirate 
                 50% 
               
               
                 number of dispenses per aspirate 
                 50 
               
               
                 number of aspirates per source plate well 
                 3 
               
               
                 per tip per batch 
               
               
                 number of total aspirate cycles per head per 
                 12 
               
               
                 batch 
               
               
                 number of spotting cycles per tip per batch 
                 2400 
               
               
                 number of spotting cycles per head per batch 
                 2400 
               
               
                 number of index cycles to ramp up and down 
                 14 
               
               
                 Total aspirate time per batch 
                 5280 
                 sec 
               
               
                 Total spotting time per batch 
                 16898 
                 sec 
               
               
                 Aspirate Serial Actions 
               
               
                 move wash station in position 
                 5 
                 sec 
               
               
                 wash tips 
                 45 
                 sec 
               
               
                 move wash station out 
                 5 
                 sec 
               
               
                 load source plate in aspirate position 
                 5 
                 sec 
               
               
                 aspirate time 
                 15 
                 sec 
               
               
                 unload source plate from aspirate position 
                 5 
                 sec 
               
               
                 Aspirate cycle time 
                 80 
                 sec 
               
               
                 Dispense Spotting Station Actions 
               
               
                 move shuttle in dispense position 
                 1 
                 sec 
               
               
                 position plate for spotting under head 
                 4 
                 sec 
               
               
                 tip firing time per high-density plate per 
                 1 
                 sec 
               
               
                 source plate 
               
               
                 reposition plate after dispense 
                 1 
                 sec 
               
               
                 Spotting Cycle Times 
                 7 
                 sec 
               
               
                 Actions 
               
               
                 load per unload source plate @ incubator 
                 40 
                 sec 
               
               
                 handling time per plate 
                 40 
                 sec 
               
               
                 Other Station Actions 
               
               
                 move shuttle in dispense position 
                 1 
                 sec 
               
               
                 unload shuttle high-density plate @ hotel 
                 4 
                 sec 
               
               
                 load high-density plate in shuttle @ hotel 
                 4 
                 sec 
               
               
                 inline QC 
                 4 
                 sec 
               
               
                 barcode reading and writing of high-density plate 
                 2 
               
               
                 Station process time per pass 
                 5 
                 sec 
               
               
                   
               
            
           
         
       
     
     Loading distribution system  800  can provide the following throughput for spotting with four 96-tip dispense devices. 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 number of 384-well source plates= 
                 16 
                 16 
               
               
                 number of unique assay= 
                 384 × 16 = 
                 6144 
               
               
                 number of tips per head= 
                 4 
                 96 
               
               
                 number of heads= 
                 4 
                 4 
               
               
                 number of total tips= 
                 96 × 4 
                 384 
               
               
                 number of passes for each high-density plate= 
                 6144/4/96 = 
                 64 
               
               
                 number of source wells per tip= 
                 6144/384 = 
                 16 
               
               
                   
               
            
           
         
       
     
     Microplate Filling 
     In some embodiments, a filling apparatus  400  can be used to fill at least some of the plurality of wells  26  of microplate  20  with one or more components of assay  1000 . It should be understood that filling apparatus  400  can comprise any one of a number of configurations. 
     In some embodiments, referring to  FIGS. 20-22(   b ), filling apparatus  400  comprises one or more assay input ports  402 , such as about 96 input ports, disposed in an input layer  404 . In some embodiments, assay input ports  402  of input layer  404  can be in fluid communication with a plurality of microfluidic channels  406  disposed in input layer  404 , an output layer  408 , or any other layer of filing apparatus  400 . In some embodiments, the plurality of microfluidic channels  406  can be formed in an underside of input layer  404  and a seal member can be placed over the underside of input layer  404 . In some embodiments, the seal member can comprise a perforation (e.g. hole) positioned over a desired location in microplate  20  to permit a discrete fluid communication passage to extend therethrough. In some embodiments, the plurality of microfluidic channels  406  can be arranged as a grouping  407  ( FIG. 20) . In some embodiments, assay input ports  402  can be positioned at a predetermined pitch (e.g. 9 mm) such that each assay input port  402  can be aligned with a center of each grouping  407 . In some embodiments, the plurality of microfluidic channels  406  can be in fluid communication with a plurality of staging capillaries  410  formed in output layer  408  ( FIGS. 21-22(   b )). 
     In some embodiments, input layer  404  and output layer  408  can be bonded or otherwise joined together to form a single unit. This bond can be made with, among other things, a double-stick tape, a laser weld, an ultrasonic weld, or an adhesive. However, it should be appreciated that the bonding or otherwise joining of input layer  404  and output layer  408  is not required. 
     During filling, assay  1000  can be put into at least one assay input port  402  and can be fluidly channeled toward at least one of the plurality of microfluidic channels  406 , first passing a surface tension relief post  418  in some embodiments. In some embodiments, surface tension relief post  418  can serve, at least in part, to evenly spread assay  1000  throughout the plurality of microfluidic channels  406  and/or engage a meniscus of assay  1000  to encourage fluid flow. Assay  1000  can be fluidly channeled through the plurality of microfluidic channels  406  and can collect in the plurality of staging capillaries  410  ( FIG. 22(   b )). Assay  1000  can then be held in the plurality of staging capillaries  410  by capillary or surface tension forces. 
     In some embodiments, as illustrated in  FIGS. 21 and 22(   a )-( b ), microplate  20  can be attached to filling apparatus  400  so that each of the plurality of staging capillaries  410  is generally aligned with each of the plurality of wells  26 . In some embodiments, filling apparatus  400  comprises alignment features  411  ( FIG. 20)  operably sized to engage corresponding alignment feature  58  on microplate  20  to, at least in part, facilitate proper alignment of each of the plurality of staging capillaries  410  with a corresponding (respective) one of the plurality of wells  26 . In some embodiments, the combined unit of filling apparatus  400  and microplate  20  can then be placed in a centrifuge. The centrifugal force of the centrifuge can, at least in part, urge assay  1000  from the plurality of staging capillaries  410  into each of the plurality of wells  26  of microplate  20 . Filling apparatus  400  can then be removed from microplate  20 . In some embodiments, microplate  20  can then receive additional reagents and/or be sealed with sealing cover  80 , or other sealing feature such as a layer of mineral oil, and then placed into high-density sequence detection system  10 . 
     In some embodiments, capillary or surface tension forces encourage flow of assay  1000  through staging capillaries  410 . In this regard, staging capillaries  410  can be of capillary size, for example, staging capillaries  410  can be formed with an exit diameter less than about 500 micron, and in some embodiments less than about 250 microns. In some embodiments, staging capillaries  410  can be formed, for example, with a draft angle of about 1-5° and can define any thickness sufficient to achieve a predetermined volume. To further encourage the desired capillary action in staging capillaries  410 , staging capillaries  410  can be provided with an interior surface that is hydrophilic, i.e., wettable. For example, the interior surface of staging capillaries  410  can be formed of a hydrophilic material and/or treated to exhibit hydrophilic characteristics. In some embodiments, the interior surface comprises native, bound, or covalently attached charged groups. For example, one suitable surface, according to some embodiments, is a glass surface having an absorbed layer of a polycationic polymer, such as poly-l-lysine. 
     Ramps 
     In some embodiments, as illustrated in  FIGS. 22(   b ) and  23 ( a )-( b ), each of the plurality of staging capillaries  410  can comprise a ramp feature  414  disposed at an entrance thereof to achieve a predetermined capillary action. It should be appreciated that ramp feature  414  can be formed on one or more edges of the entrance to each of the plurality of staging capillaries  410 . In some embodiments, ramp feature  414  can comprise a countersink lip or chamfered rim formed about the entire entrance. In some embodiments that do not employ the plurality of microfluidic channels  406 , ramp feature  414  can be used to reduce an angle between staging capillary  410  and an upper surface  456  (to be described herein) of output layer  408  to aid in capillary flow and/or exposure time to a fluid bead moving thereby. 
     Nozzles Bottom Features 
     In some embodiments, with reference to  FIGS. 22(   b ) and  24 , output layer  408  can comprise a protrusion  450  formed on an outlet  434  of staging capillary  410 . In some embodiments, protrusion  450  can be shaped to cooperate with a corresponding shape of each of the plurality of wells  26 . In some embodiments, protrusion  450  can be conically shaped to be received within circular rim portion  32  of each of the plurality of wells  26 . In some embodiments, protrusion  450  can be square-shaped to be received within square-shaped rim portion  38  of each of the plurality of wells  26 . Protrusion  450 , in some embodiments, can define a sufficiently sharp surface such that the capillary force within staging capillary  410  can retain assay  1000  and protrusion  450  can inhibit movement of assay  1000  to adjacent wells  26 . In some embodiments, protrusion  450  of output layer  408  can be positioned above microplate  20 , flush with first surface  22  of microplate  20  ( FIG. 22(   a )), or disposed within well  26  of microplate  20  ( FIG. 22(   b )). In some embodiments, protrusion  450  can define a nozzle feature that comprises a diameter that is less than the diameter of the plurality of wells  26  to aid, at least in part, in capillary retention of assay  1000  within staging capillary  410 . 
     Protrusion  450  can be provided with an exterior surface that is hydrophobic, i.e., one that causes aqueous medium deposited on the surface to bead. For example, protrusion  450  can be formed of a hydrophobic material and/or treated to exhibit hydrophobic characteristics. This can be useful, for example, to prevent spreading of a drop, formed at tip portion  1840 . A variety of known hydrophobic polymers, such as polystyrene, polypropylene, and/or polyethylene, can be utilized to obtain desired hydrophobic properties. In addition, or as an alternative, a variety of lubricants or other conventional hydrophobic films can be applied to tip portion  1840 . 
     Bottom Feature—Spacer 
     In some embodiments, as illustrated in  FIG. 24 , one or more spacer members  452  can be formed along bottom surface  429  of output layer  408  to, at least in part, achieve a desired spacing between output layer  408  and microplate  20 . In some embodiments, spacer member  452  can be formed as an elongated member ( FIG. 24 ), a post ( FIG. 107 ), one or more spaced-apart members, or the like. 
     Fluidic Patterns 
     In some embodiments, as illustrated in  FIGS. 23(   a )-( b ) and  25 ( a )( f ), the plurality of microfluidic channels  406  can have any one of a plurality of configurations for carrying assay  1000  to each of the plurality of staging capillaries  410 . In some embodiments, each of the plurality of staging capillaries  410  can be in fluid communication with only one of the plurality of microfluidic channels  406  ( FIGS. 23(   a )-( b ),  25 ( a )-( d ), and  25 ( f )) in a series-type configuration. In some embodiments, each of the plurality of staging capillaries  410  can be in fluid communication with two or more of the plurality of microfluidic channels  406  ( FIG. 25(   e )) in a multi-path or parallel-type configuration. In such parallel-type configurations, fluid can flow along the path of least resistance to fill each of the plurality of staging capillaries  410  in the least amount of time. In any configuration, the time required to fill each of the plurality of staging capillaries  410  can be reduced by reducing the length of each microfluidic channel  406 . In some embodiments, a hybrid of the series-type and the parallel-type configurations can be used. In some embodiments, as illustrated in  FIG. 25(   f ), each of the plurality of microfluidic channels  406  can be in fluid communication with only one edge of each of the plurality of staging capillaries  410  to provide pass-by and filling action simultaneously. 
     In some embodiments, each of the plurality of microfluidic channels  406  can exert, at least in part, a capillary force to draw fluid (e.g. assay  1000 ) therein to aid in reducing the time required to fill. The capillary force of each of the plurality of microfluidic channels  406  can be varied, at least in part, by varying at least the dimensional properties of the plurality of microfluidic channels  406  according to capillary principles. 
     Pressure Nodules 
     In some embodiments, as illustrated in  FIGS. 106-113 , filling apparatus  400  comprises input layer  404 , output layer  408 , and an intermediate layer  494 , or any combination thereof for filling one or more components of assay  1000  into at least some of the plurality of wells  26  in microplate  20 . 
     In some embodiments, intermediate layer  494  can be positioned and aligned between input layer  404  and output layer  408 . In some embodiments, input layer  404  comprises assay input ports  402  extending therethrough. As illustrated in  FIGS. 107 and 108 , in some embodiments, each assay input port  402  can extend through input layer  404  and terminate at an extended outlet  496 . In some embodiments, extended outlet  496  can be sized to extend from input layer  404  such that an end  498  of extended outlet  496  is spaced a predetermined distance from output layer  408  ( FIG. 108 ). Extended outlet  496  can extend through a corresponding aperture  500  ( FIG. 106 ) formed through intermediate layer  494 . 
     In some embodiments, as illustrated in  FIG. 108 , extended outlet  496  can be aligned with surface tension relief post  418  extending upward from output layer  408 . In some embodiments, an internal diameter of extended outlet  496  can be larger than an outer diameter of surface tension relief post  418  to permit surface tension relief post  418  to be at least partially received within extended outlet  496 . Surface tension relief post  418 , in some embodiments, can be sufficiently sized to facilitate even spreading of assay  1000  throughout the plurality of microfluidic channels  406  and/or engage a meniscus of assay  1000  within assay input port  402  to encourage flow. In some embodiments, extended outlet  496  and surface tension relief post  418  can cooperate to facilitate alignments of input layer  404 , output layer  408 , and intermediate layer  494 . 
     In some embodiments, intermediate member  494  comprises microfluidic channels  406  extending there along (e.g., etched or otherwise formed in one major side thereof) in fluid communication with the plurality of staging capillaries  410  in output layer  408 . For example, microfluidic channels  406 , extending along a lower surface of intermediate layer  494 , can communicate with upper-end openings of staging capillaries  410 . It should be appreciated that the particular route configuration of microfluidic channels  406  can be any one of a number of configurations selected by one skilled in the art or one of those described herein. In some embodiments, intermediate member  494  can be compliant, or resiliently deformable, to permit flexing of intermediate member  494  in response to an external force. In some embodiments, intermediate member  494  can be made of polymeric materials, such as but not limited to rubber or silicone (PDMS). 
     As illustrated in  FIGS. 107-111 , in some embodiments, input layer  404  comprises one or more nodules  502  extending from a bottom surface  504 . In some embodiments, nodules  502  can be patterned along bottom surface  504  such that each nodule  502  can engage a top surface  506  of compliant intermediate layer  494 . During centrifugation, centripetal force exerted on input layer  404  can cause nodules  502  to engage compliant intermediate layer  494  to at least partially collapse or depress a segment of intermediate layer  494  against output layer  408  to minimize fluid communication between adjacent staging capillaries  410 . In some embodiments, as illustrated in  FIGS. 109 and 110 , nodules  502  can be patterned such that each nodule  502  is positioned adjacent each of the plurality of staging capillaries  410 . For example, nodules  502  can be disposed so that each nodule aligns, or corresponds, with a respective one of staging capillaries  410 . In some embodiments, nodules  502  can be patterned over portions of microfluidic channels  406  to close microfluidic channel  406  during centrifugation. In some embodiments, as illustrated in  FIG. 111 , nodules  502  can be patterned over each of the plurality of staging capillaries  410  to seal each of the plurality of staging capillaries  410  during centrifugation. For example, upon being depressed by nodules  502  during centrifugation, segments of intermediate layer  494  can seal the upper end openings of respective, corresponding staging capillaries  410 . 
     In some embodiments, as illustrated in  FIGS. 111 and 112 , a sealing feature  508  can extend from intermediate layer  494  that can be sized to fit into the corresponding staging capillary  410  by nodule  502  acting upon intermediate layer  494 . These, and substantially equivalent, embodiments can be used to define a shut-off valve during centrifugation or anytime a force is applied to input layer  404  and/or intermediate layer  494 . 
     It should be appreciated that the physical size and/or compliancy of one of more of input layer  404 , intermediate layer  494 , nodules  502 , and sealing features  508  can be tailored to achieve a predetermined sealing engagement upon application of a predetermined amount of force. Additionally, it should be appreciated that nodules  502  and/or sealing feature  508  can be of any shape conducive to applying a force and sealing an opening, respectively, such as, but not limited to, triangular, square, or conical. 
     In some embodiments, to load each of the plurality of staging capillaries  410 , a predetermined amount of assay  1000  can be placed at each assay input port  402 . Capillary force, at least in part, can draw at least a portion of assay  1000  from assay input port  402  into microfluidic channels  406  and further fill at least some of the plurality of staging capillaries  410 . In some embodiments, once at least some of the plurality of staging capillaries  410  are filled, output layer  408  and microplate  20  can be placed into a swing-arm centrifuge. In some embodiments, the centripetal force of the swing-arm centrifuge can be sufficient to overcome the surface tension of assay  1000  in each the plurality of staging capillaries  410 , thereby forcing a metered volume of assay  1000  into each of the plurality of wells  26  of microplate  20 . In some embodiments, the centripetal force of the centrifuge can be sufficient to exert a clamping force on at least one of input layer  404  and intermediate layer  494  to fluidly seal adjacent staging capillaries  410 , either at the entrance thereof or therebetween, to prevent residual assay  1000  left in assay input port  402  or assay  1000  from an undesired one of the plurality of wells  26  of microplate  20  from overfilling a particular staging capillary. In some embodiments, an external force (e.g. mechanical, pneumatic, hydraulic, electromechanical, and the like) can be applied to exert a clamping force on at least one of input layer  404  and intermediate layer  494  to fluidly seal adjacent staging capillaries  410 , either at the entrance thereof or therebetween. 
     In some embodiments, as illustrated in  FIG. 113 , at least some of input layer  404 , intermediate layer  494 , and output layer  408  can be used in conjunction with a clamp system  511 . In some embodiments, clamp system  511  comprises a base structure  513  and one or more locking features  515  extending therefrom. In some embodiments, base structure  513  comprises at least one alignment feature  517  operably sized to engage a corresponding alignment feature  58  on microplate  20  to, at least in part, facilitate proper alignment of each of the plurality of staging capillaries  410  relative to each of the plurality of wells  26 . In some embodiments, alignment feature  517  can further engage a corresponding alignment feature  519  formed in at least one of input layer  404 , intermediate layer  494 , and output layer  408 . In some embodiments, at least some of microplate  20 , input layer  404 , intermediate layer  494 , and output layer  408  can be coupled with base structure  513  such that locking feature  515  engages input layer  404  to exert a preload on intermediate layer  494  to prevent fluid flow and/or leakage of assay  1000  prior to achieving sufficient centrifugal speed in the centrifuge. In some embodiments, a top plate  521  can be used in conjunction with base structure  513  to ensure equal pressure application across input layer  404  by locking feature  515 . 
     Venting 
     In some embodiments, as illustrated in  FIGS. 114-119 , filling apparatus  400  comprises input layer  404 , output layer  408 , and a vent layer  523 , or any combination thereof for loading assay  1000  into at least some of the plurality of wells  26  in microplate  20 . In some embodiments, output layer  408  comprises microfluidic channels  406  formed in a side thereof and extending there along in fluid communication with the plurality of staging capillaries  410  in output layer  408 . 
     In some embodiments, input layer  404  comprises assay input ports  402  extending therethrough. As illustrated in  FIGS. 115-116 , in some embodiments, each assay input port  402  can extend through input layer  404  and terminate at extended outlet  496 . In some embodiments, extended outlet  496  can be sized to extend from input layer  404  such that an end  498  of extended outlet  496  is generally flush to a top surface  525  of vent layer  523  and aligned to a flow aperture  527  extending through vent layer  523 . 
     In some embodiments, input layer  404  comprises one or more vent features  529  ( FIGS. 116-119 ). In some embodiments, vent feature  529  can be sized to have a capillary force associated therewith that is lower than a capillary force within microfluidic channels  406  and/or each of the plurality of staging capillaries  410  to reduce the likelihood of assay  1000  flow through or into vent feature  529 . In some embodiments, vent feature  529  comprises a vent hole  531  extending through input layer  404  ( FIGS. 114-118 ) and in communication with atmosphere. In some embodiments, vent hole  531  can be coupled to a chamber or manifold  533  ( FIGS. 115 and 116 ) that can couple two or more vent apertures  535  formed in vent layer  523  to atmosphere. 
     In some embodiments, vent feature  529  comprises a pressure bore  537  ( FIG. 117 ) associated with one or more of the plurality of staging capillaries  410 . In some embodiments, pressure bore  537  can be formed in input layer  404 . For example, pressure bore  537  can extend from a lower surface of input layer  404  toward, but stopping short of, an opposing surface. In some embodiments, plural pressure bores  537  are disposed in an array corresponding to an array defined by staging capillaries  410 . Pressure bores  537 , in some embodiments, can be sized to act as an air capacitor trapping a portion of air therein that can contract or expand during filling of assay  1000  into filling apparatus  400  and/or centrifuging assay  1000  into each of the plurality of wells  26 , respectively. 
     Vent feature  529 , in some embodiments, can at least partially relieve vacuum created when assay  1000  is centrifuged from each of the plurality of staging capillaries  410  into each of the corresponding plurality of wells  26  of microplate  20  and permit improved loading. In some embodiments, vent feature  529  can at least partially interrupt fluid flow between adjacent staging capillaries  410  by introducing an air gap therebetween. In some embodiments, such an air gap can provide consistent metering of assay  1000  loaded into each of the plurality of wells  26 . 
     In some embodiments, vent layer  523  can be positioned and aligned between input layer  404  and output layer  408 . In some embodiments, as illustrated in  FIG. 116 , flow aperture  527  of vent layer  523  can be aligned with surface tension relief post  418  extending upward from output layer  408 . In some embodiments, an internal diameter of flow aperture  527  can be larger than the outer diameter of surface tension relief post  418  to permit surface tension relief post  418  to be at least partially received within flow aperture  527 . Surface tension relief post  418 , in some embodiments, can be sufficiently sized to facilitate even spreading of assay  1000  throughout the plurality of microfluidic channels  406  in output layer  408  and/or engage a meniscus of assay  1000  within assay input port  402  and/or flow aperture  527  to encourage flow. In some embodiments, extended outlet  496 , flow aperture  527 , and surface tension relief post  418  can cooperate to facilitate alignments of input layer  404 , output layer  408 , and vent layer  523 . 
     As illustrated in  FIGS. 116-118 , in some embodiments, vent layer  523  can be aligned with input layer  404  and output layer  408  such that vent apertures  535  are positioned above or between each of the plurality of staging capillaries  410 . In some embodiments, vent apertures  535  can be a circular bore ( FIG. 117 ) or any other shape, such as oblong ( FIG. 118 ), to accommodate for potential misalignment between input layer  404  and vent layer  523  and/or potential misalignment between vent layer  523  and output layer  408 . 
     In some embodiments, vent layer  523  can be made of any material conducive to joining with input layer  404  and/or output layer  408 . In some embodiments, vent layer  523  can comprise PDMS, which can aid in joining vent layer  523  to input layer  404  due to the intrinsic tackiness properties of PDMS. In some embodiments, vent layer  523  can be made using a double stick adhesive tape. In such embodiments, the double stick adhesive tape can be first applied to input layer  404  and then laser cut to accurately place vent apertures  535  to simplify assembly of input layer  404  and vent layer  523 . 
     In some embodiments, to load each of the plurality of staging capillaries  410 , a predetermined amount of assay  1000  can be placed at each assay input port  402 . Such placement can be effected, for example, using an automated pipette system (e.g., a Biomek) or hand-operated single- or multi-channel pipette device (e.g., a Pipetman). Capillary force, at least in part, can draw at least a portion of assay  1000  from assay input port  402  into microfluidic channels  406  and further fill at least some of the plurality of staging capillaries  410 . In some embodiments, outlet  434  of each of the plurality of staging capillaries  410  permits venting of air within each of the plurality of staging capillaries  410  during filling. In some embodiments, once at least some of the plurality of staging capillaries  410  are filled, input layer  404 , vent layer  523 , output layer  408 , and microplate  20  can be placed into a swing-arm centrifuge. In some embodiments, the venting features  529  can reduce vacuum effects on assay  1000  during centrifugation to more easily meter a volume of assay  1000  into each of the plurality of wells  26  of microplate  20 . 
     Assay Ports on Sides 
     In some embodiments, as illustrated in  FIGS. 120-131 , filling apparatus  400  can comprise assay input ports  402  positioned within and/or upon output layer  408 . In some embodiments, as illustrated in  FIG. 120 , assay input ports  402  can be positioned at an end  420  of output layer  408 . For example, such assay input ports can be positioned along a short dimension of a major surface (e.g., a top surface) of the output layer, adjacent and parallel to an end thereof. In some embodiments, as illustrated in  FIG. 121 , assay input ports  402  can be positioned at a side  422  of output layer  408 . For example, such assay input ports can be positioned along a long dimension of a major surface (e.g., a top surface) of the output layer, adjacent and parallel to a side thereof. Still further, in some embodiments, as illustrated in  FIG. 122 , assay input ports  402  can be positioned at opposing ends  420  or opposing sides  422  (not illustrated) of output layer  408 . In some embodiments, assay input ports  402  can be positioned at opposing ends  420  or opposing sides  422  (not illustrated) of output layer  408  with a fluid interrupt  409  (e.g. wall or barrier) to fluidly isolate those assay input ports  402  on one end or side from the remaining assay input ports  402  on the other end or side. 
     As illustrated in  FIG. 123 , in some embodiments, assay input ports  402  can each comprise a fluid well  424  bound by a plurality of upstanding walls  426 . In some embodiments, fluid well  424  of each assay input port  402  can be in fluid communication with one or more corresponding microfluidic channels  406  through a throat  430  formed in fluid well  424 . For example, such a throat can be formed in a lower region of the fluid well, so as to fluidly communicate the fluid well with the microfluidic channels. Throat  430  can comprise a diameter of, for example, 2 mm or less, 1 mm or less, 0.5 mm or less, or 0.25 mm or less. In some embodiments, such as illustrated in  FIG. 123 , throat  430  comprises a reservoir in fluid communication with one or more microfluidic channel  406 . In some embodiments, surface tension relief post  418  can be disposed in throat  430  to, at least in part, evenly spread assay  1000  throughout the plurality of microfluidic channels  406  and/or engage a meniscus of assay  1000  to encourage fluid flow. Surface tension relief post can, according to some embodiments, comprise a hydrophilic surface in order to further encourage fluid flow into the throat and, thus, the microchannels. 
     In some embodiments, as illustrated in at least  FIGS. 124-131 , microfluidic channels  406  can be in fluid communication with the plurality of staging capillaries  410  extending from microfluidic channel  406 , through output layer  408 , to a bottom surface  429 . In some embodiments, bottom surface  429  can be spaced apart from first surface  22  of microplate  20  ( FIG. 124 ) or can be in contact with first surface  22  of microplate  20 . In some embodiments, each of the plurality of staging capillaries  410  can be generally aligned with a corresponding one of the plurality of wells  26  of microplate  20 . In some embodiments, a protective covering (not shown) can be disposed over microfluidic channels  406  to provide, at least in part, protection from contamination, reduced evaporation, and the like. It should be understood that such protective covering can be used with any of the various configurations set forth herein. 
     Referring to  FIGS. 125-131 , to perform a filling operation, each assay input port  402  can be at least partially filled with assay  1000  or different assays or fluids ( FIG. 125 ). At least in part through hydraulic pressure and/or capillary force, assay  1000  can flow from fluid well  424  of each assay input port  402  through throat  430  into the one or more microfluidic channels  406  ( FIG. 126 ). As assay  1000  flows across an end-opening or mouth  432  of each of the plurality of staging capillaries  410 , capillary action, at least in part, draws a metered amount of assay  1000  therein ( FIG. 127 ). Assay  1000  can continue to flow down the one or more microfluidic channels  406  until each of the plurality of staging capillaries  410  can be at least partially filled with assay  1000  ( FIG. 128 ). In some embodiments, assay  1000  in each of the plurality of staging capillaries  410  can be held therein by capillary or surface tension forces to aid in the equal metering of assay  1000  to be loaded in each of the plurality of wells  26 . In some embodiments, outlet  434  of each of the plurality of staging capillaries  410  permits venting of air within each of the plurality of staging capillaries  410  during filling. 
     As illustrated in  FIGS. 129 and 130 , in some embodiments, filling apparatus  400  can be stake cut, generally indicated at  435 , via device  436  along a portion of one or more microfluidic channels  406 . In some embodiments, stake-cutting serves to, at least in part, aid in metering of assay  1000  in each well  26  by isolating the plurality of staging capillaries  410  from any excess assay  1000  left in each assay input port  402 . This arrangement can minimize additional assay  1000  left within each assay input port  402  from overfilling each of the plurality of wells  26  during later centrifugation. In some embodiments, stake cutting can be completed through mechanical and/or thermal deformation (e.g. heat staking) of output layer  408 . It should be appreciated that a Zbig valve can be used to achieve fluid isolation between the plurality of staging capillaries  410  and assay input port  402 , such as those described in commonly-assigned U.S. patent application Ser. No. 10/336,274, filed Jan. 3, 2003 and PCT Application No. WO 2004/011147 A1. 
     As illustrated in  FIG. 132 , in some embodiments, filling apparatus  400  can comprise reduced material areas  438  disposed in output layer  408 . In some embodiments, reduced-material areas  438  comprise one or more cutout portions  440  (e.g. voids, slots, holes, grooves) formed in output layer  408  on opposing sides of microfluidic channels  406 . The use of reduced material areas  438  can provide, among other things, reduced thermal capacity in the localized areas, which can increase the rate of heat staking and/or stake cutting. In some embodiments, the elongated shape of cutout portion  440  can accommodate any misalignment of the staking tool relative to output layer  408 . In some embodiments, following staking, excess assay  1000  in assay input ports  402  and/or the upstream portion of microfluidic channels  406  relative to stake cut  435  can be removed, if desired. In some embodiments, this can be accomplished by employing a wicking member  441 , as illustrated in  FIG. 131 . 
     In some embodiments, once at least some of the plurality of staging capillaries  410  are filled, output layer  408  and microplate  20  can be placed into a swing-arm centrifuge. In some embodiments, the centripetal force of the swing-arm centrifuge can be sufficient to overcome the surface tension of assay  1000  in each the plurality of staging capillaries  410 , thereby forcing a metered volume of assay  1000  into each of the plurality of wells  26  of microplate  20  ( FIG. 133 ). 
     Referring again to  FIGS. 120-122 , filling apparatus  400  can be configured in any one of a number of configurations as desired. As described above, as illustrated in  FIG. 120 , assay input ports  402  can be positioned at end  420  of output layer  408 . When this configuration is used with a microplate comprising 6,144 wells, filling apparatus  400  can comprise, for example, eight assay input ports  402  that can each be in fluid communication with eight respective microfluidic channels  406 . Each of the eight microfluidic channels  406  can be in fluid communication with ninety-six respective staging capillaries  410 . In some embodiments, as illustrated in  FIG. 121 , assay input ports  402  can be positioned at side  422  of output layer  408 . When this configuration is used with a microplate comprising 6,144 wells, filling apparatus  400  can comprise, for example, eight assay input ports  402  that can each be in fluid communication with twelve respective microfluidic channels  406 . Each of the twelve microfluidic channels  406  can be in fluid communication with sixty-four respective staging capillaries  410 . This configuration can provide shorter channel lengths, which, in some circumstances, can have more rapid capillary filling times relative to the configuration of  FIG. 120 . 
     In some embodiments, as illustrated in  FIG. 122 , assay input ports  402  can be positioned at opposing ends  420  or opposing sides  422  (configuration not illustrated) of output layer  408 . When the configuration illustrated in  FIG. 122  is used with a microplate comprising 6,144 wells, filling apparatus  400  can comprise, for example, sixteen assay input ports  402  that can each be in fluid communication with twelve respective microfluidic channels  406 . Each of the twelve microfluidic channels  406  can be in fluid communication with thirty-two respective staging capillaries  410 . Likewise, when sixteen assay input ports  402  are positioned along opposing sides  422 , sixteen assay input ports  402  can each be in fluid communication with eight respective microfluidic channels  406 . Each of the eight microfluidic channels  406  can be in fluid communication with forty-eight respective staging capillaries  410 . These configurations can provide shorter channel lengths, which, in some circumstances, can have more rapid capillary filling times relative to the configurations of  FIGS. 120 and 121 . 
     In some embodiments, the plurality of microfluidic channels  406  can be oriented such that, during centrifugation, they are perpendicular to an axis of revolution of the centrifuge. In some embodiments, this orientation can limit the flow of assay  1000  along the plurality of microfluidic channels  406  during centrifugation. 
     Overfill Solutions 
     In some embodiments, metering a predetermined amount of assay  1000  into each of the plurality of staging capillaries  410  and finally into each of the plurality of wells  26  can be achieved using a plurality of overfill reservoirs disposed in output layer  408 . Referring to  FIGS. 134-139 , in some embodiments, filling apparatus  400  comprises fluid well  424  in fluid communication with one or more corresponding microfluidic channels  406  in fluid communication with the plurality of staging capillaries  410 . In some embodiments, at least one microfluidic channel  406  comprises one or more fluid overfill reservoir  442  in fluid communication therewith. In some embodiments, the one or more fluid overfill reservoir  442  can be a bore opened at one end (e.g., a bore extending into output layer  408  from a surface thereof; with the bore having an open upper-end and a closed bottom end.) 
     As illustrated in  FIGS. 134-139 , to perform a filling operation, each assay input port  402  can be at least partially filled with assay  1000  or other desired fluid ( FIG. 134 ). At least in part through hydraulic pressure and/or capillary force, assay  1000  can flow from fluid well  424  of each assay input port  402  into the one or more microfluidic channels  406  ( FIG. 134 ). As assay flows across an upper-end opening or mouth  432  of each of the plurality of staging capillaries  410 , capillary action, at least in part, draws a metered amount of assay  1000  therein ( FIG. 135 ). Assay  1000  can continue to flow down the one or more microfluidic channels  406  until each of the plurality of staging capillaries  410  can be at least partially filled with assay  1000  ( FIG. 136 ). In some embodiments, fluid overfill reservoir  442  can generally inhibit assay  1000  from flowing into fluid overfill reservoir  442 , at least in part because of the single opening therein generally preventing air within fluid overfill reservoir  442  from exiting. In some embodiments, fluid overfill reservoir can have a diameter equal to that of staging capillaries  410  and a depth of about 0.05 inch, or less. 
     In some embodiments, assay  1000  in each of the plurality of staging capillaries  410  can be held therein by capillary or surface tension forces to aid in the equal metering of assay  1000  to be loaded in each of the plurality of wells  26 . In some embodiments, a lower-end opening or open-air outlet  434  of each of the plurality of staging capillaries  410  permit venting of air within each of the plurality of staging capillaries  410  during filling. 
     As illustrated in  FIGS. 137 and 138  and described above, in some embodiments, filling apparatus  400  can be stake cut, generally indicated at  435 , via device  436  along a portion of one or more microfluidic channels  406 . It should be appreciated that stake-cutting or staking can be carried out, as previously described. 
     In some embodiments, once at least some of the plurality of staging capillaries  410  are filled, at least output layer  408  and microplate  20  can be placed into a swing-arm centrifuge. In some embodiments, the centripetal force of the centrifuge can be sufficient to overcome the capillary force and/or surface tension of assay  1000  in each the plurality of staging capillaries  410 , thereby forcing a metered volume of assay  1000  into each of the plurality of wells  26  of microplate  20  ( FIG. 139 ). In some embodiments, the centripetal force of the centrifuge can be sufficient to force overfill fluid (e.g. assay  1000  still remaining in microfluidic channels  406 ) into overfill reservoir  442 , thereby displacing the air within overfill reservoir  442 , rather than into the plurality of staging capillaries  410 . In some embodiments, this air can serve to isolate one staging capillary  410  from an adjacent staging capillary  410 . In some embodiments, overfill reservoir  442  can act as a reservoir for excess assay  1000 . As illustrated in  FIG. 140 , in some embodiments, overfill reservoir  442  can be disposed within output layer  408  and generally aligned with and positioned below at least one assay input port  402  in output layer  408 . 
     Microfluidic Channel Shapes 
     As illustrated in  FIGS. 141(   a )-( g ) and  142 ( a )-( g ), in some embodiments, microfluidic channels  406  can have any one or a combination of various configurations. In some embodiments, as illustrated in  FIG. 141(   a ), each microfluidic channel  406  can be in fluid communication with a pair of rows of the plurality of staging capillaries  410  via feeder channels  444 . In some embodiments, as illustrated in  FIGS. 141(   b ),  142 ( a ), and  142 ( c ), microfluidic channel  406  can be in fluid communication with a row of staging capillaries  410  that can be offset to one side of microfluidic channel  406 . In some embodiments, as illustrated in  FIGS. 141(   c )-( e ) and  142 ( d )-( f ), a cross dimension, e.g., width, of microfluidic channel  406  can vary relative to a diameter of each of the plurality of staging capillaries  410  ranging from larger than the diameter of each staging capillaries  410  to about equal to the diameter of each staging capillaries  410  to less than the diameter of each staging capillary ( FIGS. 25(   e )-( f )). In some embodiments, as illustrated in  FIGS. 141(   f ),  141 ( g ),  142 ( a ), and  142 ( b ), microfluidic channel  406  can have a generally triangular cross-section that can be either aligned with or offset from staging capillaries  410 . In some embodiments, as illustrated in  FIG. 142(   g ), microfluidic channel  406  can have a single channel portion  446  fluidly coupled to two or more rows of staging capillaries  410 . In some embodiments, single channel portion  446  comprises a centrally disposed feature  448  to, in part, aid in fluid splitting between adjacent rows of staging capillaries  410 . 
     In some embodiments, capillary or surface tension forces encourage flow of assay  1000  through microfluidic channels  406 . In this regard, microfluidic channels  406  can be of capillary size, for example, microfluidic channels  406  can be formed with a width of less than about 500 micron, and in some embodiments less than about 125 microns, less than about 100 microns, or less than about 50 microns. In some embodiments, microfluidic channels  406  can be formed, for example, with a depth of less than about 500 micron, and in some embodiments less than about 125 microns, less than about 100 microns, or less than about 20 microns. To further encourage the desired capillary action in microfluidic channels  406 , microfluidic channels  406  can be provided with an interior surface that is hydrophilic, i.e., wettable. For example, the interior surface of microfluidic channels  406  can be formed of a hydrophilic material and/or treated to exhibit hydrophilic characteristics. In some embodiments, the interior surface comprises native, bound, or covalently attached charged groups. For example, one suitable surface, according to some embodiments, is a glass surface having an absorbed layer of a polycationic polymer, such as poly-l-lysine. 
     Floating Inserts 
     In some embodiments, as illustrated in  FIGS. 143-157 , filling apparatus  400  comprises output layer  408 , a floating insert  460 , a cover  464 , port member  467 , or any combination thereof for loading assay  1000  into at least some of the plurality of wells  26  in microplate  20 . 
     In some embodiments, output layer  408  comprises one or more recessed regions or depressions  454  formed in an upper surface  456  of output layer  408 . Each depression  454  can be, in some embodiments, sized and/or shaped to receive floating insert  460  therein. In some embodiments comprising two or more depressions  454 , at least one wall  458  can be used to separate each depression  454  to define grouping  407  of staging capillaries  410  of any desired quantity and orientation. 
     In some embodiments, as illustrated in  FIG. 144 , floating insert  460  and depression  454  can together define a capillary gap  468  between a bottom surface  470  of floating insert  460  and a top surface  472  of depression  454 . In some embodiments, capillary gap  468  can result from surface variations in bottom surface  470  of floating insert  460  and/or top surface  472  of depression  454  and/or spacing gaps formed therebetween. It should be appreciated that capillary gap  468  can be quite small; therefore, the drawings of the present application may exaggerate this feature for ease of printing and understanding. In some embodiments, capillary gap  468  exhibits a capillary force sufficient to draw assay  1000  there along and to mouth  432  of each staging capillary  410 . In some embodiments, bottom surface  470  of floating insert  460  and/or top surface  472  of depression  454  can be treated and/or coated to enhance the hydrophilic properties of capillary gap  468 . In some embodiments, capillary gap  468  can be in fluid communication with an aperture  462  extend through floating insert  460 . Aperture  462  can be centrally located relative to floating insert  460  or can be located to one side and/or corner thereof. In some embodiments, aperture  462  comprises an assay receiving well  463  ( FIG. 145-157 ). In such embodiments, port member  467  is optional. 
     As illustrated in  FIG. 144 , in some embodiments, to reduce capillary force between a sidewall  474  of floating insert  460  and wall  458  of depression  454 , the thickness of floating insert  460  and the depth of depression  454  can be minimized to shorten the length of any resulting capillary channel and, thus, reduce the overall capillary force in this region. In some embodiments, as illustrated in  FIGS. 145-157 , floating insert  460  comprises a flanged base portion  490  to reduce the potential capillary surface between sidewall  474  of floating insert  460  and wall  458  of depression  454 . In some embodiments, a hydrophic surface can be employed between floating insert  460  and wall  458  of depression  454  to reduce capillary force therebetween. In some embodiments, this hydrophic surface can result from native material characteristics, treatments, coatings, and the like. 
     In some embodiments, as illustrated in  FIGS. 147-152 , floating insert  460  can be shaped to, at least in part, achieve any particular capillary and/or flow characteristics. In some embodiments, as illustrated in  FIGS. 147-149 , floating insert  460  can comprise a plurality of flow features  478  to, at least in part, extend the capillary surface to facilitate capillary flow. In some embodiments, for example, each of the plurality of flow features  478  comprises a post member  480  ( FIG. 147 ) extending orthogonally from bottom surface  470  of floating insert  460 . In some embodiments, post member  480  comprises a radiused root portion  482  to facilitate capillary flow, if desired. In some embodiments, post member  480  can be offset within the corresponding staging capillary  410  and can, if desired, contact a sidewall of staging capillary  410 . In some embodiments, each of the plurality of flow features  478  comprises a tapered member  484  ( FIGS. 148-152 ) extending from bottom surface  470  of floating insert  460 . In some embodiments, each of the plurality of staging capillaries  410  comprises a corresponding mating entrance feature  486  ( FIGS. 148 ,  150 , and  151 ) to closely conform to each flow feature  478  to define a transition capillary gap  488 . Tapered member  484  can be conically shaped ( FIGS. 148-149 ) to closely conform to the complementarily-shaped mating entrance feature  486  in staging capillary  410 . It should be appreciated that in some embodiments, the plurality of flow features  478  can further serve to individually plug or seal each corresponding capillary  410  during centrifugation ( FIG. 152 ). 
     In some embodiments, floating insert  460  can comprise any material conducive to encourage capillary action along capillary gap  468 , such as but not limited to plastic, glass, elastomer, and the like. In some embodiments, floating insert  460  can be made of at least two materials, such that an upper portion can be made of a first material and a lower portion can be made of a second material. In some embodiments, the second material can provide a desired compliancy, hydrophilicity, or any other desire property for improved fluid flow and/or sealing of staging capillaries  410 . In some embodiments, the tapered members can include a seal-facilitating film, coating, or gasket thereon. 
     In some embodiments, as seen in  FIG. 144 , cover  464  can be used, at least in part, to retain floating insert  460  within each depression  454 , if desired. In some embodiments, cover  464  comprises an aperture  466  generally aligned with an aperture  462  of floating insert  460 . In some embodiments, cover  464  comprises a pressure sensitive adhesive to, at least in part, retain floating insert  460  within depression  454 . 
     As illustrated in  FIGS. 143 and 144 , in some embodiments, port member  467  comprises assay input port  402 . In some embodiments, port member  467  can comprise a material comprising sufficient weight such that during centrifugation, the centripetal force of port member  467  exerted upon floating insert  460  and output layer  408  can aid in closing off cross-communication of fluid between adjacent staging capillaries  410 , as the upper-end openings of staging capillaries  410  can be covered and sealed by the lower surface of floating insert  460 . In some embodiments, port member  467  can be sized such that its footprint (e.g. the surface area of a bottom surface  476  of port member  467 ) can be smaller than the opening of depression  454  to aid in the exertion of centripetal force on floating insert  460  during centrifuge. 
     In some embodiments, as illustrated in  FIG. 153-155 , to load each of the plurality of staging capillaries  410 , a predetermined amount of assay  1000  can be placed at each assay input port  402  when used with port member  467  or receiving well  463 . Capillary gap  468  can be sized to provide sufficient capillary force to draw at least a portion of assay  1000  from assay input port  402  or receiving well  463  into capillary gap  468 . The capillary force of capillary gap  468  can be, at least in part, due to the non-rigid connection between floating insert  460  and output layer  408 . As illustrated in  FIG. 154 , as assay  1000  is drawn into and spreads about capillary gap  468 , each of the plurality of staging capillaries  410  in fluid communication with capillary gap  468  can begin to fill, at least in part, by capillary force as described herein. 
     In some embodiments, once at least some of the plurality of staging capillaries  410  are filled, at least output layer  408  and microplate  20  can be placed into a centrifuge. For example, the pieces can be clamped or otherwise held together, and then placed in a bucket centrifuge as a unit. In some embodiments, the centripetal force of the centrifuge can be sufficient to overcome the capillary force and/or surface tension of assay  1000  in each the plurality of staging capillaries  410 , thereby forcing a metered volume of assay  1000  into each of the plurality of wells  26  of microplate  20 . In some embodiments, the centripetal force of the centrifuge can also cause floating insert  460  to be forced and, thus, pressed against top surface  472  of depression  454 . In some embodiments, where port member  467  is installed ( FIGS. 143 and 144 ) or any additional weight member  492  ( FIGS. 156 and 157 ), this additional weight can further apply a force upon floating insert  460  to force floating insert  460  against top surface  472  of depression  454 . This force on floating insert  460  against top surface  472  of depression  454  can help to fluidly isolate each staging capillaries  410  from adjacent staging capillaries  410  for improved metering. 
     It should be appreciated that any component of filling apparatus  400 , such as input layer  404 , output layer  408 , floating insert  460 , cover  464 , port member  467 , intermediate layer  494 , vent layer  523 , etc., can comprise a plate, tile, disk, chip, block, wafer, laminate, and any combinations thereof, and the like. 
     Surface Wipe 
     As illustrated, for example, in  FIGS. 158-166 , in some embodiments, filling apparatus  400  does include the plurality of microfluidic channels  406 . In some embodiments, for example, filling apparatus  400  comprises output layer  408  and a surface wipe assembly  1800  for loading assay  1000  into at least some of the plurality of wells  26  in microplate  20 . In some embodiments, surface wipe assembly  1800  comprises one or more of a base support  1810 , a drive assembly  1812 , a funnel assembly  1814 , or any combination thereof. 
     In some embodiments, such as illustrated in  FIG. 158 , base support  1810  can be a generally planar support member operable to support microplate  20  and output layer  408  thereon. In some embodiments, base support  1810  comprises an alignment feature  1818  that can engage corresponding alignment feature  58  (refer to previous figures) of microplate  20  and/or alignment feature  519  of output layer  408  to maintain microplate  20  and output layer  408  in a predetermined alignment relative to each other and/or funnel assembly  1814 . 
     In some embodiments, drive assembly  1812  comprises a drive motor  1816 ; a guide member  1820 , coupled to or formed in base support  1810 ; a tracking member  1822 , coupled to or formed in funnel assembly  1814 ; and control system  1010 . In some embodiments, guide member  1820  and tracking member  1822  are sized and/or shaped to slidingly engage with each other to provide guiding support for funnel assembly  1814  as it moves relative to base support  1810 . In some embodiments, drive motor  1816  can be operably coupled to tracking member  1822  or base support  1810  to move tracking member  1822  relative to guide member  1820  via known drive transmission interfaces, such as mechanical drives, pneumatic drives, hydraulic drives, electromechanical drives, and the like. In some embodiments, drive motor  1816  can be controlled in response to control signals from control system  1010  or a separate control system. In some embodiments, drive motor  1816  can be operably controlled in response to a switch device controlled by a user. 
     In some embodiments, funnel assembly  1814  comprises a spanning portion  1824  generally extending above output layer  408 . In some embodiments, spanning portion  1824  can be supported on opposing ends by tracking member  1822  of drive assembly  1812  and a foot member  1826 . Tracking member  1822  and foot member  1826  can each be coupled to spanning portion  1824  via conventional fasteners in some embodiments. Foot member  1826  can be generally arcuately shaped so as to reduce the contact area between foot member  1826  and base support  1810 . In some embodiments, foot member  1826  can be made of a reduced friction material, such as Delrin®. 
     In some embodiments, spanning portion  1824  of funnel assembly  1814  comprises a slot  1828  formed vertically therethrough that can be sized and/or shaped to receive a funnel member  1830  therein. As illustrated in  FIGS. 158-166 , funnel member  1830  can comprise one or more assay chambers  1832  for receiving one or more different assays therein. It should be appreciated that drive assembly  1812  and funnel assembly  1814  can be configured to track in a direction perpendicular to that illustrated in the accompanying figures to provide an increased number of assay chambers  1832  and reduced track distances. In some embodiments, such as illustrated in  FIG. 159 , funnel member  1830  can comprise a flange portion  1834  extending about a top portion thereof. Flange portion  1834  of funnel member  1830  can be sized and/or shaped to rest upon a corresponding flange portion  1836  of slot  1828  of spanning portion  1824  to support funnel member  1830 . However, it should be appreciated that funnel member  1830  can comprise any outer profile complementary to slot  1828 . 
     Assay chambers  1832 , in some embodiments, can be shaped to provide a predetermined assay capacity for filling all of a predetermined number and/or grouping of the plurality of staging capillaries  410  in output layer  408 . In some embodiments, assay chamber  1832  comprises converging sidewalls  1838  that terminate at a tip portion  1840 . 
     In some embodiments, such as illustrated in  FIG. 160-162 , to load each of the plurality of staging capillaries  410 , a predetermined amount of assay  1000  can be placed in each assay chamber  1832 . In some embodiments, each assay chamber  1832  comprises a different assay. Assay  1000  is drawn down along sidewalls  1838  to tip portion  1840  to form a fluid bead  1842  extending from tip portion  1840  that can be in contact with upper surface  456  of output layer  408 . In some embodiments, fluid bead  1842  can be bound by a lip or wiper member  1844  extending downwardly from tip portion  1840  of funnel member  1830 . In some embodiments, wiper member  1844  can, at least in part, wipe and/or remove excess assay  1000  on upper surface  456  of output layer  408  as funnel member  1830  moves thereabout. In some embodiments, drive assembly  1812  can be actuated to advance funnel assembly  1814  across output layer  408  at a predetermined rate, as illustrated in  FIG. 161 . However, it should be appreciated that funnel assembly  1814  can be advanced manually across output layer  408 . As funnel assembly  1814  is advanced across output layer  408 , in some embodiments, fluid bead  1842  can contact the upper-end opening or entrance of each of the plurality of staging capillaries  410  and begin to fill, at least in part, by capillary force as described herein. 
     In some embodiments, such as illustrated in  FIGS. 158 and 162 , as funnel assembly  1814  continues past the last of the plurality of staging capillaries  410 , some assay  1000  can be forced off upper surface  456  of output layer  408  at an edge  1846  into at least one overflow channel  1848 . In some embodiments, once at least some of the plurality of staging capillaries  410  are filled, at least output layer  408  and microplate  20  can be placed into a centrifuge. In some embodiments, the centripetal force of the centrifuge can be sufficient to overcome the capillary force and/or surface tension of assay  1000  in each the plurality of staging capillaries  410 , thereby forcing a metered volume of assay  1000  into each of the plurality of wells  26  of microplate  20 . 
     In some embodiments, such as illustrated in  FIG. 158 , the excess assay  1000  in overflow channel  1848  can be contained using one or more reservoir pockets  1850 . In some embodiments, reservoir pocket  1850  can be in fluid communication with at least one overflow channel  1848 . In some embodiments, reservoir pocket  1850  can be deeper than overflow channel  1848  to encourage flow of assay  1000  to reservoir pocket  1850 . During centrifugation, centripetal force can further encourage assay  1000  to flow to reservoir pocket  1850 , thereby reducing the likelihood of any contamination or cross-feed between adjacent staging capillaries  410 . In some embodiments, an extended wall member  1852  can be positioned about reservoir pocket  1850  to further contain assay  1000 . 
     In some embodiments, such as illustrated in  FIGS. 163 and 164 , the excess assay  1000  in overflow channel  1848  can be contained using a reservoir trough  1854 . In some embodiments, an absorbent member  1856  can be disposed in reservoir trough  1854  to absorb excess assay  1000  therein. In some embodiments, absorbent member  1856  can be a hydrophilic fiber membrane. As illustrated in  FIG. 164 , reservoir trough  1854  can be sloped toward absorbent member  1856  to facilitate absorption of excess assay  1000 . In some embodiments, absorbent member  1856  can be removable to permit removal and relocating of the excess assay  1000  prior to centrifugation. 
     In some embodiments, such as illustrated in  FIGS. 165 and 166 , funnel member  1830  can comprise two or more discrete assay chambers  1832  for delivering one or more different assays. In such embodiments, for example, output layer  408  can comprise one or more central overflow channels  1858  extending along upper surface  456  of output layer  408  to receive at least some overflow assay  1000 . In some embodiments, central overflow channels  1858  are each disposed between each separate grouping of staging capillaries  410  served by each discrete assay chamber  1832 . In some embodiments, as illustrated in  FIG. 166 , central overflow channel  1858  can be sloped down to at least one of overflow channel  1848  ( FIG. 158 ), reservoir pocket  1850  ( FIG. 158 ), reservoir trough  1854  ( FIG. 163 ), or absorbent member  1856  ( FIG. 166 ). As illustrated in  FIG. 165 , in some embodiments, absorbent member  1856  can be sized and/or shaped to fit with an enlarged reservoir pocket  1850 . 
     Funnel Member 
     As illustrated in  FIGS. 167-180 , in some embodiments, funnel member  1830  of funnel assembly  1814  can be any one of a number of configurations sufficient to maintain fluid bead  1842  in contact with upper surface  456  of output layer  408 . In some embodiments, a predetermined shape of fluid bead  1842  and/or a predetermined flowrate of assay  1000  through tip portion  1840  can be achieved through the particular configuration of funnel member  1830 . 
     As illustrated in  FIG. 167-169 , in some embodiments, funnel member  1830  comprises one or more assay chambers  1832  in fluid communication with tip portion  1840 . As described above, in embodiments comprising two or more assay chambers  1832  ( FIG. 168 ), multiple assays can be used such that a different assay can be disposed in each assay chamber  1832 . It should be understood that any number of assay chambers  1832  can be used (e.g., 2, 4, 6, 8, 10, 12, 16, 20, 32, 64, or more). 
     In some embodiments, tip portion  1840  can be configured to define a capillary force and/or surface tension sufficient to prevent assay  1000  from exiting assay chamber  1832  prior to fluid bead  1842  engaging upper surface  456  and to permit assay  1000  to be pulled into each of the plurality of staging capillaries  410  during filling of the staging capillaries. As illustrated in  FIG. 170 , tip portion  1840  comprises a restricted orifice  1860  that is sized to increase surface tension to retain assay  1000  with assay chamber  1832 . In some embodiments, tip portion  1840  can be spaced apart from an underside surface  1862  to, at least in part, inhibit assay  1000  from collecting between funnel member  1830  and output layer  408 . In some embodiments, as illustrated in  FIG. 171 , restricted orifice  1860  can be used with wiper member  1844  to increase surface tension to retain assay  1000  and to wipe and/or remove excess assay  1000  on upper surface  456  of output layer  408 . In some embodiments, such as illustrated in  FIG. 172 , tip portion  1840  can comprise a planar cavity  1864  disposed in fluid communication with restricted orifice  1860 . In some embodiments, planar cavity  1864  can encourage the formation of wider and/or shallower fluid bead  1842  relative to similar configurations not employing planar cavity  1864 . In some configurations, the wider and/or shallower fluid bead  1842  can, at least in part, prolong the time fluid bead  1842  is in contact with each of the plurality of staging capillaries  410 . 
     As illustrated in  FIG. 173 , in some embodiments, funnel member  1830  can comprise wiper  1844  spaced apart from tip portion  1840  to wipe and/or remove excess assay  1000  on upper surface  456  of output layer  408 . In some embodiments, wiper  1844  can extend a distance from underside surface  1862  of funnel member  1830  equal to about a distance from underside surface  1862  to a distal end of tip portion  1840 . As illustrated in  FIGS. 174-176 , each tip portion  1840  associated with each assay chamber  1832  can be offset relative to adjacent tip portions  1840 . In some embodiments, this offset relationship between adjacent tip portions  1840  can permit the plurality of staging capillaries  410  to be closely spaced with reduced likelihood for crosstalk between adjacent fluid beads  1842 . 
     Still referring to  FIGS. 174-176 , in some embodiments, restricted orifice  1860  comprises an elongated slot  1866  ( FIG. 174 ) generally extending from one edge of tip portion  1840  to the opposing edge to define an elongated fluid bead  1842 . However, in some embodiments, restricted orifice  1860  comprises one or more apertures  1868 . In some embodiments, the reduced cross-sectional area of apertures  1868  relative to that of elongated slot  1866  can serve to withstand a fluid head pressure exerted by assay  1000  in assay chamber  1832  that would otherwise overcome the surface tension of fluid bead  1842  exiting elongated slot  1866  and possibly lead to premature discharge of assay  1000 . In some embodiments, the restricted orifice  1860  can be collinear as well as offset as illustrated in ( FIG. 174 ). 
     In some embodiments, such as illustrated in  FIGS. 177-179 , funnel member  1830  can comprise an internal siphon passage  1870  to, at least in part, control the flowrate of assay  1000  from restricted orifice  1860 . In some embodiments, funnel member  1830  comprises a main chamber  1872  fluidly coupled to a delivery chamber  1874  via siphon passage  1870 . In some embodiments, siphon passage  1870  can be positioned along a bottom of main chamber  1872 . Siphon passage  1870  can comprise an upturned section  1876  that can require assay  1000  in main chamber  1872  to flow, at least in part, against the force of gravity. In some embodiments, main chamber  1872  and delivery chamber  1874  can be fluidly coupled at the top thereof by a top chamber  1878 . When main chamber  1872  is filled at least partially above top chamber  1878 , the excess assay  1000  can flow across top chamber  1878  into delivery chamber  1874 . During filling, as the level of assay  1000  drops below the bottom surface of top chamber  1878  and assay  1000  flows from restricted orifice  1860 , assay  1000  within delivery chamber  1874  can be replaced through the siphoning action of siphon passage  1870  at the bottom of main chamber  1872 . This arrangement can reduce the fluid head pressure exerted at restricted orifice  1860 . Accordingly, the fluid head pressure exerted at restricted orifice  1860  can be generally to about the fluid head pressure of assay  1000  contained in delivery chamber  1874 . 
     In some embodiments, as illustrated in  FIGS. 179 and 180 , funnel member  1830  can be formed with a two- or more-piece construction. As illustrated in  FIG. 179 , funnel member  1830  can comprise a first section  1880  and a second section  1882 . First section  1880  can comprise one or more desired features. For example, as illustrated in  FIG. 179 , upturned section  1876  of  FIG. 178  can be formed in first section  1880 . First section  1880  and second section  1882  can then be joined or otherwise mated along a generally vertical joining line  1884  ( FIG. 178 ) to form funnel member  1830 . In some embodiments, first section  1880  and second section  1882  can be joined or otherwise mated along a generally horizontal joining line  1886  ( FIG. 180 ). In some embodiments, first section  1880  and second section  1882  can be made from different materials to achieve a predetermined performance. In some embodiments, second section  1882  can be made of an elastomer to provide enhance flexibility to accommodate for variations in output layer  408  and enhanced wiping performance of wiper member  1844 . 
     Surface Treatment 
     In some embodiments, portions of filling apparatus  400  that are intended to contact assay  1000 , such as assay input ports  402 , microfluidic channels  406 , the plurality of staging capillaries  410 , and the like, can be hydrophilic. Likewise, in some embodiments, surfaces not intended to contact assay  1000  can be hydrophobic. 
     In some embodiments, filling apparatus  400  comprises a treatment to increase surface energy thereof to improve flow and/or capillary action of any surface of filling apparatus  400  exposed to assay  1000 , such as assay input ports  402 , microfluidic channels  406 , staging capillaries  410 , microfluidic channels  406 , depression  454 , upper surface  456 , etc. In some embodiments, surface energy can be improved, for example, when using a polymer material in the manufacture of filling apparatus  400 , through surface modification of the polymer material via Michael addition of acrylamide or PEO-acrylate onto laminated surface; surface grafting of acrylamide or PEO-acrylate via atom transfer radical polymerization (ARTP); surface grafting of acrylamide via Ce(IV) mediated free radical polymerization; surface initiated living radical polymerization on chloromethylated surface; coating of negatively charged polyelectrolytes; plasma CVD of acrylic acid, acrylamide, and other hydrophilic monomers; or surface adsorption of an ionic or non-ionic surfactant. In some embodiments, surfactants, such as those set forth in Tables 2 and 3, can be used. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Surfactants for Coating 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Hydrophile- 
               
               
                   
                   
                   
                   
                 Lipophile 
               
               
                   
                   
                   
                   
                 Balance 
               
               
                   
                 No. 
                 Name 
                 MW 
                 (HLB) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 Tetronic 901 
                 4700 
                 3 
               
               
                   
                 2 
                 Tetronic 1107 
                 1500 
                 24 
               
               
                   
                 3 
                 Tetronic 1301 
                 6800 
                 2 
               
               
                   
                 4 
                 Poly(styrene-b-ethylene oxide) 
                 Mn: 3600-67000 
               
               
                   
                 5 
                 Poly(stryrene-b-sodium acrylate) 
                 Mn: 1800-42500 
               
               
                   
                 6 
                 Triton X-100 
                   
                 13.5 
               
               
                   
                 7 
                 Triton X-100 reduced 
               
               
                   
                 8 
                 Tween 20 
                 1228 
                 16.7 
               
               
                   
                 9 
                 Tween 85 
                 1839 
                 11 
               
               
                   
                 10 
                 Span 83 
                 1109.56 
                 3.7 
               
               
                   
                 11 
                 Span 80 
                 428.62 
                 4.3 
               
               
                   
                 12 
                 Span 40 
                 402.58 
                 6.7 
               
               
                   
                   
               
            
           
           
               
            
               
                 Tetronic: 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 Triton X-100: 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 Triton X-100 reduced: 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 Tween: 
               
               
                 Poly(oxyethylene) sorbitan monolauate 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Surfactants for Wetting Polypropylene 
               
               
                   
               
             
            
               
                 Acids: 
               
            
           
           
               
               
            
               
                 Dodecyl sulfate, Na salt 
                 CH 2 (CH 2 ) 11 OSO 3   − Na +   
               
               
                 Octadecyl sulfate, Na salt 
                 CH 3 (CH 2 ) 17 OSO 3   − Na +   
               
            
           
           
               
            
               
                 Quaternary ammonium compounds: 
               
            
           
           
               
               
            
               
                 Cetyltrimethylammonium bromide 
                 CH 3 (CH 2 ) 15 N + (CH 3 ) 3  Br −   
               
               
                 Octadecyltrimethylammonium bromide 
                 CH 3 (CH 2 ) 17 N + (CH 3 ) 3 Br −   
               
            
           
           
               
            
               
                 Ethers: 
               
            
           
           
               
               
            
               
                 Brij-52 
                 CH 3 (CH 2 ) 15 (OCH 2 CH 2 ) 2 OH 
               
               
                 Brij 56 
                 CH 3 (CH 2 ) 15 (OCH 2 CH 2 ) 10 OH 
               
               
                 Brij 58 
                 CH 3 (CH 2 ) 15 (OCH 2 CH 2 ) 20 OH 
               
               
                 Brij 72 
                 CH 3 (CH 2 ) 17 (OCH 2 CH 2 ) 2 OH 
               
               
                 Brij 76 
                 CH 3 (CH 2 ) 17 (OCH 2 CH 2 ) 10 OH 
               
               
                 Brij 78 
                 CH 3 (CH 2 ) 17 (OCH 2 CH 2 ) 20 OH 
               
            
           
           
               
            
               
                 Esters: 
               
            
           
           
               
               
            
               
                 Poly(ethylene glycol) monolaurate 
                 CH 3 (CH 2 ) 10 CO(OCH 2 CH 2 ) 4 • 5 OH 
               
               
                 Poly(ethylene glycol) distearate 
                 CH 3 (CH 2 ) 16 —CO—(OCH 2 ) 9 —O—CO—(CH 2 ) 16 CH 3   
               
               
                 Poly(ethylene glycol)dioleate 
                 CH 3 (CH 2 ) 7 CH═CH(CH 2 ) 7 —CO—(OCH 2 ) 9 — 
               
               
                   
                 —O—CO—(CH 2 ) 7 CH═CH(CH 2 ) 7 CH 3   
               
               
                   
               
            
           
         
       
     
     In some embodiments, filling apparatus  400  can comprise polyolefins; poly(cyclic olefins); polyethylene terephthalate; poly(alkyl(meth)acrylates); polystyrene; poly(dimethyl siloxane); polycarbonate; structural polymers, for example, poly(ether sulfone), poly(ether ketone), poly(ether ether ketone), and liquid crystalline polymers; polyacetal; polyamides; polyimides; poly(phenylene sulfide); polysulfones; poly(vinyl chloride); poly(vinyl fluoride); poly(vinylidene fluoride); copolymers thereof; and mixtures thereof. 
     In some embodiments, a co-agent can be employed to enhance the hydrophilicity and/or improve the shelf life of filling apparatus  400 . Co-agents can be, for example, a water-soluble or slightly water-soluble homopolymer or copolymers prepared by monomers comprising, for example, (meth)acrylamide; N-methyl (methyl)acrylamide, N,N-dimethyl (methyl)acrylamide, N-ethyl (meth)acrylamide, N-n-propyl(meth)acrylamide, N-iso-propyl(meth)acrylamide, N-ethyl-N-methyl (meth)acrylamide, N,N-diethyl (meth)acrylamide, N-hydroxymethyl (meth)acrylamide, N-(3-hydroxypropyl) (meth)acrylamide, N-vinylformamide, N-vinylacetamide, N-methyl-N-vinylacetamide, vinyl acetate that can be hydrolyzed to give vinylalcohol after polymerization, 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl(meth)acrylate, N-vinypyrrolidone, poly(ethylene oxide) (meth)acrylate, N-(meth)acryloxysuccinimide, N-(meth)acryloylmorpholine, N-2,2,2-trifluoroethyl (meth)acrylamide, N-acetyl(meth)acrylamide, N-amido(meth)acrylamide, N-acetamido (meth)acrylamide, N-tris(hydroxymethyl)methyl (meth)acrylamide, N-(methyl)acryloyltris(hydroxymethyl)methylamine, (methyl)acryloylurea, vinyloxazolidone, vinylmethyloxazolidone, and combinations thereof. In some embodiments, the co-agent can be poly(acrylic acid-co-N,N-dimethylacrylamide) or poly(N N-dimethyl acrylamide-co-styrene sulfonic acid). 
     Microplate Sealing Cover 
     In some embodiments, such as illustrated in  FIGS. 26 and 27 , sealing cover  80  can be generally disposed across microplate  20  to seal assay  1000  within each of the plurality of wells  26  of microplate  20  along a sealing interface  92  (see  FIGS. 4 ,  5 ,  26 , and  27 ). That is, sealing cover  80  can seal (isoloate) each of the plurality of wells  26  and its contents (i.e. assay  1000 ) from adjacent wells  26 , thus maintaining sample integrity between each of the plurality of wells  26  and reducing the likelihood of cross contamination between wells. In some embodiments, sealing cover  80  can be positioned within an optional depression  94  ( FIG. 30 ) formed in main body  28  of microplate  20  to promote proper positioning of sealing cover  80  relative to the plurality of wells  26 . 
     In some embodiments, sealing cover  80  can be made of any material conducive to the particular processing to be done. In some embodiments, sealing cover  80  can comprise a durable, generally optically transparent material, such as an optically clear film exhibiting abrasion resistance and low fluorescence when exposed to an excitation light. In some embodiments, sealing cover  80  can comprise glass, silicon, quartz, nylon, polystyrene, polyethylene, polycarbonate, copolymer cyclic olefin, polycyclic olefin, cellulose acetate, polypropylene, polytetrafluoroethylene, metal, and combinations thereof. 
     In some embodiments, sealing cover  80  comprises an optical element, such as a lens, lenslet, and/or a holographic feature. In some embodiments, sealing cover  80  comprises features or textures operable to interact with (e.g., by interlocking engagement) circular rim portion  32  or square-shaped rim portion  38  of the plurality of wells  26 . In some embodiments, sealing cover  80  can provide resistance to distortion, cracking, and/or stretching during installation. In some embodiments, sealing cover  80  can comprise water impermeable-moisture vapor transmission values below 0.5 (cc-mm)/(m2-24 hr-atm). In some embodiments, sealing cover  80  can maintain its physical properties in a temperature range of 4° C. to 99° C. and can be generally free of inclusions (e.g. light blocking specks) greater than 50 μm, scratches, and/or striations. In some embodiments, sealing cover  80  can comprise a liquid such as, for example, oil (e.g., mineral oil). 
     In some embodiments, such sealing material can comprise one or more compliant coatings and/or one or more adhesives, such as pressure sensitive adhesive (PSA) or hot melt adhesive. In some embodiments, a pressure sensitive adhesive can be readily applied at low temperatures. In some embodiments, the pressure sensitive adhesive can be softened to facilitate the spreading thereof during installation of sealing cover  80 . In some embodiments, such sealing maintains sample integrity between each of plurality of wells  26  and prevents wells cross-contamination of contents between wells  26 . In some embodiments, adhesive  88  exhibits low fluorescence. 
     In some embodiments, the sealing material can provide sufficient adhesion between sealing cover  80  and microplate  20  to withstand about 2.0 lbf per inch or at least about 0.9 lbf per inch at 95° C. In some embodiments, the sealing material can provide sufficient adhesion at room temperature to contain assay  1000  within each of the plurality of wells  26 . This adhesion can inhibit sample vapor from escaping each of the plurality of wells  26  by either direct evaporation or permeation of water and/or assay  1000  through sealing cover  80 . In some embodiments, the sealing material maintains adhesion between sealing cover  80  and microplate  20  in cold storage at 2° C. to 8° C. range (non-freezing conditions) for 48 hours. 
     In some embodiments, in order to improve sealing of the plurality of wells  26  of microplate  20 , various treatments to microplate  20  can be used to enhance the coupling of sealing cover  80  to microplate  20 . In some embodiments, microplate  20  can be made of a hydrophobic material or can be treated with a hydrophobic coating, such as, but not limited to, a fluorocarbon, PTFE, or the like. The hydrophobic material or coating can reduce the number of water molecules that compete with the sealing material on sealing cover  80 . As discussed above, grooves  52 ,  54  can be used to provide seal adhesion support on the outer edges of sealing cover  80 . In these embodiments, for example, a pressure chamber gasket can be sealed against grooves  52 ,  54  for improved sealing. 
     Turning now to  FIG. 28 , in some embodiments, sealing cover  80  can comprise multiple layers, such as a friction reduction film  82 , a base stock  84 , a compliant layer  86 , a pressure sensitive adhesive  88 , and/or a release liner  90 . In some embodiments, friction reduction film  82  can be Teflon or a similar friction reduction material that can be peeled off and removed after sealing cover  80  is applied to microplate  20  and before microplate  20  is placed in high-density sequence detection system  10 . In some embodiments, base stock  84  can be a scuff resistant and water impermeable layer with low to no fluorescence. While in some embodiments, compliant layer  86  can be a soft silicone elastomer or other material known in the art that is deformable to allow pressure sensitive adhesive  88  to conform to irregular surfaces of microplate  20 , increase bond area, and resist delamination of sealing cover  80 . In some embodiments, pressure sensitive adhesive  88  and compliant layer  86  can be a single layer, if the pressure sensitive adhesive exhibit sufficient compliancy. Release liner  90  is removed prior to coupling pressure sensitive adhesive  88  to microplate  20 . 
     Compatibility of Cover and Assay 
     In some embodiments, adhesive  88  can selected so as to be compatible with assay  1000 . For example, in some embodiments adhesive  88  is free of nucleases, DNA, RNA and other assay components, as discussed below. In some embodiments, sealing cover  80  comprises one or more materials that are selected so as to be compatible with detection probes in assay  1000 . In some embodiments, adhesive layer  88  is selected for compatibility with detection probes. 
     Methods of matching a detection probe with a compatible sealing cover  80  include, in some embodiments, varying compositions of sealing cover  80  by different weight percents of components such as polymers, crosslinkers, adhesives, resins and the like. These sealing covers  80  can then be tested as a function of their corresponding fluorescent intensity level for different dyes. In such embodiments, comparison can be analyzed at room temperature as well as at elevated temperatures typically employed with PCR. Comparisons can be analyzed over a period of time and in some embodiments, the time period can be, for example, up to 24 hours. Data can be collected for each of the varying compositions of sealing cover  80  and plotted such that fluorescence intensity of the dye is on the X-axis and time is on the Y-axis. Some embodiments of the present teachings include a method of testing compatibility of the detection probe comprising an oligonucleotide and a fluorophore to a composition of a sealing cover. In such embodiments, the method includes depositing a quantity of the fluorophore into a plurality of containers, providing a plurality of sealing covers that have different compositions and sealing the containers with the sealing covers. Methods also include exciting the fluorophore in each of the containers and then measuring an emission intensity from the fluorophore in each of the containers. In such embodiments, the method can also include an evaluation of the emission intensity from the fluorophore of each of the containers and then a determination of which sealing cover composition is compatible with the fluorophore. In some embodiments, the method includes holding a temperature of the containers constant. The method can include measuring the emission intensity from the fluorophore in each container over a period of time, for example, as long as about 24 hours. In some embodiments, the method includes heating the containers to a temperature above about 20° C., optionally to a temperature from about 55° C. to about 100° C. In some embodiments, the method includes cycling the temperature of the plurality of containers. The temperature of the containers can be cycled according to a typical PCR temperature profile. Table  4  shows exemplary data that can be generated for such a comparison. In this example, a dye is evaluated by comparing it at non-heated and heated temperatures to a cyclic olefin copolymer (COC) and glue material with varying percentages of a crosslinker. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Percentage of Flourescence Signal Loss 
               
            
           
           
               
               
            
               
                   
                 Percentage of Fluorescence Signal Loss Post 
               
               
                   
                 Incubation with Dye (20 hrs; 59° C.) 
               
            
           
           
               
               
               
            
               
                   
                 Fresh Material 
                 Material Heated 
               
               
                 Sealing Cover Composition 
                 (Room Temperature) 
                 (24 hrs; 70° C.) 
               
               
                   
               
               
                 Control (No COC, glue, 
                  0% Loss 
                  0% Loss 
               
               
                 or crosslinker) 
               
               
                 COC/Glue/0% crosslinker 
                  0% Loss 
                  0% Loss 
               
               
                 COC/Glue/0.5% crosslinker 
                 87% Loss 
                 76% Loss 
               
               
                 COC/Glue/1% crosslinker 
                 86% Loss 
                 12.5% Loss   
               
               
                 COC/Glue/3% crosslinker 
                 55% Loss 
                  0% Loss 
               
               
                 COC/Glue/5% crosslinker 
                 97% Loss 
                 95% Loss 
               
               
                   
               
            
           
         
       
     
     In some embodiments, kits are provided, comprising, for example, a sealing cover  80  and one or more compatible detection probes that are compatible (e.g., emission intensity does not degrade when in contact) with sealing cover  80 . In some embodiments, a kit can comprise one or more detection probes that are compatible (e.g., do not degrade over time when in contact) with adhesive  88  of sealing cover  80 . Kits may comprise a group of detection probes that are compatible with sealing cover  80  comprising adhesive  88  and microplate  20 . In some embodiments, the present teachings include methods for matching a group of detection probes that are compatible with sealing cover  80  and spotting into at least some of plurality of wells  26  of microplate  20 . 
     Microplate Sealing Cover Roll 
     As can be seen in  FIGS. 181 and 182 , in some of the embodiments, sealing cover  80  can be configured as a roll  512 . The use of sealing cover roll  512  can provide, in some embodiments, and circumstances, improved ease in storage and application of sealing cover  80  on microplate  20  when used in conjunction with a manual or automated sealing cover application device, as discussed herein. In some embodiments, sealing cover roll  512  can be manufactured using a laminate comprising a protective liner  514 , a base stock  516 , an adhesive  518 , and/or a carrier liner  520 . During manufacturing, protective liner  514  can be removed and discarded. Base stock  516  and adhesive  518  can then be kiss-cut, such that base stock  516  and adhesive  518  are cut to a desired shape of sealing cover  80 , yet carrier liner  520  is not cut. Excess portions of base stock  516  and adhesive  518  can then be removed and discarded. In some embodiments, base stock  516  can be a scuff resistant and water impermeable layer with low to no fluorescence. 
     In some embodiments, carrier liner  520  can then be punched or otherwise cut to a desired shape and finally the combination of carrier liner  520 , base stock  516 , and adhesive  518  can be rolled about a roll core  522  (see  FIG. 182 ). Roll core  522  can be sized so as not to exceed the elastic limitations of base stock  516 , adhesive  518 , and/or carrier liner  520 . In some embodiments, adhesive  518  is sufficient to retain base stock  516  to carrier liner  520 , yet permit base stock  516  and adhesive  518  to be released from carrier liner  520  when desired. In some embodiments, base stock  516 , adhesive  518 , and carrier liner  520  are rolled upon roll core  522  such that base stock  516  and adhesive  518  face toward roll core  522  to protect base stock  516  and adhesive  518  from contamination and reduce the possibility of premature release. 
     As can be seen in  FIG. 182 , in some embodiments, such a desired shape of carrier liner  520  can comprise a plurality of drive notches  524  formed along and slightly inboard of at least one of the elongated edges  526 . The plurality of drive notches  524  can be shaped, sized, and spaced to permit cooperative engagement with a drive member to positively drive sealing cover roll  512  and aid in the proper positioning of sealing cover  80  relative to microplate  20 . In the some embodiments, the desired shape of carrier liner  520  can further comprise a plurality of staging notches  528  to be used to permit reliable positioning of sealing cover  80 . In some embodiments, the plurality of staging notches  528  can be formed along at least one elongated edge  526 . In some embodiments, the plurality of staging notches  528  can be shaped and sized to permit detection by a detector, such as an optical detector, mechanical detector, or the like. An end/start of roll notch or other feature  530  can further be used in some embodiments to provide notification of a first and/or last sealing cover  80  on sealing cover roll  512 . Similar to the plurality of staging notches  528 , end/start of roll notch  530  can be shaped and sized to permit detection by a detector, such as an optical detector, mechanical detector, or the like. It should be appreciated that the foregoing notches and features can have other shapes than those set forth herein or illustrated in the attached figures. It should also be appreciated that other features, such as magnetic markers, non-destructive markers (e.g. optical and/or readable markers), or any other indicia may be used on carrier liner  520 . To facilitate such detection with an optical detector to avoid physical contact, in some embodiments, carrier liner  520  can be opaque. However, in some embodiments, carrier liner  520  can be generally opaque only near elongated edges  526  with generally clear center sections  532  to aid in in-process adhesive inspection. 
     Sealing Cover Applicator 
     In some embodiments, sealing cover  80  can be laminated onto microplate  20  using a hot roller apparatus  540 , as illustrated in  FIG. 29 . In some embodiments, hot roller apparatus  540  comprises a heated top roller  542  heated by a heating element  544  and an unheated bottom roller  546 . A first plate guide  548  can be provided for guiding microplate  20  into hot roller apparatus  540 , while similarly a second plate guide  550  can be provided for guiding microplate  20  out of hot roller apparatus  540 . 
     During sealing, sealing cover  80  can be placed on top of microplate  20  and the combination can be fed into hot roller apparatus  540  such that sealing cover  80  is in contact with first plate guide  548 . As sealing cover  80  and microplate  20  pass and engage heated top roller  542 , heat can be applied to sealing cover  80  to laminate sealing cover  80  to microplate  20 . This laminated combination can then exit hot roller apparatus  540  as it passes second plate guide  550 . In some embodiments, the heat from heated top roller  542  reduces the viscosity of the adhesive of sealing cover  80  to allow the adhesive to better adhere to microplate  20 . 
     In some embodiments, hot roller apparatus  540  can variably control the amount of heat applied to sealing cover  80 . In this regard, sufficient heat can be supplied to provide adhesive flow or softening of the adhesive of sealing cover  80  without damaging assay  1000 . In some embodiments, hot roller apparatus  540  can variably control a drive speed of heated top roller  542  and unheated bottom roller  546 . In some embodiments, hot roller apparatus  540  can variably control a clamping force between heated top roller  542  and unheated bottom roller  546 . By varying these parameters, optimal sealing of sealing cover  80  to microplate  20  can be achieved with minimal negative effects to assay  1000 . 
     Manual Sealing Cover Applicator 
     In some embodiments, sealing cover  80  can be laminated onto microplate  20  using a manual sealing cover applicator  552 , such as illustrated in  FIG. 183 . In some embodiments, manual sealing cover applicator  552  can be used in conjunction with a fixture  554 , such as illustrated in  FIG. 184 . In some embodiments, fixture  554  can comprise a generally planar substrate  556  comprising a recessed portion  558 . Recessed portion  558 , in some embodiments, can be longitudinally aligned with generally planar substrate  556  and sized to receive microplate  20  therein. In some embodiments, fixture  554  can comprise an alignment feature  560  that can be complementary to alignment feature  58  on microplate  20 . In some embodiments, alignment feature  560  can comprise a corner chamfer, a pin, a slot, a cut corner, an indentation, a graphic, a nub, a protrusion, and/or other unique feature that can be capable of interfacing with alignment feature  58  or other feature of microplate  20 . In some embodiments, fixture  554  can comprise one or more recesses  562  formed in generally planar substrate  556  to permit, among other things, improved grasping of microplate  20  for ease of insertion and withdrawal of microplate  20  from fixture  554 . In some embodiments, one or more recesses  562  can be positioned along opposing ends of microplate  20 . 
     Referring now to FIGS.  183  and  185 - 187 , in some embodiments, manual sealing cover applicator  552  comprises a hinged housing  564  sized to receive sealing cover roll  512  therein. In some embodiments, hinged housing  564  comprises a base section  566  and at least one cover section  568 . In some embodiments, at least one cover section  568  can be pivotally coupled to base section  566  about axis  570 . In some embodiments, at least one cover section  568  comprises a pair of apertures  572  (only one illustrated) formed in sidewalls  574  that can each be sized to receive a pin  576  extending from an applicator roller  578  to permit pivotal movement of at least one cover section  568  relative to base section  566 . In some embodiments, a latch member  580  can be used to releasably couple base section  566  to at least one cover section  568 . Latch member  580  can be pivotally coupled to one of base section  566  and at least one cover section  568  and positionable in a locked position ( FIG. 186 ), coupling base section  566  and at least one cover section  568 , and an unlocked position ( FIG. 187 ), permitting relative pivotal movement of base section  566  and at least one cover section  568 . 
     As illustrated in  FIGS. 185-187 , in some embodiments, base section  566  comprises at least one of applicator roller  578 , a support structure  582 , a roll hub  584 , a stretcher  586 , a plane assembly  588 , an intermediate roller  590 , a drive roller assembly  592 , a pressure roller  594 , and a waste gate  596 . In some embodiments, applicator roller  578  can comprise a generally cylindrical member comprising the pair of pins  576  disposed on opposing ends thereof along axis  570 . In some embodiments, the pair of pins  576  can engage support structure  582  to permit rotating movement of applicator roller  578  relative thereto. In some embodiments, applicator roller  578  can be made of, at least in part, a compliant material to permit applicator roller  578  to accommodate variations in fixture  554  and/or microplate  20 . 
     In some embodiments, roll hub  584  can be fixedly coupled to support structure  582  to support sealing cover roll  512  thereon and permit relative rotation therebetween. In some embodiments, roll hub  584  comprises a pair of friction legs  598  extending outwardly from tangential sections  600  of a central portion  602 . In some embodiments, the pair of friction legs  598  can each extend along only a portion of roll hub  584 . The pair of friction legs  598  can be sized to frictionally engage an inner surface of roll core  522  of sealing cover roll  512  to provide drag and/or positively retain sealing cover roll  512  on roll hub  584 . 
     In some embodiments, stretcher  586  comprises a bracket portion  604  and an engaging portion  606 . In some embodiments, bracket portion  604  can be fixedly coupled to support structure  582  to provide a generally rigid support. In some embodiments, engaging portion  606  comprises a mounting section  608  and one or more finger members  610  extending from mounting section  608 . The one or more finger members  610  can comprise an upturned end  612  to form an engaging corner  614  to contact sealing cover roll  512  as it passes thereby. In some embodiments, mounting section  608  can be fixedly coupled to bracket portion  604  via conventional fasteners and/or a tab member interface  616  ( FIG. 185 ). 
     Still referring to  FIGS. 185-187 , in some embodiments, plane assembly  588  comprises a plate member  618  and a plane roller  620  rotatably coupled to plate member  618  along axis  622 . In some embodiments, plane roller  620  can be a generally cylindrical member comprising a pair of pins  624  disposed on opposing ends thereof along axis  622 . In some embodiments, the pair of pins  624  can engage apertures formed in plate member  618  to permit rotating movement of plane roller  620  relative thereto. In some embodiments, plane roller  620  can be made of, at least in part, a compliant material to permit plane roller  620  accommodate variations in fixture  554  and/or microplate  20 . In some embodiments, plane roller  620  can carry carrier liner  520  of sealing cover roll  512 . In some embodiments, plane roller  620  can be sized to apply a force on a backside of carrier liner  520  and, consequently, on sealing cover  80  to adhere sealing cover  80  to microplate  20  during application. In some embodiments, carrier liner  520  can then travel along plate member  618  to intermediate roller  590 . It should be appreciated that plane roller  620  can comprise posts (not illustrated) formed thereon to engage the plurality of drive notches  524  formed on some embodiments of carrier liner  520  to aid in alignment. 
     In some embodiments, intermediate roller  590  can comprise a generally cylindrical member comprising a pair of pins  626  disposed on opposing ends thereof along axis  628 . In some embodiments, the pair of pins  626  can engage apertures formed in support structure  582  to permit rotating movement of intermediate roller  590  relative thereto. In some embodiments, intermediate roller  590  can be comprises of, at least in part, a compliant material to permit intermediate roller  590  to accommodate variations in fixture  554  and/or microplate  20 . In some embodiments, intermediate roller  590  can carry carrier liner  520  of sealing cover roll  512 . In some embodiments, intermediate roller  590  can be tapered along its longitudinal length to a reduced cross-section area at about a longitudinal midpoint of intermediate roller  590 . This tapered configuration can aid in maintaining carrier liner  520  generally centered on intermediate roller  590 . In some embodiments, intermediate roller  590  can be sized to apply a force on a backside of carrier liner  520  and, consequently, on sealing cover  80  to adhere sealing cover  80  to microplate  20  during application. 
     As best seen in  FIG. 185 , in some embodiments, drive roller assembly  592  comprises at least one knob portion  630  disposed on at least one end of a drive roller  632 . In some embodiments, drive roller  632  can comprise a generally cylindrical member comprising a pair of pins  634  (illustrated hidden in  FIG. 185 ) disposed on opposing ends thereof along axis  636 . In some embodiments, the pair of pins  634  can engage apertures formed in support structure  582  to permit rotating movement of drive roller  632  relative thereto. In some embodiments, the pair of pins  634  can further engage the at least one knob portion  630 . In some embodiments, a pair of knob portions  630  can be used and disposed on opposing ends of drive roller  632  to permit both left-handed and right-handed operation. Knob portion  630  can be manually manipulated by a user to manually advance carrier liner  520  of sealing cover roll  512 . In some embodiments, drive roller  632  can be comprised of, at least in part, a compliant material to permit drive roller  632  to accommodate variations in fixture  554  and/or microplate  20 . In some embodiments, drive roller  632  can be sized to apply a force on a backside of carrier liner  520  and, consequently, on sealing cover  80  to adhere sealing cover  80  to microplate  20  during application. 
     In some embodiments, drive roller  632  can be sized to operably engage pressure roller  594  to receive carrier liner  520  of sealing cover roll  512  therebetween (see  FIG. 185 ). In some embodiments, pressure roller  594  can be a generally cylindrical member comprising a pair of pins  638  disposed on opposing ends thereof along axis  640 . In some embodiments, the pair of pins  638  can engage apertures formed in a support bracket  642  to permit rotating movement of pressure roller  594  relative thereto. In some embodiments, support bracket  642  can be fixedly mounted to or integrally formed with at least one cover section  568 . In some embodiments, pressure roller  594  can be biased to apply a force against drive roller  632  to, at least in part, positively grab, and/or advance carrier liner  520 . 
     Finally, in some embodiments, carrier liner  520  of sealing cover roll  512  can be fed from a lower portion of sealing cover roll  512  forward along a top side of plate member  618 . Carrier liner  520  can then be fed around plane roller  620 , along an bottom side of plate member  618 , around intermediate roller  590 , between pressure roller  594  and drive roller  632 , and finally out of waste gate  596 . 
     In some embodiments, during operation, a user can manually manipulate at least one knob portion  630  until an edge of sealing cover  80  can be advanced to a predetermined seal position. In some embodiments, manual sealing cover applicator  552  can then be placed on top of fixture  554  having microplate  20  mounted thereon. In some embodiments, the user can then apply a downward force on, at least in part, a handle member  640  and push/pull manual sealing cover applicator  552  from one end of microplate  20  to an opposing end of microplate  20 . This motion and the construction of manual sealing cover applicator  552  causes sealing cover  80  to engage and be mounted to microplate  20 . In some embodiments, the downward force applied to manual sealing cover applicator  552  activates adhesive  518 . This motion, in some embodiments, serves to expel the waste (i.e. carrier liner  520  having no sealing cover  80 ) out of waste gate  596 . 
     In some embodiments, sealing cover roll  512  can be loaded in manual sealing cover applicator  552  by positioning latch member  580  in the unlocked position ( FIG. 187 ) and pivoting at least one cover section  568  upward. Sealing cover roll  512  can then be place on roll hub  584 . Carrier liner  520  can then be routed through manual sealing cover applicator  552  as described above.). In some embodiments, closing of the at least one cover section  568  causes pressure roller  594  to apply a force on carrier liner  520 . In some embodiments, drive roller  632  and/or knob section  630  can be ratcheted to maintain carrier liner  520  under tension. 
     It should be appreciated that this arrangement can provide reduced possibility of sealing cover application defects, improved sealing cover placement accuracy, reduced operator skill, and faster sealing cover application. 
     Automated Sealing Cover Applicator—Roll 
     In some embodiments, as illustrated in  FIGS. 188-192 , sealing cover  80  can be laminated onto microplate  20  using an automated sealing cover applicator  1100 . In some embodiments, automated sealing cover applicator  1100  comprises a housing  1102  sized to receive sealing cover roll  512  therein. In some embodiments, housing  1102  can comprise a base section  1104  and cover section  1106  connectable therewith. In some embodiments, cover section  1106  can comprise an opening  1108  for receiving a sealing cover cassette  1110  therein. 
     Referring now to  FIGS. 189 and 190 , in some embodiments, base section  1104  comprises at least one of a microplate tray assembly  1112 , a tray drive system  1114 , a sealing cover drive system  1116  for at least in part alignment control of sealing cover roll  512 , a heated roller assembly  1118 , and an applicator control system  1120 . 
     In some embodiments, microplate tray assembly  1112  comprises a generally planar tray member  1122  that can be movable between an extended position ( FIGS. 188-190 ) and a retracted position. In some embodiments, generally planar tray member  1122  comprises a recessed portion  1124 . Recessed portion  1124 , in some embodiments, can be sized to receive microplate  20  therein. In some embodiments, microplate tray assembly  1112  comprises an alignment feature  1126  that can be complementary to alignment feature  58  on microplate  20 . In some embodiments, alignment feature  1126  can a corner chamfer, a pin, a slot, a cut corner, an indentation, a graphic, a nub, a protrusion, or other unique feature that can be capable of interfacing with alignment feature  58  or other feature of microplate  20 . In some embodiments, microplate tray assembly  1112  comprises one or more recesses  1128  formed in generally planar tray member  1122  to permit, among other things, improved grasping of microplate  20  for ease of insertion and withdrawal of microplate  20  from microplate tray assembly  1112 . In some embodiments, one or more recesses  1128  can be positioned along opposing ends of microplate  20 . In some embodiments, generally planar tray member  1122  comprises a uniquely sized and/or shaped insert  1130  that can be fastened within recessed portion  1124  to accommodate varying sizes of microplates or other devices. 
     As can be seen in  FIG. 190 , in some embodiments, microplate tray assembly  1112  can be moved between the extended position and the retracted position via tray drive system  1114 . In some embodiments, tray drive system  1114  comprises at least one of a drive motor  1132  and a drive track member  1134 . In some embodiments, drive track member  1134  can be a threaded member, such as but not limited to a worm gear, threadedly engaging a receiver  1136  fixedly coupled to microplate tray assembly  1112 . Drive motor  1132  can be actuated by a control switch and/or applicator control system  1120  to rotatably turn drive track member  1134 . In turn, microplate tray assembly  1112  can travel relative to drive track member  1134  between the extended and retracted positions. During such travel, microplate tray assembly  1112  can be guided via at least one guide member  1137  mounted within base section  1104 . It should be appreciated that tray drive system  1114  comprises a cable drive system, a track drive system, a rack and pinion system, a hydraulic system, a pneumatic system, a solenoid system, or the like. 
     In some embodiments, as illustrated in  FIGS. 189-192 , sealing cover cassette  1110  comprises at least one of a support structure  1138 , a cover member  1140 , a roll hub  1142 , a plane roller  1144 , at least one feed roller  1146 , a sprocket drive member  1148 , and a waste gate  1150 . 
     In some embodiments, roll hub  1142  can be fixedly coupled to support structure  1138  to support sealing cover roll  512  thereon and permit relative rotation therebetween. In some embodiments, roll hub  1142  comprises pair of friction legs  598  extending outwardly from tangential sections  600  of central portion  602  as discussed herein. In some embodiments, roll hub  1142  can comprise a cylindrical support member  1152 . 
     In some embodiments, plane roller  1144  can be a generally cylindrical member rotatably supported by support structure  1138  to permit rotating movement of plane roller  1144  relative thereto. In some embodiments, plane roller  1144  can be made of, at least in part, a compliant material to permit plane roller  1144  to accommodate variations in microplate tray assembly  1112  and/or microplate  20 . In some embodiments, plane roller  1144  can be sized and/or positioned to engage microplate tray assembly  1112  and/or microplate  20  to apply a compressing force upon sealing cover  80  and microplate  20  to impart at least an initial sealing engagement. 
     In some embodiments, the at least one feed roller  1146  can comprise a pair of cylindrical members rotatably supported by support structure  1138  to permit rotating movement of feed roller  1146  relative thereto. In some embodiments, feed rollers  1146  can be made of a material to, at least in part, positively grab and/or advance carrier liner  520 . Feed roller  1146  can also be configured to impart a drag force on carrier liner  520  opposing a driving force by sprocket drive member  1148  to ensure carrier liner  520  and sealing cover  80  disposed thereon are generally flat between feed roller  1146  and sprocket drive member  1148 . 
     As best seen in  FIG. 185 , in some embodiments, sprocket drive member  1148  can be a generally cylindrical member comprising at least one sprocket portion  1154  disposed on at least one end of a support rod  1156  ( FIG. 189 ) rotatable about an axis  1157 . In some embodiments, a pair of sprocket portions  1154  can be provided such that each of the pair of sprocket portions  1154  can be disposed on opposing ends of support rod  1156 . In some embodiments, support rod  1156  can be rotatably coupled to support structure  1138 . The pair of sprocket portions  1154  can each comprise a plurality of engaging portions  1158  that are each sized and spaced to enmesh with each of the plurality of drive notched  524  formed on carrier liner  520  of sealing cover roll  512 . 
     In some embodiments, sprocket drive member  1148  can be driven by sealing cover drive system  1116 . In some embodiments, sealing cover drive system  1116  can comprise a drive motor  1160  ( FIG. 189 ) enmeshingly engaging a drive gear  1162  ( FIG. 191 ) fixed coupled at an end of support rod  1156  of sprocket drive member  1148  ( FIG. 191 ). In some embodiments, drive motor  1160  can be actuated by a control switch and/or applicator control system  1120  to rotatably turn sprocket drive member  1148  and drive carrier liner  520  of sealing cover roll  512 . In some embodiments, drive motor  1160  can be fixedly mounted within base section  1104 . In some embodiments, a vibration isolation member  1164  can be disposed between drive motor  1160  and a support structure  1166  within base section  1104 . 
     As best seen in  FIG. 192 , in some embodiments, carrier liner  520  of sealing cover roll  512  can be fed from sealing cover roll  512  downward between feed roller  1146  and around sprocket drive members  1148  and out waste gate  1150 . To aid in initial feeding of carrier liner  520  around sprocket drive members  1148 , a guide wall  1168  can be provided to direct an end of carrier liner  520  toward waste gate  1150 . 
     In some embodiments, as illustrated in  FIGS. 190 and 192 , sealing cover cassette  1110  can further comprise a latch system  1170  for operably coupling sealing cover cassette  1110  to cover section  1106 . In some embodiments, latch system  1170  comprises a lip member  1172  disposed on one end of cover member  1140  and at least one biasing members  1174 . As best seen in  FIG. 192 , lip member  1172  can engage an underside of cover section  1106 . Similarly, at least one biasing member  1174  can be generally U-shaped and have a retaining feature  1177  that can be sized to engage an underside of cover section  1106 . In this regarding, at least one biasing member  1174  can impart a locking force such that retaining feature  1177  remains engaged with the underside of cover section  1106  until a user overcomes the biasing force to disengage retaining feature  1177  from cover section  1106 . To install sealing cover cassette  1110  into cover section  1106 , one can simply insert lip member  1172  under cover section  1106  and pivot a front end of sealing cover cassette  1110  downward until the at least one biasing member  1174  engages cover section  1106 . This motion can further engage drive gear  1162  with drive motor  1160 . 
     As illustrated in  FIG. 190 , in some embodiments, heated roller assembly  1118  can be used to apply at least one of heat and pressure to sealing cover  80  and/or microplate  20  as tray generally planar tray member  1122  passed therebelow. In some embodiments, heat and/or pressure can be used to activate adhesive  518  on sealing cover  80  to effect sealing interface  112 . In some embodiments, heated roller assembly  1118  comprises a heated roller  1178  rotatably supported within a removable housing  1180 . In some embodiments, heated roller  1178  can be heated internally via a heating member  1182  and/or heated externally via a heating device  1184 . In some embodiments, heating member  1182  and/or heating device  1184  can be controlled by applicator control system  1120 . It should be appreciated that heated roller assembly  1118  can be manufactured as a sub-assembly to permit easy retrofitting of existing automated sealing cover applicators  1100  for use with heat sensitive adhesives. It should also be appreciated that in some embodiments, heating device  1184  can serve as a convective and/or indirect heater of sealing cover  80  as microplate  20  passes therebelow. In such embodiments, heated roller  1178  can be eliminated. 
     In some embodiments, applicator control system  1120  can be operable to control tray drive system  1114  and/or sealing cover drive system  1116  to apply sealing cover  80  to microplate  20 . Applicator control system  1120  comprises an electrical circuit operable to output various control signals to drive motor  1132  and/or drive motor  1160  in response to a program mode of operation and/or data input. In some embodiments, applicator control system  1120  can receive data input from at least one sensor disposed in automated sealing cover applicator  1100 , such as, but not limited to, a tray drive sensor for detecting encumbered operation of microplate tray assembly  1112 , a sealing cover drive sensor for detecting encumbered operation of sealing cover cassette  1110 , a sealing cover position sensor for detecting one of the plurality of staging notches  528  formed in carrier liner  520 , an end/start of roll sensor for detecting end/start of roll notch  530 , a temperature sensor for detecting a temperature of heated roller  1178 , or any other sensor for detecting a desired operating parameter of automated sealing cover applicator  1100 . In some embodiments, applicator control system  1120  can be response to at least one of a power switch  1186 , a tray activation button  1188 , and/or a seal application button  1190  ( FIG. 188 ). Still further, in some embodiments, applicator control system  1120  can output a control status indicia  1192  that can include, but is not limited to, a TEMP alert indicia, a SEAL EMPTY alert indicia, a TRAY JAM alert indicia, a SEAL JAM alert indicia, a POWER alert indicia, a READY alert indicia, or the like. In some embodiments, the TEMP alert indicia can be used to indicate when a desired temperature has been reached. In some embodiments, the SEAL EMPTY alert indicia can be used to indicate when sealing cover roll  512  is at or near empty of sealing covers  80 . In some embodiments, the TRAY JAM alert indicia can be used to indicate when microplate tray assembly  1112  is encumbered. In some embodiments, the SEAL JAM alert indicia can be used to indicate when at least one sealing cover  80  is encumbered. 
     It should be appreciated that this arrangement can provide reduced possibility of sealing cover application defects, improved sealing cover placement accuracy, reduced operator skill, and faster sealing cover application. 
     Automated Sealing Cover Applicator—Single Sheet 
     Turning now to  FIGS. 193-201 , in some embodiments, automated sealing cover applicator  1100  comprises a single sheet applicator assembly  1194 . In some embodiments, single sheet applicator assembly  1194  comprises at least one of a plate member  1196 , a cartridge receiving assembly  1198 , a sealing cover cartridge  1200 , and a planer drive system  1202 . 
     As can be seen in  FIGS. 195 and 197 , in some embodiments, sealing cover cartridge  1200  comprises at least one of a top cover  1204 , a bottom cover  1206 , a separator  1208 , at least one wheel member  1210 , and a sealing cover carrier assembly  1212 . In some embodiments, sealing cover carrier assembly  1212  comprises a carrier liner  1214  and a sealing cover  80  disposed on carrier liner  1214 . In some embodiments, carrier liner  1214  can be sized larger than sealing cover  80  to define a flap  1216  along a leading edge of carrier liner  1214 . In some embodiments, carrier liner  1214  can be similar in material to carrier liner  520 . 
     In some embodiments, top cover  1204  can be generally planar in construction and comprises a pair of feed slots  1218  formed along a leading edge  1220  thereof. The pair of feed slots  1218  can be sized to reveal a portion of sealing cover carrier assembly  1212 , specifically flap  1216 , for later use in dispensing sealing cover  80 . 
     In some embodiments, bottom cover  1206  can be generally planar in construction and can comprise a pair of feed slots  1222  formed along a leading edge  1224  thereof. The pair of feed slots  1222  can be sized to generally align with the pair of feed slots  1218  of top cover  1204  to reveal a portion of sealing cover carrier assembly  1212 , specifically flap  1216 , for later use in dispensing sealing cover  80 . 
     In some embodiments, separator  1208  can be generally planar in construction and can be sized to be generally received within top cover  1204  and bottom cover  1206 . In some embodiments, separator  1208  can comprise at least one rib  1226  extending about a periphery of separator  1208  and/or traversing thereabout to support sealing cover carrier assembly  1212  thereon. Separator  1208  can further comprise at least one coupling member  1228  for retaining at least one wheel member  1210 . In some embodiments, the at least one coupling member  1228  can be a C-shaped members sized to engage and retain a reduced cross-section portion  1230  of at least one wheel member  1210 . In some embodiments, the outer diameter of the at least one coupling member  1228  can be less than the outer diameter the at least one wheel member  1210  to reduce interference between the at least one coupling member  1228  and sealing cover carrier assembly  1212 . 
     In some embodiments, top cover  1204 , separator  1208 , and bottom cover  1206  can be coupled together to encapsulate sealing cover carrier assembly  1212  and sealing cover  80  therein, as illustrated in  FIG. 196 . Bottom cover  1206  can comprise at least one mounting stud  1232  formed on an interior side thereof. Top cover  1204  and separator  1208  can comprise at least one aperture  1234  generally aligned with the at least one mounting stud  1232  to receive a threaded fastener therethrough. However, it should be appreciate that other coupling systems, such as a snap-lock interface, can be used. As illustrated in  FIG. 196 , in some embodiments, a slot  1236  can be formed between top cover  1204  and bottom cover  1206 . Slot  1236  can be generally aligned with a tangent of sealing cover carrier assembly  1212  such that as carrier liner  1214  can be driven about the at least one wheel member  1210 , sealing cover  80  can be encouraged to delaminate from carrier liner  1214  and be urged from sealing cover cartridge  1200  for application upon microplate  20 . 
     As best seen in  FIGS. 193 ,  194 , and  198 - 201 , in some embodiments, sealing cover  80  can be urged from sealing cover cartridge  1200  for application upon microplate  20  by first inserting sealing cover cartridge  1200 , having sealing cover  80  disposed therein, into cartridge receiving assembly  1198 . In some embodiments, cartridge receiving assembly  1198  comprises a removable cartridge support  1238 . Removable cartridge support  1238  can be sized to receive sealing cover cartridge  1200  therein for insertion into automated sealing cover applicator  1100 . Automated sealing cover applicator  1100  comprises an opening  1240  formed in a cover section  1242 . In some embodiments, cover section  1242  can have an inwardly-extending angled lip portion  1244 . Angled lip portion  1244  can support and retain an adjustable handle member  1246  via a fastener  1247 . In some embodiments, adjustable handle member  1246  comprises a grasping portion  1248  and an urging member  1250  disposed on an opposing end of adjustable handle member  1246  relative to grasping portion  1248 . In some embodiments, urging member  1250  can be operable to engage a backside of removable cartridge support  1238  and urge sealing cover cartridge  1200  toward planer drive system  1202 . 
     In some embodiments, planer drive system  1202  comprises a generally triangular mounting block  1252  and at least one drive roller  1254  mounted thereto that can be sized and generally aligned with at least one feed slot  1218 , 1222  to operably engage flap  1216  of carrier liner  1214  to drive sealing cover carrier assembly  1212  and urge sealing cover  80  out of slot  1236 . In some embodiments, at least one drive roller  1254  can be operably driven via a drive motor, such as drive motor  1160 , through a gear assembly  1256  ( FIG. 194 ). 
     With particular reference to  FIGS. 198-201 , planer drive system  1202  can further comprise a plane roller  1258 . In some embodiments, plane roller  1258  can be a generally cylindrical member rotatably supported by support structure  1166  to permit rotating movement of plane roller  1258  relative thereto. In some embodiments, plane roller  1258  can be made of, at least in part, a compliant material to permit plane roller  1258  to accommodate variations in microplate tray assembly  1112  and/or microplate  20 . In some embodiments, plane roller  1258  can be sized and/or positioned to engage microplate tray assembly  1112  and/or microplate  20  to apply a compressing force upon sealing cover  80  and microplate  20  to impart at least an initial sealing engagement. In some embodiments, plane roller  1258  can be heated. 
     During operation, in some embodiments, sealing cover carrier assembly  1212 , carrying a single sealing cover  80 , can be preloaded or loaded by a user into sealing cover cartridge  1200  such that flap  1216  of carrier liner  1214  can be exposed through at least one feed slot  1218 ,  1222 . This arrangement can provide reduced contamination of sealing cover  80  and microplate  20 . As illustrated in  FIG. 198 , sealing cover cartridge  1200  can then be loaded into removable cartridge support  1238  and inserted into opening  1240  of cover section  1242  until urging member  1250  engages removable cartridge support  1238  such that flap  1216  can be urged against at least one drive roller  1254  of planer drive system  1202 . Microplate  20  can be loaded into microplate tray assembly  1112 . As illustrated in  FIG. 199 , microplate tray assembly  1112  can then be either manually or automatically driven into automated sealing cover applicator  1100 . At least one drive roller  1254  can then be actuated at a predetermined time to drive flap  1216  of carrier liner  1214  about at least one wheel member  1210 . However, because of, at least in part, the radius of the at least one wheel member  1210 , sealing cover  80  can be delaminated from carrier liner  1214  and urged out of slot  1236 , as illustrated in  FIG. 200 . Finally, sealing cover  80  can generally engage microplate  20  and plane roller  1258  applies a compressing force upon sealing cover  80  and microplate  20  to impart at least an initial sealing engagement between sealing cover  80  and microplate  20 . This arrangement can provide reduced possibility of sealing cover application defects, improved sealing cover placement accuracy, reduced operator skill, and faster sealing cover application. 
     Thermocycler System 
     With reference to  FIGS. 30-44 ,  47 , and  48 , in some embodiments, thermocycler system  100  comprises at least one thermocycler block  102 . Thermocycler system  100  provides heat transfer between thermocycler block  102  and microplate  20  during analysis to vary the temperature of a sample to be processed. It should be appreciated that in some embodiments thermocycler block  102  can also provide thermal uniformity across microplate  20  to facilitate accurate and precise quantification of an amplification reaction. In some embodiments, a control system  1010  ( FIGS. 30 ,  41 , and  42 ) can be operably coupled to thermocycler block  102  to output a control signal to regulate a desired thermal output of thermocycler block  102 . In some embodiments, the control signal of control system  1010  can be varied in response to an input from a temperature sensor (not illustrated). 
     In some embodiments, thermocycler block  102  comprises a plurality of fin members  104  ( FIGS. 42 and 44 ) disposed along a side thereof to dissipate heat. In some embodiments, thermocycler block  102  comprises at least one of a forced convection temperature system that blows hot and cool air onto microplate  20 ; a system for circulating heated and/or cooled gas or fluid through channels in microplate  20 ; a Peltier thermoelectric device; a refrigerator; a microwave heating device; an infrared heater; or any combination thereof. In some embodiments, thermocycler system  100  comprises a heating or cooling source in thermal connection with a heat sink. In some embodiments, the heat sink can be configured to be in thermal communication with microplate  20 . In some embodiments, thermocycler block  102  continuously cycles the temperature of microplate  20 . In some embodiments, thermocycler block  102  cycles and then holds the temperature for a predetermined amount of time. In some embodiments, thermocycler block  102  maintains a generally constant temperature for performing isothermal reactions upon or within microplate  20 . 
     Multiple Thermocyclers 
     In some embodiments, a plurality of thermocycler blocks  102  can be employed to thermally cycle a plurality of microplates  20  to permit higher throughput of microplates  20  through high-density sequence detection system  10 . In some embodiments, each of the plurality of thermocycler blocks  102  can thermally cycle a separate microplate  20  to increase the overall duty cycle of detection system  300  and, in turn, high-density sequence detection system  10 . In other words, during a typical PCR analysis, temperature cycles are used, at least in part, to denature (at a high temperature, e.g, about 95° C.) and then extend (at a low temperature, e.g., about 60° C.) a DNA target. Conventional detection systems can then measure a resultant emission while at the low temperature. However, as can be appreciated, during these temperature cycles, conventional detection systems are idle until the next low temperature portion of the cycle. For instance, in cases where about 40 temperature cycles are completed over a 2-hour period, the conventional detection system is active to measure the resultant emission about 40 times. The remaining time the conventional detection system is idle. Therefore, it should be appreciated that conventional thermocycler systems limit the duty cycle of conventional excitation systems and/or conventional detection systems. 
     In some embodiments, for example, the plurality of thermocycler blocks  102  can be synchronized to provide offset temperature cycles. In some embodiments, the plurality of thermocycler blocks  102  can be synchronized to maximize or provide at or near 100% usage of detection system  300 . The exact number of thermocycler blocks  102  to be used is, at least in part, dependent on the time required to measure all the samples on a single thermocycler and the degree of time offset between the cycling profiles of each thermocycler system. 
     In some embodiments, detection system  300  can comprise a driving device to position detection system  300  and, in some embodiments, excitation system  200  above one of the plurality of thermocycler blocks  102  to measure a resultant emission from the corresponding microplate  20 . In some embodiments, detection system  300  can comprise a movable mirror to permit measurement of the resultant emission of multiple microplates  20  from a fixed position. In some embodiments, each of the plurality of thermocycler blocks  102  can be positioned on a carousel or track system for movement relative to detection system  300 . It should be appreciated that any system, in addition to those described herein, can be used to permit detection of resultant emission from one or more microplates  20  positioned on the plurality of thermocycler blocks  102  by a single detection system  300  to increase the duty cycle thereof. 
     Thermal Compliant Pad 
     With reference to  FIG. 33 , thermal compliant pad  140  can be disposed between thermocycler block  102  and any adjacent component, such as microplate  20  or a sealing cover  80 . It should be understood that thermal compliant pad  140  is optional. Thermal compliant pad  140  can better distribute heating or cooling through a contact interface between thermocycler block  102  and the adjacent component. This arrangement can reduce localized hot spots and compensate for surface variations in thermocycler block  102 , thereby providing improved thermal distribution across microplate  20 . 
     Pressure Clamp System 
     As will be further described herein, according to some embodiments, pressure clamp system  110  can apply a clamping force upon sealing cover  80 , microplate  20 , and thermocycler block  102  to, at least in part, operably seal assay  1000  within the plurality of wells  26  during thermocycling and further improve thermal communication between microplate  20  and thermocycler block  102 . Pressure clamp system  110  can be configured in any one of a number of orientations, such as described herein. Additionally, pressure clamp system  110  can comprise any one of a number of components depending upon the specific orientation used. Therefore, it should be understood that variations exist that are still regarded as being within the scope of the present teachings. 
     Transparent Bag 
     As illustrated in  FIGS. 30-33 , in some embodiments, pressure clamp system  110  can comprise an inflatable transparent bag  116  positioned between and in engaging contact with a transparent window  112  and sealing cover  80 . In the embodiment illustrated in  FIG. 30 , transparent window  112  and thermocycler block  102  are fixed in position against relative movement. Inflatable transparent bag  116  comprises an inflation/deflation port  118  that can be fluidly coupled to a pressure source  122 , such as an air cylinder, which can be controllable in response to a control input from a user or control system  1010 . It should be understood that in some embodiments inflatable transparent bag  116  can comprise a plurality of inflation/deflation ports to facilitate inflation/deflation thereof. 
     Upon actuation of pressure source  122 , pressurized fluid, such as air, can be introduced into inflatable transparent bag  116 , thereby inflating transparent bag  116  in order to exert a generally uniform force upon transparent window  112  and upon sealing cover  80  and microplate  20 . In some embodiments, such generally uniform force can serve to provide a reliable and consistent sealing engagement between sealing cover  80  and microplate  20 . This sealing engagement can substantially prevent water evaporation or contamination of assay  1000  during thermocycling. In some embodiments, inflatable transparent bag  116  can be part of the transparent window  112 , thereby forming a bladder. 
     Still referring to  FIG. 30 , it should be appreciated that in some embodiments transparent window  112 , inflatable transparent bag  116 , and sealing cover  80  permit free transmission therethrough of an excitation light  202  generated by an excitation system  200  and the resultant fluorescence emission. Transparent window  112 , inflatable transparent bag  116 , and sealing cover  80  can be made of a material that is non-fluorescent or of low fluorescence. In some embodiments, transparent window  112  can be comprised of Vycor®, fused silica, quartz, high purity glass, or combination thereof. By way of non-limiting example, window  112  can be comprised of Schott Q2 quartz glass. In some embodiments, window  112  can be from about ¼ to about ½ inch thick; e.g., in some embodiments, about ⅜ inch thick. In some embodiments, a broadband anti-reflective coating can be applied to one or both sides of window  112  to reduce glare and reflections. In some embodiments, the transparent window  112  can comprise optical elements such as a lens, lenslets, and/or a holographic feature. 
     In some embodiments, as illustrated in  FIG. 31 , transparent window  112  can be movable to exert a generally uniform force upon transparent bag  116  and, additionally, upon sealing cover  80  and microplate  20 . In this embodiment as in others, transparent bag  116  can comprise a fixed internal amount of fluid, such as air. Transparent window  112  can be movable using any moving mechanism (not illustrated), such as an electric drive, mechanical drive, hydraulic drive, or the like. 
     Pressure Chamber 
     In some embodiments, as illustrated in  FIGS. 34-40 , pressure clamp system  110  can further employ a pressure chamber  150  in place of transparent bag  116 . 
     Pressure chamber  150  can be a pressurizable volume generally defined by transparent window  112 , a frame  152  that can be coupled to transparent window  112 , and a circumferential chamber seal  154  disposed along an edge of frame  152 . Circumferential chamber seal  154  can be adapted to engage a surface to define the pressurizable, airtight, or at least low leakage, pressure chamber  150 . Transparent window  112 , frame  152 , circumferential chamber seal  154 , and the engaged surface bound the actual volume of pressure chamber  150 . Circumferential chamber seal  154  can engage one of a number of surfaces that will be further discussed herein. A port  120 , in fluid communication with pressure chamber  150  and pressure source  122 , can provide fluid to pressure chamber  150 . 
     In the interest of brevity, it should be appreciated that the particular configuration and arrangement of sealing cover  80  and microplate  20  illustrated in  FIGS. 34-40  can be similar to that illustrated in  FIGS. 30-33 . 
     In some embodiments, as illustrated in  FIGS. 34 and 36 , circumferential chamber seal  154  can be positioned such that it engages a portion of sealing cover  80 . A downward force from transparent window  112  can be exerted upon microplate  20  to maintain a proper thermal engagement between microplate  20  and thermocycler block  102 . Additionally, such downward force can further facilitate sealing engagement of sealing cover  80  and microplate  20 . Still further, pressure chamber  150  can then be pressurized to exert a generally uniform force upon sealing cover  80  and sealing interface  92 . Such generally uniform force can provide a reliable and consistent sealing engagement between sealing cover  80  and microplate  20 . This sealing engagement can reduce water evaporation or contamination of assay  1000  during thermocycling. 
     With particular reference to  FIG. 37 , it should be appreciated that in some embodiments circumferential chamber seal  154  of pressure chamber  150  can be positioned to engage thermocycler block  102 , rather than microplate  20 . Microplate  20  can be positioned within pressure chamber  150 . As pressure chamber  150  is pressurized, force is exerted upon sealing cover  80 , thereby providing a sealing engagement between sealing cover  80  and microplate  20 . 
     In some embodiments, as illustrated in  FIG. 39 , to improve thermal contact between microplate  20  and thermocycler block  102 , optional posts  156  can be employed. Optional posts  156  can be adapted to be coupled with transparent window  112  and downwardly extend therefrom. Optional posts  156  can then engage at least one of microplate  20  or sealing cover  80  to ensure proper contact between microplate  20  and thermocycler block  102  during thermocycling. 
     Inverted Orientation 
     In some embodiments, as illustrated in  FIGS. 27 ,  32 ,  35 ,  41 ,  44 ,  47 , and  48 , microplate  20  can be inverted such that each of the plurality of wells  26  is generally inverted, such that the opening of each of the plurality of wells  26  is directed downwardly. Among other things, this arrangement can provide improved fluorescence detection. As illustrated in  FIG. 27 , this inverted arrangement causes assay  1000  to collect adjacent sealing cover  80  and, thus, addresses the occurrence of condensation effecting fluorescence detection and improves optical efficiency, because assay  1000  is now disposed adjacent to the opening of each of the plurality of wells  26 . 
     In some embodiments, as illustrated in  FIG. 32 , thermocycler block  102  remains stationary and is positioned above microplate  20  and transparent window  112  is positioned below microplate  20 . Inflatable transparent bag  116  can then be positioned in engaging contact between transparent window  112  and sealing cover  80 . It should be appreciated that transparent window  112 , inflatable transparent bag  116 , and sealing cover  80  can permit free transmission therethrough of excitation light  202  generated by excitation system  200  positioned below transparent window  112  and the resultant fluorescence therefrom. In some embodiments, detection system  300  can be positioned below microplate  20  to detect such fluorescence generated in response to excitation light  202  of excitation system  200 . 
     In some embodiments, as illustrated in  FIG. 35 , microplate  20  can be positioned in an inverted orientation, similar to that described in connection with  FIG. 32 , and further employ pressure chamber  150 . Circumferential chamber seal  154  can then be positioned such that it engages a portion of sealing cover  80 . A force from transparent window  112  can be exerted upon microplate  20  to maintain a proper thermal engagement between microplate  20  and thermocycler block  102  and sealing engagement between sealing cover  80  and microplate  20 . Pressure chamber  150  can then be pressurized to exert a generally uniform force across sealing cover  80 . 
     Vacuum Channels 
     As illustrated in  FIG. 38 , some embodiments can comprise a vacuum assist system  170 . In this regard, in some embodiments, port  120  can be eliminated. Vacuum assist system  170  can comprise a pressure/vacuum source  172  fluidly coupled to at least one vacuum channel  174 , which extends throughout thermocycler block  102 . Vacuum channel  174  can comprise grooves or, alternatively or in addition, can comprise a porous or permeable section of thermocycler block  102 . Vacuum channel  174  can be evacuated so as to form a vacuum within a volume  176  defined by transparent window  112 , an O-ring  178 , and thermocycler block  102 . Upon actuation of pressure source  172 , a vacuum can be formed in vacuum channel  174 . This vacuum can vacate volume  176  causing outside air pressure to exert a clamping force on transparent window  112 , thereby clamping sealing cover  80  against microplate  20  to ensure a proper seal and further clamping microplate  20  to thermocycler block  102  to ensure a proper thermal contact. It should be understood that in some embodiments vacuum assist system  170  can be formed in transparent window  112 . 
     Relief Port 
     Turning now to  FIG. 40 , in some embodiments a relief port  158  can be in fluid communication with pressure chamber  150 . Relief port  158  can be operable to slowly bleed gas in pressure chamber  150  and/or simultaneously remove water vapor from pressure chamber  150  to reduce condensation. Removal of water vapor can, in some circumstances, improve fluorescence detection. Relief port  158  can be used in connection with any of the embodiments described herein. 
     Window Heating Device 
     In some embodiments, as illustrated in  FIG. 41 , transparent window  112  can comprise a heating device  160 . Heating device  160  can be operable to heat transparent window  112 , which in turn heats each of the plurality of wells  26  to reduce the formation of condensation within each of the plurality of wells  26 . In some cases, condensation can reduce optical performance and, thus, reduce the efficiency and/or stability of fluorescence detection. 
     In some embodiments, heating device  160  can comprise a layer member  162  that can be laminated to transparent window  112 . In some embodiments, layer member  162  can comprise a plurality of heating wires (not illustrated) distributed uniformly throughout layer member  162 , which can each be operable to heat an adjacent area. In some embodiments, layer member  162  can be an indium tin oxide coating that is applied uniformly across transparent window  112 . A pair of bus bars  164  can be disposed on opposing ends of transparent window  112 . Electrical current can then be applied between bus bars  164  to heat the indium tin oxide coating, which provides a consistent and uniform heat across transparent window  112  without interfering with fluorescence transmission. Bus bars  164  can be controlled in response to control system  1010 . In some embodiments, heating device  160  can be on both sides of transparent window  112 . 
     Clamp Mechanism 
     In some embodiments, as seen in  FIGS. 202-206 , pressure chamber  150  can be used with a clamp mechanism  1400  (best illustrated in  FIGS. 204-206 ). Clamp mechanism  1400  can retain pressure chamber  150  in a clamped position against thermocycler system  100 . 
     Turning now to  FIGS. 202 and 203 , one of some embodiments of pressure chamber  150  is illustrated. A chamber body  1402  has a first side  1404  and a second side  1406 . In some embodiments, chamber body  1402  can be formed from aluminum or other materials such as steel, stainless steel, standard plastic, or fiber-reinforced plastic compound, such as a resin or polymer, and mixtures thereof. An opening  1408  extends through first side  1404  and second side  1406 . 
     A chamber cover  1410  has an opening  1412  surrounded by circumferential chamber seal  154 . Circumferential chamber seal  154  can have a peripheral lip that  1413  that defines a sealing plane abutting sealing cover  80  of microplate  20 . In some embodiments, peripheral lip  1413  can be positioned radially inward of a periphery of opening  1412 . A reactive surface  1415  can span between opening  1412  and peripheral lip  1413 . Reactive surface  1415  can react to fluid pressure in pressure chamber  150  by increasingly urging peripheral lip  1413  against sealing cover  80  as the fluid pressure increases from zero to about 25 pounds per square inch (PSI). In some embodiments, chamber cover  1410  is formed from stainless steel. In some embodiments, a gasket  1414  ( FIG. 203 ) can fit in a groove  1416  formed in a periphery of opening  1408  and provide a seal between chamber cover  1410  and chamber body  1402 . Chamber cover  1410  can be as thin as practicable and have a lower thermal mass than said chamber body to reduce heat flow between microplate  20  and chamber body  1402 . In some embodiments, frame  152  (also seen in  FIG. 35 ) can comprise chamber cover  1410  and chamber body  1402 . 
     In some embodiments, a thin film heater  1418  can be positioned on chamber cover  1410  to further reduce heat flow into chamber body  1402 . Thin film heater  1418  can have a heater signal input  1420  to receive heater power from control system  1010 . In some embodiments, a thermocouple  1422  can be positioned on chamber cover  1410  and provide a cover temperature signal  1424 , by way of non-limiting example, via leads or other signal transmission medium, to control system  1010 . Thermocouple  1422  can comprise, by way of non-limiting example, a type E, type J, type K, or type T thermocouple. Control system  1010  can use cover temperature signal  1424  to control heater power applied to thin film heater  1418  and thereby reduce temperature differences across microplate  20 . In some embodiments, thin film heater  1418  can have a power dissipation of at least 50 watts. 
     In some embodiments, circumferential chamber seal  154  can be molded from a silicone material. In some embodiments, circumferential chamber seal  154  can be insert-molded with chamber cover  1410 . An alignment ring  1426  can be fastened to chamber body  1402  through chamber cover  1410 , and secure chamber cover  1410  to second side  1406 . Microplate  20  can fit within an inner periphery of alignment ring  1426 . Alignment ring  1426  can locate microplate  20  with respect to thermocycler system  100 . In some embodiments, an alignment feature  1428  can interface with alignment feature  58  of microplate  20 . In some embodiments, recesses  1430  can be formed in the inner periphery of alignment ring  1426 . Recesses  1430  reduce a contact area between alignment ring  1426  and microplate  20  and can thereby reduce heat flow between microplate  20  and alignment ring  1426 . 
     On first side  1404 , a flange  1432  can protrude radially inward from the periphery of opening  1408  and support a window seal  1434 . In some embodiments, flange  1432  can be about ¼″ wide. A surface of transparent window  112  can abut window seal  1434 . In some embodiments, for example when window seal  1434  is a non-adhesive type seal, a window-retaining ring  1436  can be secured to chamber body  1402  and clamp transparent window  112  against window seal  1434 . A connector  1438  can provide a connection to port  120  ( FIGS. 34-37 ,  39 - 40 ) that is in fluid communication with the internal volume of pressure chamber  150 . 
     At least one catch  1440  can be positioned on frame  152 . In some embodiments, a pair of catches  1440  can be positioned on opposing sides of a perimeter of frame  152 . Each of the pair of catches  1440  can have a centering feature  1442 . 
     Referring now to  FIGS. 204-206 , thermocycler system  100  and clamp mechanism  1400  are illustrated fixedly mounted to a support structure  1444 . In some embodiments, support structure  1444  can be generally planar in construction and adapted to be mounted within housing  1008  ( FIG. 1 ). Clamp mechanism  1400  can be movable to between a locked condition ( FIG. 204 ) and an unlocked condition ( FIG. 205 ) and can be adapted to selectively clamp pressure chamber  150  against thermocycler system  100 . An opening can be provided in support structure  1444  to allow contact between pressure chamber  150  and thermocycler system  100 . In the locked condition, clamp mechanism  1400  can secure pressure chamber  150  in a clamped position against thermocycler system  100 . In the clamped position, circumferential chamber seal  154  can be pressed against sealing cover  80  (best seen in  FIG. 203 ). In the unlocked condition, clamp mechanism  1400  can allow pressure chamber  150  to be moved to an unclamped position away from thermocycler system  100 . In some embodiments, the unclamped position can provide a gap of ⅜ inch between thermocycler block  102  ( FIG. 203 ) and microplate  20 . In some embodiments, clamp mechanism  1400  can be actuated manually. In other embodiments, clamp mechanism  1400  can be actuated by pneumatics, hydraulics, electric machines and/or motors, electromagnetics, or any other suitable means. 
     In some embodiments, clamp mechanism  1400  can have a clamp frame  1446  fixedly mounted to support structure  1444 . An over-center link  1448  can pivot about a first end  1450  that can be pivotally connected to clamp frame  1446 . A bellcrank  1452  can pivot about a pivot pin  1454  connected to clamp frame  1446 . A lever arm  1456  can have a clamp end  1458  pivotally connected to an input end  1460  of bellcrank  1452 . Lever arm  1456  can have an intermediate portion  1462  pivotally connected to a second end  1464  of over-center link  1448 . An input end  1466  of lever arm  1456  can be pivotally connected to a telescoping end  1468  of a pneumatic cylinder  1470 . A ball joint  1472  can pivotally connect telescoping end  1468  to input end  1466 . A mounting end  1474  of pneumatic cylinder  1470  can pivotally connect to support structure  1444 . In various other embodiments, mounting end  1474  of pneumatic cylinder  1470  can pivotally connect to clamp frame  1446 . Bellcrank  1452  can have a clamp end  1476 . A clamp pin  1478  can project from clamp end  1476  and engage centering feature  1442  when clamp mechanism  1400  is in the locked condition. It should be appreciated that the clamp mechanism  1400  on one side of thermocycler system  100  has been described. A second clamp mechanism  1401  can be positioned on the other side of thermocycler system  100  ( FIG. 206 ). Second clamp mechanism  1401  can be symmetrical with the side just described and operate similarly. A transverse member  1479  can connect lever arm  1456  to the lever arm of the other side. 
     Operation of the clamp assembly  1400  embodiment illustrated in  FIGS. 204-206  will now be described. Pneumatic cylinder  1470  can be movable between an extended condition ( FIG. 205 ) and a contracted condition ( FIGS. 204 and 206 ). As pneumatic cylinder  1470  moves to the contracted condition, it can cause lever arm  1456  to pivot as indicated by a curved arrow A. Lever arm  1456  can in turn cause bellcrank  1452  to pivot as indicated by a curved arrow B, thereby moving clamp pin  1478  towards centering feature  1442 . Clamp pin  1478  can then become centered in centering feature  1442 . As bellcrank  1452  completes rotating in the direction of arrow B, it can cause clamp pin  1478  to move chamber  150  from an unclamped position towards the clamped position against thermocycler assembly  100 . This can cause circumferential chamber seal  154  to press against microplate  20  (best seen in  FIG. 203 ). A clamping pressure between chamber seal  154  and microplate  20  can be adjusted by varying the pivot location of first end  1450  of over-center link  1448 . In some embodiments, an adjustment mechanism  1477 , such as, by way of non-limiting example, a screw, can be used to vary the pivot location as indicated by arrows A ( FIG. 205 ). 
     Moving clamp mechanism  1400  to the unlocked condition will now be described. As pneumatic cylinder  1470  moves to the extended condition, it can cause lever arm  1456  to pivot in a direction opposite curved arrow A. Lever arm  1456  can in turn cause bellcrank  1452  to pivot in a direction opposite curved arrow B, thereby relieving the clamping pressure between clamp pin  1478  and catch  1440 . Clamp pin  1478  can then disengage from centering feature  1442 . As bellcrank  1452  completes rotating in the direction opposite curved arrow B, it can cause clamp pin  1478  to move away from catch  1440 , allowing chamber  150 , with microplate  20 , to move to the unclamped position away from thermocycler system  100 . 
     In some embodiments, a pair of rails  1480  can be used to traverse pressure chamber  150  between a thermocycler position adjacent thermocycler system  100  ( FIG. 204 ) and a loading position away from thermocycler system  100  ( FIG. 205 ). In some embodiments, the loading position can be external of housing  1008 . In such embodiments, housing  1008  has an aperture that allows pressure chamber  150  and rails  1480  to pass therethrough. In some embodiments, a position sensor  1487  can be positioned on support structure  1440  and provide a position signal indicative of pressure chamber  150  being in the thermocycler position. In some embodiments, position sensor can be of an infrared, limit switch, contactless proximity, or ultrasonic type. Rails  1480  can be slidably mounted to support structure  1444 . In some embodiments, optical sensor  1491  can read marking indicia  94  ( FIG. 16 ) on microplate  20  as it is moved to the thermocycler position. Optical sensor  1491  can provide a marking data signal indicative of marking indicia  94  to control system  1010 . 
     In some embodiments, rails  1480  can be telescoping rails. Rails  1480  can be moved manually or can be motorized. In some motorized embodiments, a rack gear  1482  can be positioned on at least one of rails  1480 . A rotating actuator  1484  can be adapted with a pinion gear  1486  that engages rack gear  1482 . Rotating actuator  1484  can rotate in response to control signals from control system  1010 . In some embodiments, rotating actuator  1484  can be an electric motor, such as a stepper motor. For example, actuator  1484  can be a Vexta PK245-02AA stepper motor available from Oriental Motor U.S.A. Corp. In other embodiments, rotating actuator  1484  can be pneumatic or hydraulic. Pressure chamber  150  can be attached between rails  1480 . 
     In some embodiments, a lost motion mechanism  1488  can be positioned between rails  1480  and pressure chamber  150 . Lost motion mechanism  1488  can allow pressure chamber  150  limited perpendicular movement with respect to rails  1480 . The limited perpendicular movement facilitates moving pressure chamber  150  between the clamped and unclamped positions as clamp assembly  1400  moves between the locked and unlocked conditions, respectively. 
     In some embodiments, lost motion mechanism  1488  can include shoulder bolts  1490  threaded into rails  1480 . Catches  1440  can have through holes  1492  that slidingly engage shoulder bolts  1490 . In some embodiments, springs  1494  can be positioned between catches  1440  and rails  1480 . Springs  1494  can bias pressure chamber  140  toward the unclamped position and facilitate moving it away from thermocycler assembly  100  when clamp assembly  1400  moves to the unlocked condition. 
     Pneumatic System 
     Referring now to  FIGS. 207 and 208 , a pneumatic system  1500  is illustrated in accordance with some embodiments. Pneumatic system  1500  can provide pneumatic control for various pneumatic devices used in sequence detection system  10 . By way of non-limiting example, the pneumatic devices can include, alone or in any combination, pressure chamber  150 , pneumatic cylinders  1470 , and vacuum source  172 . 
     An input coupling  1502  can provide a connection point for a supply of compressed fluid, such as, by way of non-limiting example, air, but can also comprise nitrogen, argon, or helium. Input coupling  1502  can be accessible from an exterior of housing  1008  ( FIG. 1 ). In some embodiments, a pressure relief valve  1504  can be in fluid communication with input coupling  1502 . In some embodiments, pressure relief valve  1504  can have a maximum pressure of 120 PSI. In some embodiments, a particle filter  1506  can be in fluid communication with pressure relief valve  1504 . In some embodiments, a condensation separator  1508  can be in fluid communication with particle filter  1508 . Alternatively, condensation separator  1508  can be in fluid communication with pressure relief valve  1504 . Particle filter  1506  and condensation separator  1508  can provide a conditioned fluid supply  1510  to a remainder of pneumatic system  1500 . 
     In some embodiments, a first pressure regulator  1512  can be in fluid communication with conditioned fluid supply  1510 . First pressure regulator  1512  can provide a first fluid supply  1516  to a chamber pressurization subsystem  1518  and/or to other subsystems. 
     In chamber pressurization subsystem  1518 , a check valve  1520  can be connected in series with first pressure regulator  1512 . Check valve  1520  can reduce a risk of depressurization of the internal volume of pressure chamber  150  in the event conditioned fluid supply  1510  is interrupted. A ballast tank  1522  can be in fluid communication with the first fluid supply  1516  and increase a fluid volume of chamber pressurization subsystem  1518 . The increased volume can reduce pressure variations of the first fluid supply  1516 . Ballast tank  1522  can also provide a fluid reserve to help maintain pressure in the event first fluid supply  1516  is interrupted. One side of a charge valve  1524  can be in fluid communication with the first fluid supply  1516 . The other side of charge valve  1524  can be in fluid communication with the internal volume of pressure chamber  150 . A flexible fluid line can connect chamber pressurization subsystem  1518  to connector  1438  of chamber  150 . Charge valve  1524  can be controlled by control system  1010  in accordance with a method described later herein. In some embodiments, charge valve  1524  can be a part number MKH0NBG49A available from ______. 
     A pressure sensor  1526  can be in fluid communication with the internal volume of pressure chamber  150  and can provide a chamber pressure signal  1527  to control system  1010 . In some embodiments, pressure sensor  1526  can be a part number MPS-P6N-AG available from Parker-Hannifin Corp. A chamber pressure relief valve  1528  can be in fluid communication with the internal volume of pressure chamber  150  and establish a maximum pressure that can be applied thereto. In some embodiments, the maximum pressure of  1528  chamber pressure relief valve can be less than, or equal to, 30 PSI. 
     Pressurization subsystem  1518  can also comprise a release valve  1530  in fluid communication with the internal volume of pressure chamber  150 . The other side of release valve  1530  can be vented to atmosphere. Release valve  1530  can be controlled by control system  1010  in accordance with a method described later herein. In some embodiments, release valve  1530  can be a part number MKH0NBG49A available from ______. In some embodiments, the charge and release valves  1524 ,  1530  can maintain chamber pressure at about 18 PSI while the microplate temperature is greater than 40 degrees Celsius. This combination of pressure and temperature conditions can help reduce a possibility of pressure within wells  26  overcoming the chamber pressure and causing wells  26  to leak between sealing cover  80 . A first silencer  1532  can be in fluid communication with the other side of release valve  1530  to reduce noise as fluid is vented. 
     In some embodiments, a second pressure regulator  1534  can be in fluid communication with conditioned fluid supply  1510 . Second pressure regulator  1534  can provide a second fluid supply  1536  to a cylinder control subsystem  1538 . Second pressure regulator  1540  can also provide second fluid supply  1536  to a vacuum control subsystem  1540 . A pressure transducer  1542  can be in fluid communication with second fluid supply  1536  and provide a pressure signal  1544  to control system  1010 . In some embodiments, pressure transducer  1542  can comprise a part number MPS-P6N-AG available from Parker-Hannifin Corp. In some embodiments, second fluid supply  1536  is greater than, or equal to, 50 PSI. 
     In cylinder control subsystem  1538 , a cylinder valve  1546  can have a pressure port  1548 , an exhaust port  1550 , a first port  1552 , and a second port  1554 . Cylinder valve  1546  can be referred to as a 3-position, 2-port valve, commonly referred to as a 3/2 valve. In some embodiments, cylinder valve  1546  can comprise a part number P2MISGEE2CV2DF7 available from ______ or a part number B360BA549C available from ______. Pressure port  1548  can be in fluid communication with second fluid supply  1536 . Exhaust port  1550  can be vented to atmosphere. Cylinder silencer  1556  can be in fluid communication with exhaust port  1550  to reduce noise when fluid is vented from pneumatic cylinder  1470 . First port  1552  can be in fluid communication with first port  1558  of pneumatic cylinder  1470 . Second port  1554  can be in fluid communication with second port  1559  of pneumatic cylinder  1470 . Cylinder valve  1546  can be manually controlled. In some embodiments, cylinder valve  1546  is a servovalve controlled by control system  1010  in accordance with a method described later herein. 
     Cylinder valve  1546  can have three positions that route fluid between ports  1548 - 1554 . A first position can route pressure port  1548  to first port  1552  and route second port  1554  to exhaust port  1550 . A second position can block pressure port  1548  and route first and second ports  1552 ,  1554  to exhaust port  1550 . A third position can route pressure port  1548  to second port  1554  and route first port  1552  to exhaust port  1550 . The first, second, and third positions of cylinder valve  1546  can be referred to as the lock, release, and unlock positions, respectively. 
     When cylinder valve  1546  is in the lock position, fluid routing through cylinder valve  1546  can cause pneumatic cylinder  1470  to move to the contracted condition, thereby moving clamp mechanism  1400  to the locked condition ( FIG. 204 ). When cylinder valve  1546  is in the unlock position, the fluid routing through cylinder valve  1546  can cause pneumatic cylinder  1470  to move to the extended condition, thereby moving clamp mechanism  1400  to the unlocked condition ( FIG. 205 ). When cylinder valve  1546  is in the release position, the fluid routing through cylinder valve  1546  can cause pneumatic cylinder  1470  to be freely extended or contracted by an outside influence, thereby allowing clamp mechanism  1400  to be manually moved between the closed and open positions. It should be noted that over-center link  1448  can maintain clamp mechanism in the locked condition when cylinder valve  1546  is moved to the release position. A first limit switch  1560  can sense, either directly or indirectly, when pneumatic cylinder  1470  is in the extended condition and provide a corresponding signal  1562  to control system  1010 . A second limit switch  1564  can be used to sense, either directly or indirectly, when pneumatic cylinder  1470  is in the contracted condition and provide a corresponding signal  1566  to control system  1010 . In some embodiments, first and second limits switches  1560 ,  1564  can be integral to pneumatic cylinder  1470 . In some embodiments, pneumatic cylinder  1470  can be a Parker-Hannifin Corp. SRM Series pneumatic cylinder with piston sensing capability. In some embodiments, pneumatic cylinder  1470  can be a part number L06DP-SRMBSY400 from Parker-Hannifin Corp. 
     In some embodiments, vacuum control system  1540  selectively actuates vacuum source  172 . Vacuum generated by vacuum source  172  can be provided to thermocycler system  100  or other systems. Vacuum control system  1572  can comprise a vacuum control valve  1568 . In some embodiments, vacuum control valve  1568  can comprise a part number P2MISDEE2CV2BF7 available from ______. 
     Vacuum control valve  1568  can have a pressure port  1570 , an exhaust port  1572 , a first port  1574 , and a second port  1576 . Vacuum control valve  1568  can be referred to as a 3-position, 2-port valve, commonly referred to as a 3/2 valve. Pressure port  1570  can be in fluid communication with second fluid supply  1536 . In some embodiments, exhaust port  1572  can be blocked. In other embodiments, exhaust port  1572  can be vented to atmosphere. First port  1574  can be in fluid communication with vacuum source  172 . Second port  1576  can be blocked in some embodiments having exhaust port  1572  vented to atmosphere. In other embodiments, second port  1576  can be vented to atmosphere. Vacuum control valve  1568  can be manually controlled. In some embodiments, vacuum control valve  1568  is a servovalve controlled by control system  1010  in accordance with a method described later herein. 
     Vacuum control valve  1568  can have three positions that route fluid between ports  1570 - 1576 . A first position can route pressure port  1570  to first port  1574 , and can block exhaust port  1572  and second port  1576 . A second position can block pressure port  1570 , and route first and second ports  1574 ,  1576  through exhaust port  1572 . A third position can route pressure port  1570  to second port  1576 , and block first port  1574  and exhaust port  1572 . The first, second, and third positions of vacuum control valve  1568  can also be referred to as the vacuum on, vacuum off, and vent positions, respectively. 
     When vacuum control valve  1568  is in the vacuum on position, the fluid routing through vacuum control valve  1568  can flow through vacuum source  172 . Vacuum source  172  generates a vacuum in response thereto that can be fluidly coupled to the thermocycler system  100  or other systems. When vacuum control valve  1568  is in the vacuum off position, second fluid supply  1536  is disconnected from vacuum source  172  and vacuum source  172  can be routed to atmosphere through exhaust port  1572  and/or second port  1576 . When vacuum control valve  1568  is in the vent position, second fluid supply  1536  can be purged to atmosphere through second port  1576 . 
     Referring now to  FIG. 209 , a method  1580  is illustrated, according to some embodiments, for clamping pressure chamber  150  to thermocycler system  100 . Method  1580  can be executed by control system  1010  when pressure chamber  150  is placed in proximity to thermocycler block  102 . Method  1580  can begin in step  1582  and can proceed to decision step  1584  to determine whether pressure chamber  150  is properly located within clamp mechanism  1400 . Position signal  1489  ( FIG. 204 ) can be used to make the determination. When pressure chamber  150  is properly located, method  1580  can proceed to step  1586  and move cylinder valve  1546  to the lock position. Method  1580  can then proceed to decision step  1588  and determine whether pneumatic cylinder  1470  has moved to the contracted condition, thereby placing clamp mechanism  1400  in the locked condition. Decision step  1588  can make the determination by using signal  1566  ( FIG. 207 ) from second limit switch  1570 . Method  1580  can execute decision step  1588  until pneumatic cylinder  1470  moves to the contracted condition. Method  1580  can then proceed to step  1590  and can perform a leak test  1590  as described later herein. Method  1580  can then proceed to decision step  1592  and determine, from results of leak test  1590 , whether leak test  1590  passed. If leak test  1590  passed, then method  1580  can proceed to step  1594  and exit. If leak test  1590  failed, then method  1580  can proceed to step  1610  and release chamber  150  according to a method described later herein. 
     Returning to decision step  1584 , if method  1580  determines that chamber  150  is improperly located within clamp mechanism  1400 , then method  1580  can proceed to step  1596 . In step  1596 , method  1580  can indicate that chamber  150  is improperly located within clamp mechanism  1400 . Method  1580  can then proceed to method  1610  and assure clamp mechanism  1400  is in the unlocked condition. Method  1580  can indicate the improper location of chamber  150  though, by way of example, a buzzer, lamp, writing to a computer memory in control system  1010 , or any other suitable means. 
     Referring now to  FIG. 210 , method  1590  is illustrated, according to some embodiments of the invention, for performing the leak test on chamber  150 . Method  1590  can be executed by control system  1010  when chamber  150  is in the clamped position. Method  1590  can begin at step  1591  and can proceed to step  1593 . In step  1593 , method  1590  can pressurize chamber  150  by opening charge valve  1524  and closing release valve  1530  ( FIG. 207 ). Method  1590  can then proceed to decision step  1595  and determine a chamber leak rate of pressure chamber  150 . In one of some embodiments, the chamber leak rate can be determined by determining a difference in air pressure, as indicated by pressure transducer  1526 , over a predetermined amount of time. In one example, the chamber leak rate can be expressed in units of PSI/minute. In decision step  1595 , method  1590  can compare the chamber leak rate to a predetermined leak rate. If the chamber leak rate is less than the predetermined leak rate, method  1590  can proceed to step  1598 , indicating that the leak test has passed. Method  1590  can then proceed to step  1600  and open charge valve  1524  to connect ballast tank  1536  to the internal volume of pressure chamber  150 . In step  1600 , method  1590  can also provide an indication to control system  1010  that thermocycling can begin. 
     Returning now to decision step  1595 , if the chamber leak rate is greater than, or equal to, the predetermined leak rate, method  1590  can proceed to step  1602 , indicating that the leak test has failed. Method  1590  can then proceed to step  1604  and indicate the failure though, by way of example, a buzzer, lamp, writing to the computer memory in control system  1010 , or any other suitable means. Method  1590  can exit at step  1606  from either step  1600  or step  1604 . 
     Referring now to  FIG. 211 , method  1610  of unclamping pressure chamber  150  from thermocycler system  100  is illustrated according to one of several embodiments. Method  1610  can be executed by control system  1010 . In some embodiments, method  1612  can be called by method  1580 . Method  1610  can also be executed after thermocycling is completed. Method  1610  can begin in step  1612  and then can proceed to step  1614 . In step  1614 , method  1610  can move cylinder valve  1546  to the unlock position, which can cause pneumatic cylinder  1470  to begin moving to the extended condition and changing clamp mechanism to the unlocked condition. Method  1610  can then proceed to decision step  1616  and determine whether pneumatic cylinder  1470  has moved to the extended condition. Decision step  1616  can make the determination by using signal  1562  ( FIG. 207 ) from first limit switch  1560 . Method  1610  can execute decision step  1616  until pneumatic cylinder  1470  moves to the extended condition. Method  1610  can then proceed to step  1618  and exit. 
     Excitation System 
     In some embodiments, as illustrated in  FIGS. 42-49 , excitation system  200  generally comprises a plurality of excitation lamps  210  generating excitation light  202  in response to control signals from control system  1010 . Excitation system  200  can direct excitation light  202  to each of the plurality of wells  26  or across the plurality of wells  26 . In some embodiments, excitation light  202  can be a radiant energy comprising a wavelength that permits detection of photo-emitting detection probes in assay  1000  disposed in at least some of the plurality of wells  26  of microplate  20  by detection system  300 . 
     By way of background, it should be understood that the quantitative analysis of assay  1000 , in some embodiments, can involve measurement of the resultant fluorescence intensity or other emission intensity. In some embodiments of the present teachings, fluorescence from the plurality of wells  26  on microplate  20  can be measured simultaneously using a CCD camera. In an idealized optical system, if all of the plurality of wells  26  have the same concentration of dye, each of the plurality of wells  26  would produce an identical fluorescence signal. In some prior conventional designs, wells near the center of the microplate may appear significantly brighter (i.e. output more signal) than those wells near the edge of the microplate, despite the fact that all of the wells may be outputting the same amount of fluorescence. There are several reasons for this condition in some current designs-vignetting, shadowing, and the particular illumination/irradiance profile. 
     With respect to vignetting, camera lenses can collect more light from the center of the frame relative to the edges. This can reduce the efficiency of certain prior, conventional detection systems. Additionally, in certain prior, conventional designs, the irradiance profile is sometimes not uniform. Most commercially available irradiance sources have a greater irradiance value (watts/meter 2 ) near the center compared to the edges of the irradiance zone. In PCR, it has been found that for a given dye, until the dye saturates or bleaches, the amount of fluorescence can be proportional to the irradiance of the illumination source. Therefore, if the excitation light is brighter at the center, then the fluorescence signal from a well near the edge of the irradiance zone would be less than an identical well near the center. Shadowing can occur due to the depth of the wells. Unless the excitation light is perpendicular to the microplate, some part of the well may not be properly illuminated. In other words, the geometry of the well may block some of the light from reaching the bottom of the well. In addition, the amount of fluorescence emitted, which can be collected, may vary from center to edge. As should be appreciated by one skilled in the art, noise sources are often constant across the field of view of the camera. Therefore, for wells near the edges of microplate  20  that output a smaller amount of fluorescence, the signal to noise ratio can be adversely effected, thereby reducing the efficiency of high-density sequence detection system  10 . As illustrated in  FIG. 50 , a graph illustrates the relative intensity or light transmission versus well location on a plate. As can be seen from the graph, the effects of vignetting and shadowing causes the light intensity to drop off along the edges of the field of view of the plate. 
     The present teachings, at least in part, address these effects so that identical wells output generally identical fluorescence irrespective of their location on microplate  20 . By using the profile from  FIG. 50 , the optimum irradiance profile can be calculated. With reference to  FIG. 51 , a corresponding irradiance profile, represented by a dashed line, can provide a higher irradiance along the edges. This irradiance profile, when coupled with the effects of vignetting and shadowing, creates generally uniform signal strength across all of the plurality of wells  26  of microplate  20 . 
     Excitation Sources 
     In some embodiments, as illustrated in  FIGS. 42-49 , the plurality of excitation lamps  210  of excitation system  200  can be fixedly mounted to a support structure  212 . In some embodiments, the plurality of excitation lamps  210  can be removably mounted to support structure  212  to permit convenient interchange, exchange, replacement, substitution, or the like. In some embodiments, support structure  212  can be generally planar in construction and can be adapted to be mounted within housing  1008  ( FIG. 1 ). The plurality of excitation lamps  210  can be arranged in a generally circular configuration and directed toward microplate  20  to promote uniform excitation of assay  1000  in each of the plurality of wells  26 . The present teachings permit a generally uniform excitation that is substantially free of shadowing. In some embodiments, the plurality of excitation lamps  210  can be arranged in a generally circular configuration about an aperture  214  formed in support structure  212 . Aperture  214  permits the free transmission of fluorescence therethrough for detection by detection system  300 , as described herein. 
     In some embodiments, as illustrated in  FIGS. 52-56 , each of the plurality of excitation lamps  210  can be configured to achieve the desired irradiance profile. In some embodiments, as seen schematically in  FIG. 52 , each of the plurality of excitation lamps  210  can comprise a lens  216 . Lens  216  can be shaped to provide a desired irradiance profile (see  FIG. 51 ). The exact shape of lens  216  can depend, at least in part, upon one or more of the desired irradiance profile at microplate  20 , the illumination/irradiance profile at each of the plurality of excitation lamps  210 , and the size and position of microplate  20  relative to the plurality of excitation lamps  210 . The shape of lens  216  can be calculated in response to the particular application using commercially available software, such as ZEMAX and/or ASAP. 
     In some embodiments, as seen schematically in  FIG. 53 , each of the plurality of excitation lamps  210  can comprise a mirror  218 . Mirror  218  can be shaped to provide a desired irradiance profile (see  FIG. 51 ). The exact shape of mirror  218  can be dependent, at least in part, upon the desired irradiance profile at microplate  20 , the illumination/irradiance profile at each of the plurality of excitation lamps  210 , and the size and position of microplate  20  relative to the plurality of excitation lamps  210 . The shape of mirror  218  can be calculated in response to the particular application using commercially available software, such as ZEMAX and/or ASAP. 
     In some embodiments, as illustrated in  FIG. 54 , each of the plurality of excitation lamps  210  can comprise a combination of lens  216  and mirror  218  to achieve the desired irradiance profile. Again, lens  216  and mirror  218  can be calculated in response to the particular application using commercially available software, such as ZEMAX and/or ASAP. 
     Turning now to  FIG. 55 , in some embodiments, each of the plurality of excitation lamps  210  can be aligned such that their optical centers converge on a single point  220 . Additionally, in some embodiments, a desired irradiance profile (see  FIG. 51 ) can be achieved by directing each of the plurality of excitation lamps  210  at a predetermined location  222   a - 222   n  on microplate  20 , as illustrated in  FIG. 56 . In some embodiments, each of the plurality of excitation lamps  210  can comprise lens  216  and/or mirror  218  and can further be aligned as illustrated in  FIG. 56  to achieve more complex irradiance profiles. As can be appreciated, employing any of the above techniques described herein can provide improved irradiance across microplate  20 , thereby improving the resultant signal to noise ratio of the plurality of wells  26  along the edge of microplate  20 . 
     It is anticipated that the plurality of excitation lamps  210  can be any one of a number of sources. In some embodiments, the plurality of excitation lamps  210  can be a laser source having a wavelength of about 488 nm, an Argon ion laser, an LED, a halogen bulb, or any other known source. In some embodiments, the LED can be a MR16 from Opto Technologies (Wheeling Ill.; http://www.optotech.com/MR16.htm). In some embodiments, the LED can be provided by LumiLEDS. In some embodiments, the halogen bulb can be a 75 W, 21 V DC lamp or a 50 W, 12 V DC lamp. 
     As discussed above, each of the plurality of excitation sources  210  can be removably coupled to support structure  212  to permit convenient interchange, exchange, replacement, substitution, or the like thereof. In some embodiments, the particular excitation source(s) employed can be selected by one skilled in the art to exhibit desired characteristics, such as increased power, better efficiency, improved uniformity, multi-colors, or having any other desired performance criteria. In embodiments employing multi-color and/or multi-wavelength excitation sources, additional detection probes and/or dyes can be used to, in some circumstances, increase throughput of high-density sequence detection system  10  by including multiple assays in each of the plurality of wells  26 . 
     In some embodiments, the temperature of the plurality of excitation lamps  210  can be controlled to decrease the likelihood of intensity and spectral shifts. In such embodiments, the temperature control can be, for example, a cooling device. In some embodiments, the temperature control can maintain each of the plurality of excitation lamps  210  at an essentially constant temperature. In some embodiments, the intensity can be controlled via a photodiode feedback system, utilizing pulse width modulation (PWM) control to modulate the power of the plurality of excitation lamps  210 . In some embodiments, the PWM can be digital. In some embodiments, shutters can be used to control each of the plurality of excitation lamps  210 . It should be appreciated that any of the excitation assemblies  200  illustrated in  FIGS. 42-49  and described above can be interchanged with each other. 
     Detection Systems 
     In some embodiments, as illustrated in  FIGS. 42-44 ,  47 , and  48 , detection system  300  can be used to detect and/or gather fluorescence emitted from assay  1000  during analysis. In some embodiments, detection system  300  can comprise a collection mirror  310 , a filter assembly  312 , and a collection camera  314 . After excitation light  202  passes into each of the plurality of wells  26  of microplate  20 , assay  1000  in each of the plurality of wells  26  can be illuminated, thereby exciting a detection probe disposed therein and generating an emission (i.e. fluorescence) that can be detected by detection system  300 . 
     In some embodiments, collection mirror  310  can collect the emission and/or direct the emission from each of the plurality of wells  26  towards collection camera  314 . In some embodiments, collection mirror  310  can be a 120 mm-diameter mirror having ¼ or ½ wave flatness and 40/20 scratch dig surface. In some embodiments, filter assembly  312  comprises a plurality of filters  318 . During analysis, microplate  20  can be scanned numerous times—each time with a different filter  318 . 
     In some embodiments, collection camera  314  comprises a multi-element photo detector  324 , such as, but not limited to, charge coupled devices (CCDs), diode arrays, photomultiplier tube arrays, charge injection devices (CIDs), CMOS detectors, and avalanche photodiodes. In some embodiments, the emission from each of the plurality of wells  26  can be focused on collection camera  314  by a lens  316 . In some embodiments, collection camera  314  is an ORCA-ER cooled CCD type available from Hamamatsu Photonics. In some embodiments, lens  316  can have a focal length of 50 mm and an aperture of 2.0. In some embodiments, collection camera  314  can be mounted to, and prealigned with, lens  316 . 
     In some embodiments, detection system  300  can comprise a light separating element, such as a light dispersing element. Light dispersing element can comprise elements that separate light into its spectral components, such as transmission gratings, reflective gratings, prisms, beam splitters, dichroic filters, and combinations thereof that are can be used to analyze a single bandpass wavelength without spectrally dispersing the incoming light. In some embodiments, with a single bandpass wavelength light dispersing element, a detection system can be limited to analyzing a single bandpass wavelength. Therefore, one or more light detectors, each comprising a single bandpass wavelength light dispersing element, can be provided. 
     In some embodiments, as seen in  FIG. 212 , an alignment mount  320  can mate collection camera  314  and lens  316 . Alignment mount  320  can provide a mechanism to adjust an axial alignment and a distance between an optic assembly  322  and multi-element photo detector  324 . Lens  316  can receive optic assembly  322  and can mount to a mounting face  326  of a base plate  328 . Base plate  328  can have an aperture  330  formed therein that can allow light to pass from optic assembly  322  to multi-element photo detector  324 . In some embodiments, base plate  328  can be formed from a metal, such as steel, stainless steel, or aluminum. 
     Collection camera  314  can contain multi-element photo detector  324  and can mount to a camera mounting plate  332 . Mounting plate  332  can have an aperture  334  that can align with aperture  330 . Mounting plate  332  can have a face  336  generally parallel to a mating face  338  of base plate  328 . In some embodiments, mounting plate  332  can be formed from a metal, such as steel, stainless steel, or aluminum. At least one resilient member  340  can attach to mounting plate  332  and to base plate  328 . Resilient member  340  can be formed, by non-limiting example, from a spring and/or other elastic structure. Resilient member  340  can provide a bias force that urges face  336  towards mating face  338 . A planarity adjustment feature, such as, by way of non-limiting example, at least one setscrew  342 , can be positioned between face  336  and mating face  338 . At least one setscrew  342  can apply a force opposite the bias force provided by resilient member  340  and maintain face  336  in a spaced relationship from mating face  338 . 
     In some embodiments, at least one set screw  342  can have a thread pitch between 80 and 100 threads per inch (TPI), inclusive. In some embodiments, at least one setscrew  342  can be a ball-end type. In some embodiments, three setscrews  342  can be radially spaced around mounting plate  332 . In some embodiments, the planarity adjustment feature can comprise cams, motorized screws, fluid-containing bags, or inclined planes. In some embodiments, the space between face  336  and mating face  338  can be less than ⅛ inch. In some embodiments, a light blocking gasket  344  can be positioned in the space between face  336  and mating face  338 . In some embodiments, light blocking gasket  344  can be formed from closed cell foam. Light blocking gasket  344  can have apertures formed therein that align with apertures  330  and  334 , and with the planarity adjustment feature. 
     In some embodiments, at least one of collection camera  314  and lens  316  can have a mount comprising a threaded mount or a bayonet mount. The threaded mount can comprise, for example, a C-mount or a CS-mount. The bayonet mount can comprise, for example, an F-mount or a K-mount. In some embodiments, collection camera  314  can be mounted to mounting plate  332  using a mounting ring  346  and a retaining ring  348 . In some embodiments, mounting plate  332  can be formed from a metal, such as steel, stainless steel, or aluminum. Collection camera  314  can be secured to mounting ring  346 . Mounting ring  346  can fit into a groove  350  formed around a periphery of aperture  334 . Retaining ring  348  can fasten to mounting plate  332  and can cover at least a portion of groove  350  and a portion of mounting ring  346 , thereby retaining mounting ring  346  within groove  350 . In some embodiments, retaining ring  348  can be formed from a metal, such as steel, stainless steel, or aluminum. In some embodiments, a concentricity adjustment feature, such as at least one set screw  352 , can protrude radially into groove  350  and can press against an outer periphery  354  of mounting ring  346 . The concentricity adjustment feature can locate mounting ring  350  in an x-y plane of groove  350 . The x-y plane can be illustrated by a coordinate system  356 . In some embodiments, at least one setscrew  352  can have a thread pitch between 80 TPI and 100 TPI, inclusive. In some embodiments, at least one setscrew  352  can be a ball-end type. The concentricity adjustment feature in other embodiments can include cams, motorized screws, fluid-containing bags, and/or inclined planes. 
     A line segment  358  can represent an image plane of optic assembly  322 . An arrow  360  can be centered on optic assembly  322  and normal to its image plane  358 . A line segment  362  can represent an image plane of multi-element photo detector  324 . An arrow  364  can be centered on multi-element photo detector  324  and normal to its image plane  362 . 
     In operation, the planarity adjustment feature, such as at least one set screw  342 , can be used to tilt mounting plate  332  such that image plane  362  can become parallel with image plane  322 . The planarity adjustment feature can also used to adjust the distance between optic assembly  322  and multi-element photo detector  324 . 
     The concentricity adjustment feature, such as at least one setscrew  352 , can translate mounting ring  346  in the x-y plane. Translating mounting ring  346  can adjust arrow  364  concentrically with arrow  360 . 
     In some embodiments, alignment features  368  can align base plate  328  with support structure  212 . Locations of alignment features  368  and dimensions of alignment mount  320  can be selected to place the arrow  360  concentric with a center of microplate  20 . Locations of alignment features  356  and dimensions of alignment mount  320  can be selected to place image plane  358  in parallel with an image plane of microplate  20 . In some embodiments having collection mirror  310  (of  FIGS. 42 and 43 ), locations of alignment features  356  and dimensions of alignment mount  320  can be selected to place image plane  358  perpendicular with the image plane of microplate  20 . In some embodiments, base plate  328  can include a foot plate  366 . By way of non-limiting example, alignment features  368  can comprise any combination of dowels and keys. 
     Control System 
     In some embodiments, control system  1010  can be operable to control various portions of high-density sequence detection system  10  and to collect data. In such embodiments, control system  1010  can comprise software and devices operable to collect and analysis data; control operation of electrical, mechanical, and optical portions of high-density sequence detection system  10 ; and thermocycling. In some embodiments, such data analysis can comprise organizing, manipulating, and reporting of data and derived results to determine relative gene expression within assay  1000 , between various test samples, and across multiple test runs. 
     In some embodiments, control system  1010  can archive data within a database, database retrieval, database analysis and manipulation, and bioinformatics. In some embodiments, control system  1010  can be operable to analyze raw data and among other actions, control operation of high-density sequence detection system  10 . Such analysis of raw data can comprise compensating for point spread (PSF), background or base emissions, a unique intensity profile, optical crosstalk, detector and/or optical path variability and noise, misalignment, or movement during operation. This can be accomplished, in some embodiments, by utilizing internal controls in several of the plurality of wells  26 , as well as calibrating high-density sequence detection system  10 . In some embodiments, data analysis can comprise difference imaging, such as comparing an image from one point in time to an image at a different point in time, or image subtracting. In some embodiments, data analysis can comprise curve fitting based on a specific gene or a gene set. Still further, in some embodiments, data analysis can comprise using no template control (NTC) background or baseline correction. In some embodiments, data analysis can comprise error estimation using confidence values derived in terms of CT. See U.S. Patent Application No. 60/517,506 filed Nov. 4, 2003 and U.S. Patent Application No. 60/519,077 (Attorney Docket No. AB 5043) filed Nov. 10, 2003. 
     In some embodiments, the present teachings can provide a method for reducing signal noise from an array of pixels of a segmented detector for biological samples. The signal noise comprises a dark current contribution and readout offset contribution. The method can comprise providing a substantially dark condition for the array of pixels, wherein the dark condition comprises being substantially free of fluorescent light emitted from the biological samples, providing a first output signal from a binned portion of the array of pixels by collecting charge for a first exposure duration, transferring the collected charge to an output register and reading out the register, wherein transferring of the collected charge from the binned pixels comprises providing a gate voltage to a region near the binned pixels to move collected charge from the binned pixels, and wherein the collected charge can be transferred in a manner that causes the collected charge to be shifted to the output register, providing a second output signal from each pixel by collecting charge for a second exposure duration, transferring the collected charge to the output register, and reading out the register, providing a third output signal by resetting and reading out the output register, determining the dark current contribution and the readout offset contribution from the first output signal, the second output signal, and the third output signal. 
     In some embodiments, the present teachings can provide a method of characterizing signal noise associated with operation of a charge-coupled device (CCD) adapted for analysis of biological samples, wherein the signal noise comprises a dark current contribution, readout offset contribution, and spurious change contribution. The method can comprise providing a plurality of first data points associated with first outputs provided from the CCD under a substantially dark condition during a first exposure duration, providing a plurality of second data points associated with second outputs provided from the CCD under the substantially dark condition during a second exposure duration wherein the second duration is different from the first duration, providing a plurality of third data points associated with third outputs provided from a cleared output register of the CCD without comprising charge transferred thereto, determining the dark current contribution per unit exposure time by comparing the first data points and the second data points, determining the readout offset contribution from the third data points, and determining the spurious charge contribution based on the dark current contribution and the readout offset contribution. See U.S. patent application Ser. No. 10/913,601 filed Aug. 5, 2004; U.S. patent application Ser. No. 10/660,460 filed Sep. 11, 2003, and U.S. patent application Ser. No. 10/660,110 filed Sep. 11, 2003. 
     Methods of Use and Analysis 
     Polynucleotide Amplification 
     In some embodiments, a high-density sequence detection system or components thereof are used for the amplification of polynucleic acids, such as by PCR. Briefly, by way of background, PCR can be used to amplify a sample of target Deoxyribose Nucleic Acid (DNA) for analysis. Typically, the PCR reaction involves copying the strands of the target DNA and then using the copies to generate additional copies in subsequent cycles. Each cycle doubles the amount of the target DNA present, thereby resulting in a geometric progression in the number of copies of the target DNA. The temperature of a double-stranded target DNA is elevated to denature the DNA, and the temperature is then reduced to anneal at least one primer to each strand of the denatured target DNA. In some embodiments, the target DNA can be a cDNA. In some embodiments, primers are used as a pair—a forward primer and a reverse primer—and can be referred to as a primer pair or primer set. In some embodiments, the primer set comprises a 5′ upstream primer that can bind with the 5′ end of one strand of the denatured target DNA and a 3′ downstream primer that can bind with the 3′ end of the other strand of the denatured target DNA. Once a given primer binds to the strand of the denatured target DNA, the primer can be extended by the action of a polymerase. In some embodiments, the polymerase can be a thermostable DNA polymerase, for example, a Taq polymerase. The product of this extension, which sometimes may be referred to as an amplicon, can then be denatured from the resultant strands and the process can be repeated. Temperatures suitable for carrying out the reactions are well known in the art. Certain basic principles of PCR are set forth in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188, each issued to Mullis et al. 
     In some embodiments, PCR can be conducted under conditions allowing for quantitative and/or qualitative analysis of one or more target DNA. Accordingly, detection probes can be used for detecting the presence of the target DNA in an assay. In some embodiments, the detection probes can comprise physical (e.g., fluorescent) or chemical properties that change upon binding of the detection probe to the target DNA. Some embodiments of the present teaching can provide real time fluorescence-based detection and analysis of amplicons as described, for example, in PCT Publication No. WO 95/30139 and U.S. patent application Ser. No. 08/235,411. 
     In some embodiments, assay  1000  can be a homogenous polynucleotide amplification assay, for coupled amplification and detection, wherein the process of amplification generates a detectable signal and the need for subsequent sample handling and manipulation to detect the amplified product is minimized or eliminated. Homogeneous assays can provide for amplification that is detectable without opening a sealed well or further processing steps once amplification is initiated. Such homogeneous assays  1000  can be suitable for use in conjunction with detection probes. For example, in some embodiments, the use of an oligonucleotide detection probe, specific for detecting a particular target DNA can be included in an amplification reaction in addition to a DNA binding agent of the present teachings. Homogenous assays among those useful herein are described, for example, in commonly assigned U.S. Pat. No. 6,814,934. 
     In some embodiments, methods are provided for detecting a plurality of targets. Such methods include those comprising forming an initial mixture comprising an analyte sample suspected of comprising the plurality of targets, a polymerase, and a plurality of primer sets. In some embodiments, each primer set comprises a forward primer and a reverse primer and at least one detection probe unique for one of the plurality of primer sets. In some embodiments, the initial mixture can be formed under conditions in which one primer elongates if hybridized to a target. 
     In some embodiments, the location of a fluorescent signal on a solid support, such as microplate  20 , can be indicative of the identity of a target comprised by the analyte sample. In some embodiments, a plurality of detection probes are distributed to identify loci of at least some of the plurality of wells  26  of microplate  20 . A signal deriving from a detection probe, such as, for example, an increase in fluorescence intensity of a fluorophore at a particular locus can be detected if an amplification product binds to a detection probe and is then amplified. The location of the locus can indicate the identity of the target, and the intensity of the fluorescence can indicate the quantity of the target. 
     In some embodiments, reagents are provided comprising a master mix comprising at least one of catalysts, initiators, promoters, cofactors, enzymes, salts, buffering agents, chelating agents, and combinations thereof. In some embodiments, reagents can include water, a magnesium catalyst (such as MgCl 2 ), polymerase, a buffer, and/or dNTP. In some embodiments, specific master mixes can comprise AmpliTaq® Gold PCR Master Mix, TaqMan® Universal Master Mix, TaqMan® Universal Master Mix No AmpErase® UNG, Assays-by-DesignSM, Pre-Developed Assay Reagents (PDAR) for gene expression, PDAR for allelic discrimination and Assays-On-Demand®, (all of which are marketed by Applied Biosystems). However, the present teachings should not be regarded as being limited to the particular chemistries and/or detection methodologies recited herein, but may employ Taqman®; Invader®; Taqman Gold®; protein, peptide, and immuno assays; receptor binding; enzyme detection; and other screening and analytical methodologies. 
     In some embodiments, high-density sequence detection system  10  is operable for analysis of materials (e.g., polynucleotides) comprising or derived from genetic materials from organisms. In some embodiments, such materials comprise or are derived from substantially the entire genome of an organism. In some embodiments, such organisms include, for example, humans, mammals, mice,  Arabidopsis  or any other plant, bacteria, fungi, or animal species. In some embodiments, assay  1000  comprises at least one of a homogenous solution of a DNA sample, at least one primer set for detection of a polynucleotide comprising or derived from such genetic materials, at least one detection probe, a polymerase, and a buffer. In some embodiments, assay  1000  comprises at least one of a plurality of different detection probes and/or primer sets to perform multiplex PCR, which can be particularly useful when analyzing a whole genome having, for example, about 30,000 different genes. In some embodiments, analysis of substantially the entire genome of an organism is conducted on a single microplate  20 , or on multiple microplates (e.g., two, three, four or more) each comprising subparts of such materials comprising or derived from the genetic materials of the organism. In some embodiments using multiple microplates, a plurality of plates contain a plurality of assay  1000  having essentially identical materials and a plurality of assay  1000  having different materials. In some embodiments, a plurality of plates do not contain assay  1000  having essentially identical materials. In some embodiments, microplate  20  comprises a fixed subset of a genome. It should also be recognized that the present teachings can be used in connection with genotyping, gene expression, or other analysis. 
     In various some embodiments, the microplate can be covered with a sealing liquid prior to performance of analysis or reaction of assay  1000 . For example, in some embodiments, a sealing liquid is applied to the surface of a microplate comprising reaction spots comprising an assay  1000  for amplification of polynucleotides. In some embodiments, a sealing liquid can be a material which substantially covers the material retention regions (e.g., reaction spots) on the microplate so as to contain materials present in the material retention regions, and substantially prevent movement of material from one reaction region to another reaction region on the substrate. In some embodiments, the sealing liquid can be any material which is not reactive with assay  1000  under normal storage or usage conditions. In some embodiments, the sealing liquid can be substantially immiscible with assay  1000 . In some embodiments, the sealing liquid can be transparent, have a refractive index similar to glass, have low or no fluorescence, have a low viscosity, and/or be curable. In some embodiments the sealing liquid can comprise a flowable, curable fluid such as a curable adhesive selected from the group consisting of: ultra-violet-curable and other light-curable adhesives; heat, two-part, or moisture activated adhesives; and cyanoacrylate adhesives. In some embodiments, the sealing liquid can be selected from the group consisting of mineral oil, silicone oil, fluorinated oils, and other fluids which are substantially non-miscible with water. 
     In some embodiments, the sealing liquid can be a fluid when it is applied to the surface of the microplate and in some embodiments, the sealing liquid can remain fluid throughout an analytical or chemical reaction using the microplate. In some embodiments, the sealing liquid can become a solid or semi-solid after it is applied to the surface of the microplate. 
     Other Amplification Methods 
     As should be appreciated from the discussion above, the present teachings can find utility in a wide variety of amplification methods, such as PCR, Reverse Transcription PCR(RT-PCR), Ligation Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), self-sustained sequence replication (3SR), strand displacement activation (SDA), Q (3replicase) system, isothermal amplification methods, and other known amplification method or combinations thereof. Additionally, the present teachings can find utility for use in a wide variety of analytical techniques, such as ELISA; DNA and RNA hybridizations; antibody titer determinations; gene expression; recombinant DNA techniques; hormone and receptor binding analysis; and other known analytical techniques. Still further, the present teachings can be used in connection with such amplification methods and analytical techniques using not only spectrometeric measurements, such as absorption, fluorescence, luminescence, transmission, chemiluminescence, and phosphorescence, but also colorimetric or scintillation measurements or other known detection methods. It should also be appreciated that the present teachings may be used in connection with microcards and other principles, such as set forth in U.S. Pat. Nos. 6,126,899 and 6,124,138. 
     In some embodiments, the reagents can comprise first and second oligonucleotides effective to bind selectively to adjacent, contiguous regions of target DNA and that can be ligated covalently by a ligase enzyme or by chemical means. Such oligonucleotide ligation assays (OLA) are described, for example, in U.S. Pat. No. 4,883,750; and Landegren, U., et al.,  Science  241:1077 (1988). In this approach, the two oligonucleotides (oligonucleotides) are reacted with the target under conditions effective to ensure specific hybridization of the oligonucleotides to their targets. When the oligonucleotides have base-paired with their targets, such that confronting end subunits in the oligonucleotides are base paired with immediately contiguous bases in the target, the two oligonucleotides can be joined by ligation, e.g., by treatment with ligase. After the ligation step, microplate  20  is heated to dissociate unligated detection probes, and the presence of ligated, target-bound detection probe is detected by reaction with an intercalating dye or by other means. The oligonucleotides for OLA can also be designed to bring together a fluorescer-quencher pair, as discussed above, leading to a decrease in a fluorescence signal when the analyte sequence is present. In some embodiments of the OLA ligation method, the concentration of a target region from an analyte polynucleotide can be increased, if desired, by amplification with repeated hybridization and ligation steps. Simple additive amplification can be achieved using the analyte polynucleotide as a target and repeating denaturation, annealing, and ligation steps until a desired concentration of the ligated product is achieved. 
     In other embodiments, the ligated product formed by hybridization and ligation can be amplified by ligase chain reaction (LCR). In this approach, two complementary sets of sequence-specific oligonucleotide detection probes are employed for each target DNA. One of the two sets of sequence-specific oligonucleotide detection probes comprises first and second oligonucleotides designed for sequence-specific binding to adjacent, contiguous regions of a first strand of target DNA. The second of the two sets of sequence-specific oligonucleotide detection probes comprises first and second oligonucleotides designed for sequence-specific binding to adjacent, contiguous regions of a second strand of target DNA. With continued cycles of denaturation, reannealing, and ligation in the presence of the two complementary oligonucleotide sets, the target DNA is amplified exponentially, allowing small amounts of target DNA to be detected and/or amplified. In a further modification, the oligonucleotides for OLA or LCR assay bind to adjacent regions in a target that are separated by one or more intervening bases, and ligation is effected by reaction with (i) a DNA polymerase, to fill in the intervening single stranded region with complementary nucleotides, and (ii) a ligase enzyme to covalently link the resultant bound oligonucleotides. 
     Detection Probes 
     In some embodiments, a detection probe comprises a moiety that facilitates detection of a nucleic acid sequence, and in some embodiments, quantifiably. In some embodiments, a detection probe can comprise, for example, a fluorophore such as a fluorescent dye, a hapten such as a biotin or a digoxygenin, a radioisotope, an enzyme, or an electrophoretic mobility modifier. In some embodiments, the level of amplification can be determined using a fluorescently labeled oligonucleotide. In some embodiments, a detection probe can comprise a fluorophore further comprising a fluorescence quencher. 
     In some embodiments, a detection probe can comprise a fluorophore and can be, for example, a 5′-exonuclease assay probe such as a TaqMan® probe (marketed by Applied Biosystems), a stem-loop Molecular Beacon (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 , Nature Biotechnology  14:303-308 (1996); Vet et al.,  Proc Natl Acad Sci USA.  96:6394-6399 (1999)), a stemless or linear molecular beacon (see., e.g., PCT Patent Publication No. WO 99/21881), a Peptide Nucleic Acid (PNA) Molecular Beacon™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), a linear PNA Molecular Beacon (see, e.g., Kubista et al.,  SPIE  4264:53-58 (2001)), a flap endonuclease probe (see, e.g., U.S. Pat. No. 6,150,097), a Sunrise®/Amplifluor® probe (see, e.g., U.S. Pat. No. 6,548,250), a stem-loop and duplex Scorpion™ probe (see, e.g., Solinas et al.,  Nucleic Acids Research  29:E96 (2001), and U.S. Pat. No. 6,589,743), a bulge loop probe (see, e.g., U.S. Pat. No. 6,590,091), a pseudo knot probe (see, e.g., U.S. Pat. No. 6,589,250), a cyclicon (see, e.g., U.S. Pat. No. 6,383,752), an MGB Eclipse™ probe (Marketed by Epoch Biosciences), a hairpin probe (see, e.g., U.S. Pat. No. 6,596,490), a peptide nucleic acid (PNA) light-up probe, a self-assembled nanoparticle probe, or a ferrocene-modified probe described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al.,  Methods  25:463-471 (2001); Whitcombe et al.,  Nature Biotechnology  17:804-807 (1999); Isacsson et al.,  Molecular Cell Probes  14:321-328 (2000); Svanvik et al.,  Anal. Biochem.  281:26-35 (2000); Wolffs et al.,  Biotechniques  766:769-771 (2001), Tsourkas et al.,  Nucleic Acids Research  30:4208-4215 (2002); Riccelli et al.,  Nucleic Acids Research  30:4088-4093 (2002); Zhang et al., Sheng Wu Hua Xue Yu Sheng Wu Li Xue Bao (Shanghai) ( Acta Biochimica et Biophysica Sinica ) 34:329-332 (2002); Maxwell et al.,  J. Am. Chem. Soc.  124:9606-9612 (2002); Broude et al.,  Trends Biotechnol.  20:249-56 (2002); Huang et al.,  Chem. Res. Toxicol.  15:118-126 (2002); Yu et al.,  J. Am. Chem. Soc  14:11155-11161 (2001). In some embodiments, a detection probe can comprise a sulfonate derivative of a fluorescent dye, a phosphoramidite form of fluorescein, or a phosphoramidite forms of CY5. Detection probes among those useful herein are also disclosed, for example, in U.S. Pat. Nos. 5,188,934, 5,750,409, 5,847,162, 5,853,992, 5,936,087, 5,986,086, 6,020,481, 6,008,379, 6,130,101, 6,140,500, 6,140,494, 6,191,278, and 6,221,604. Energy transfer dyes among those useful herein include those described in U.S. Pat. Nos. 5,728,528, 5,800,996, 5,863,727, 5,945,526, 6,335,440, 6,849745, U.S. Patent Application Publication No. 2004/0126763 A1, PCT Publication No. WO 00/13026A1, PCT Publication No. WO 01/19841A1, U.S. Patent Application Ser. No. 60/611,119, filed Sep. 16, 2004, and U.S. patent application Ser. No. 10/788,836, filed Feb. 26, 2004. In some embodiments, a detection probe can comprise a fluorescence quencher such as a black hole quencher (marketed by Metabion International AG), an Iowa Black™ quencher (marketed by Integrated DNA Technologies), a QSY quencher (marketed by Molecular Probes), and Dabsyl and Eclipse™ Dark Quenchers (marketed by Epoch). 
     In some embodiments, a detection probe can comprise a fluorescent dye. In such embodiments, the fluorescent dye can comprise at least one of rhodamine green (R110), 5-carboxyrhodamine, 6-carboxyrhodamine, N,N′-diethyl-2′,7′-dimethyl-5-carboxy-rhodamine (5-R6G), N,N′-diethyl-2′,7′-dimethyl-6-carboxyrhodamine (6-R6G), 5-carboxy-2′,4′,5′,7′,-4,7-hexachlorofluorescein, 6-carboxy-2′,4′,5′,7′,4,7-hexachloro-fluorescein, 5-carboxy-2′,7′-dicarboxy-4′,5′-dichlorofluorescein, 6-carboxy-2′,7′-dicarboxy-4′,5′-dichlorofluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-5-carboxyfluorescein, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-6-carboxy-fluorescein, 1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein, or those dyes set forth in Table 5. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Absor- 
                   
                   
               
               
                   
                 bance 
                 Emission 
                 Extinction 
               
               
                 Fluorescent Dye 
                 (nm) 
                 (nm) 
                 Coefficient 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 5-Fluorescein 1   
                 495 
                 520 
                 73000 
               
               
                 5-Carboxyfluorescein (5-FAM ™) 1   
                 495 
                 520 
                 83000 
               
               
                 6-Carboxyfluorescein (6-FAM ™) 1   
                 495 
                 520 
                 83000 
               
               
                 6-Carboxyhexachlorofluorescein 
                 535 
                 556 
                 73000 
               
               
                 (6-HEX ™) 1   
               
               
                 6-Carboxytetrachlorofluorescein 
                 521 
                 536 
                 73000 
               
               
                 (6-TET ™) 1   
               
               
                 JOE ™ 1   
                 520 
                 548 
                 73000 
               
               
                 LightCycler ® Red 640 2   
                 625 
                 640 
               
               
                 LightCycler ® Red 705 2   
                 685 
                 705 
               
               
                 Oregon Green ® 488 1   
                 496 
                 516 
                 76000 
               
               
                 Oregon Green ® 500 1   
                 499 
                 519 
                 84000 
               
               
                 Oregon Green ® 514 1   
                 506 
                 526 
                 85000 
               
               
                 BODIPY ® FL-X 1   
                 504 
                 510 
                 70000 
               
               
                 BODIPY ® FL 1   
                 504 
                 510 
                 70000 
               
               
                 BODIPY ®-TMR-X 1   
                 544 
                 570 
                 56000 
               
               
                 BODIPY ® R6G 1   
                 528 
                 547 
                 70000 
               
               
                 BODIPY ® 650/665 1   
                 650 
                 665 
                 101000 
               
               
                 BODIPY ® 564/570 1   
                 563 
                 569 
                 142000 
               
               
                 BODIPY ® 581/591  1   
                 581 
                 591 
                 136000 
               
               
                 BODIPY ® TR-X 1   
                 588 
                 616 
                 68000 
               
               
                 BODIPY ® 630/650 1   
                 625 
                 640 
                 101000 
               
               
                 BODIPY ® 493/503 1   
                 500 
                 509 
                 79000 
               
               
                 5-Carboxyrhodamine 6G 1   
                 524 
                 557 
                 102000 
               
               
                 5(6)-Carboxytetramethylrhodamine 
                 546 
                 576 
                 90000 
               
               
                 (TAMRA) 1   
               
               
                 6-Carboxytetramethylrhodamine 
                 544 
                 576 
                 90000 
               
               
                 (TAMRA) 1   
               
               
                 5(6)-Carboxy-X-Rhodamine (ROX) 1   
                 576 
                 601 
                 82000 
               
               
                 6-Carboxy-X-Rhodamine (ROX) 1   
                 575 
                 602 
                 82000 
               
               
                 AMCA-X (Coumarin) 1   
                 353 
                 442 
                 19000 
               
               
                 Texas Red ®-X 1   
                 583 
                 603 
                 116000 
               
               
                 Rhodamine Red ™-X 1   
                 560 
                 580 
                 129000 
               
               
                 Marina Blue ® 1   
                 362 
                 459 
                 19000 
               
               
                 Pacific Blue ™ 1   
                 416 
                 451 
                 37000 
               
               
                 Rhodamine Green ™-X 1   
                 503 
                 528 
                 74000 
               
               
                 7-diethylaminocoumarin-3-carboxylic 
                 432 
                 472 
                 56000 
               
               
                 acid 1   
               
               
                 7-methoxycoumarin-3-carboxylic 
                 358 
                 410 
                 26000 
               
               
                 acid 1   
               
               
                 Cy3 ® 3   
                 552 
                 570 
                 150000 
               
               
                 Cy3B ® 3   
                 558 
                 573 
                 130000 
               
               
                 Cy3 ® 3   
                 643 
                 667 
                 250000 
               
               
                 Cy5.5 ® 3   
                 675 
                 694 
                 250000 
               
               
                 DY-505 4   
                 505 
                 530 
                 85000 
               
               
                 DY-550 4   
                 553 
                 578 
                 122000 
               
               
                 DY-555 4   
                 555 
                 580 
                 100000 
               
               
                 DY-610 4   
                 606 
                 636 
                 140000 
               
               
                 DY-630 4   
                 630 
                 655 
                 120000 
               
               
                 DY-633 4   
                 630 
                 659 
                 120000 
               
               
                 DY-636 4   
                 645 
                 671 
                 120000 
               
               
                 DY-650 4   
                 653 
                 674 
                 77666 
               
               
                 DY-675 4   
                 674 
                 699 
                 116666 
               
               
                 DY-676 4   
                 674 
                 699 
                 84000 
               
               
                 DY-681 4   
                 691 
                 708 
                 125000 
               
               
                 DY-700 4   
                 702 
                 723 
                 96000 
               
               
                 DY-701 4   
                 706 
                 731 
                 115000 
               
               
                 DY-730 4   
                 734 
                 750 
                 113000 
               
               
                 DY-750 4   
                 747 
                 776 
                 45700 
               
               
                 DY-751 4   
                 751 
                 779 
                 220000 
               
               
                 DY-782 4   
                 782 
                 800 
                 102000 
               
               
                 Cy3.5 ® 3   
                 581 
                 596 
                 150000 
               
               
                 EDANS 1   
                 336 
                 490 
                 5700 
               
               
                 WellRED D2-PA 5   
                 750 
                 770 
                 170000 
               
               
                 WellRED D3-PA 5   
                 685 
                 706 
                 224000 
               
               
                 WellRED D4-PA 5   
                 650 
                 670 
                 203000 
               
               
                 Pyrene 
                 341 
                 377 
                 43000 
               
               
                 Cascade Blue ™ 1   
                 399 
                 423 
                 30000 
               
               
                 Cascade Yellow ™ 1   
                 409 
                 558 
                 24000 
               
               
                 PyMPO 1   
                 415 
                 570 
                 26666 
               
               
                 Lucifer Yellow 1   
                 428 
                 532 
                 11000 
               
               
                 NBD-X 1   
                 466 
                 535 
                 22000 
               
               
                 Carboxynapthofluorescein 1   
                 598 
                 668 
                 42000 
               
               
                 Alexa Fluor ® 350 1   
                 346 
                 442 
                 19000 
               
               
                 Alexa Fluor ® 405 1   
                 401 
                 421 
                 35000 
               
               
                 Alexa Fluor ® 430 1   
                 434 
                 541 
                 16000 
               
               
                 Alexa Fluor ® 488 1   
                 495 
                 519 
                 71000 
               
               
                 Alexa Fluor ® 532 1   
                 532 
                 554 
                 81000 
               
               
                 Alexa Fluor ® 546 1   
                 556 
                 573 
                 104000 
               
               
                 Alexa Fluor ® 555 1   
                 555 
                 565 
                 150000 
               
               
                 Alexa Fluor ® 568 1   
                 578 
                 603 
                 91300 
               
               
                 Alexa Fluor ® 594 1   
                 590 
                 617 
                 73000 
               
               
                 Alexa Fluor ® 633 1   
                 632 
                 647 
                 100000 
               
               
                 Alexa Fluor ® 647 1   
                 650 
                 665 
                 239000 
               
               
                 Alexa Fluor ® 660 1   
                 663 
                 690 
                 132000 
               
               
                 Alexa Fluor ® 680 1   
                 679 
                 702 
                 184000 
               
               
                 Alexa Fluor ® 700 1   
                 702 
                 723 
                 192000 
               
               
                 Alexa Fluor ® 750 1   
                 749 
                 775 
                 240000 
               
               
                 Oyster 556 ® 6   
                 556 
                 570 
                 15000 
               
               
                 Oyster 645 ® 6   
                 645 
                 666 
                 250000 
               
               
                 Oyster 656 ® 6   
                 656 
                 674 
                 220000 
               
               
                 5(6)-Carboxyeosin 1   
                 521 
                 544 
                 95000 
               
               
                 Erythrosin 1   
                 529 
                 544 
                 90000 
               
               
                   
               
               
                   1 Marketed by Molecule Probes; 
               
               
                   2 Marketed by Roche Applied Science; 
               
               
                   3 Marketed by Amersham Biosciences; 
               
               
                   4 Marketed by Synthegen, LLC; 
               
               
                   5 Marketed by Beckman Coulter, Inc.; 
               
               
                   6 Marketed by Denovo Biolabels; 
               
            
           
         
       
     
     In some embodiments, amplified sequences can be detected in double-stranded form by a detection probe comprising an intercalating or a crosslinking dye, such as ethidium bromide, acridine orange, or an oxazole derivative, for example, SYBR Green® (marketed by Molecular Probes, Inc.), which exhibits a fluorescence increase or decrease upon binding to double-stranded nucleic acids. In some embodiments, a detection probe comprises SYBR Green® or Pico Green® (marketed by Molecular Probes, Inc.). 
     In some embodiments, a detection probe can comprise an enzyme that can be detected using an enzyme activity assay. An enzyme activity assay can utilize a chromogenic substrate, a fluorogenic substrate, or a chemiluminescent substrate. In some embodiments, the enzyme can be an alkaline phosphatase, and the chemiluminescent substrate can be (4-methoxyspiro[1,2-dioxetane-3,2′(5′-chloro)-tricyclo[3.3.1.13,7]decan]-4-yl) phenylphosphate. In some embodiments, a chemiluminescent alkaline phosphatase substrate can be CDP-Star® chemiluminescent substrate or CSPD® chemiluminescent substrate (marketed by Applied Biosystems). 
     In some embodiments, the present teachings can employ any of a variety of universal detection approaches involving real-time PCR and related approaches. For example, the present teachings contemplate embodiments in which an encoding ligation reaction is performed in a first reaction vessel (such as for example, an eppendorf tube), and a plurality of decoding reactions are then performed in microplate  20  described herein. For example, a multiplexed oligonucleotide ligation reaction (OLA) can be performed to query a plurality of target DNA, wherein each of the resulting reaction products is encoded with, for example, a primer portion, and/or, a universal detection portion. By including a distinct primer pair in each of plurality of wells  26  of microplate  20  corresponding to the primers sequences encoded in the OLA, a given encoded target DNA can be amplified by that distinct primer pair in a given well of plurality of wells  26 . Further, a universal detection probe (such as, for example, a nuclease cleavable TaqMan® probe) can be included in each of plurality of wells  26  of microplate  20  to provide for universal detection of a single universal detection probe. Such approaches can result in a universal microplate  20 , with its attendant benefits including, among other things, one or more of economies of scale, manufacturing, and/or ease-of-use. The nature of the multiplexed encoding reaction can comprise any of a variety of techniques, including a multiplexed encoding PCR pre-amplification or a multiplexed encoding OLA. Further, various approaches for encoding a first sample with a first universal detection probe, and a second sample with a second universal detection probe, thereby allowing for two sample comparisons in a single microplate  20 , can also be performed according to the present teachings. Illustrative embodiments of such encoding and decoding methods can be found for example in PCT Publication No. WO2003US0029693 to Aydin et al., PCT Publication No. WO2003US0029967 to Andersen et al., U.S. Provisional Application Nos. 60/556,157 and 60/630,681 to Chen et al., U.S. Provisional Application No. 60/556,224 to Andersen et al., U.S. Provisional Application No. 60/556,162 to Livak et al., and U.S. Provisional Application No. 60/556,163 to Lao et al. 
     Single Nucleotide Polymorphism (SNP) 
     In some embodiments, the detection probes can be suitable for detecting single nucleotide polymorphisms (SNPs). A specific example of such detection probes comprises a set of four detection probes that are identical in sequence but for one nucleotide position. Each of the four detection probes comprises a different nucleotide (A, G, C, and T/U) at this position. The detection probes can be labeled with probe labels capable of producing different detectable signals that are distinguishable from one another, such as different fluorophores capable of emitting light at different, spectrally resolvable wavelengths (e.g., 4-differently colored fluorophores). In some embodiments, for example SNP analysis, two colors can be used for two known variants. 
     In some embodiments, at least one of the forward primer and the reverse primer can further comprise a detection probe. A detection probe (or its complement) can be situated within the forward primer between the first primer sequence and the sequence complementary to the target DNA, or within the reverse primer between the second primer sequence and the sequence complementary to the target DNA. A detection probe can comprise at least about 10 nucleotides up to about 70 nucleotides and, more particularly, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 50 nucleotides, or about 60 nucleotides. In some embodiments, a detection probe (or its complement) can further comprise a Zip-Code™ sequence (marketed by Applied Biosystems). In some embodiments, a detection probe can comprise an electrophoretic mobility modifier, such as a nucleobase polymer sequence that can increase the size of a detection probe, or in some embodiments, a non-nucleobase moiety that increases the frictional coefficient of the detection probe, such as those mobility modifier described in commonly-owned U.S. Pat. Nos. 5,514,543, 5,580,732, 5,624,800, and 5,470,705 to Grossman. A detection probe comprising a mobility modifier can exhibit a relative mobility in an electrophoretic or chromatographic separation medium that allows a user to identify and distinguish the detection probe from other molecules comprised by the sample. In some embodiments, a detection probe comprising a sequence complementary to a detection probe and an electrophoretic mobility modifier can be, for example, a ZipChute™ detection probe (marketed by Applied Biosystems). In these embodiments, hybridization of a detection probe with an amplicon, followed by electrophoretic analysis, can be used to determine the identity and quantity of the target DNA. 
     RT-PCR 
     In some embodiments, the present teaching provide methods and apparatus for Reverse Transcriptase PCR(RT-PCR), which include the amplification of a Ribonucleic Acid (RNA) target. In some embodiments, assay  1000  can comprise a single-stranded RNA target, which comprises the sequence to be amplified (e.g., an mRNA), and can be incubated in the presence of a reverse transcriptase, two primers, a DNA polymerase, and a mixture of dNTPs suitable for DNA synthesis. During this process, one of the primers anneals to the RNA target and can be extended by the action of the reverse transcriptase, yielding an RNA/cDNA doubled-stranded hybrid. This hybrid can be then denatured and the other primer anneals to the denatured cDNA strand. Once hybridized, the primer can be extended by the action of the DNA polymerase, yielding a double-stranded cDNA, which then serves as the double-stranded target for amplification through PCR, as described herein. RT-PCR amplification reactions can be carried out with a variety of different reverse transcriptases, and in some embodiments, a thermostable reverse-transcriptions can be used. Suitable thermostable reverse transcriptases can comprise, but are not limited to, reverse transcriptases such as AMV reverse transcriptase, MuLV, and Tth reverse transcriptase. 
     Amplifications for MicroRNA and Small Interfering RNA 
     In some embodiments, assay  1000  can be an assay for the detection of RNA, including small RNA. Detection of RNA molecules can be, in various circumstances, very important to molecular biology, in research, industrial, agricultural, and clinical settings. Among the types of RNA that are of interest in some embodiments are, for example, naturally occurring and synthetic regulatory RNAs such as small RNA molecules (Lee, et al., Science 294: 862-864, 2001; Ruvkun, Science 294: 797-799; Pfeffer et al., 304: Science 734-736, 2004; Ambros, Cell 107: 823-826, 2001; Ambros et al., RNA 9: 277-279, 2003; Carrington and Ambros,  Science  301: 336-338, 2003; Reinhart et al.,  Genes Dev.  16: 1616-1626, 2002 Aravin et al., Dev. Cell 5: 337-350, 2003, Tuschel et al., Science 294: 853-858, 2001; Susi P. et al., Plant Mol. Biol. 54: 157-174, 2004; Xie et al., PLoS Biol. 2: E104, 2004). Small RNA molecules, such as, for example, micro RNAs (miRNA), short interfering RNAs (siRNA), small temporal RNAs (stRNA) and short nuclear RNAs (snRNA), can be, typically, less than about 40 nucleotides in length and can be of low abundance in a cell. With appropriate detection probes, high-density sequence detection system  10  can detect miRNA expression found in, for instance, cell samples taken at different stages of development. In some embodiments, coexpression patterns can be analyzed across microplate  20  with TaqMan sensitivity, specificity, and dynamic range. In some embodiments, such methods obviate the need for running further assays to validate the expression levels. In some embodiments, high-density sequence detection system  10  can be used to validate that siRNA molecules have successfully, post-translationally regulated the gene expression patterns of interest. In some embodiments, such methods may be useful during the manipulation of gene expression patterns using siRNAs in order to elucidate gene function and/or interrelationships amongst genes. In some embodiments, gene expression patterns can be introduced into living cells, cellular assays can be seen on high-density sequence detection system  10  and can reveal gene functions. In some embodiments, analysis for small RNA can be run on high-density sequence detection system  10  allowing for a high number of simultaneous assays  1000  on a single sample with performance that obviates the need for secondary assays to validate the gene expression results. 
     In some embodiments, the methods of the present teachings can include forming a detection mixture comprising a detection probe set ligation sequence, and a primer set. In such embodiments, any detection probe set ligation sequence comprised by the detection mixture can be amplified using PCR on high-density sequence detection system  10  and thereby form an amplification product. In such embodiments, detection of amplification of any detection probe ligation sequence of an analyte. In some embodiments, detection of amplification by high-density sequence detection system  10  can comprise detection of binding of a detection probe to a detection probe hybridization sequence comprised by a probe set ligation sequence or an amplification product thereof. In some configurations, detecting can comprise contacting a PCR amplification product such as an amplified probe set ligation sequence with a detection probe comprising a label under hybridizing conditions. 
     Pre-Amplification and Multiplex Methods 
     In some embodiments for amplification of a polynucleotide, assay  1000  can comprise a preamplification product, wherein one or more polynucleotides in an analyte has been amplified prior to being deposited in at least one of the plurality of wells  26 . In some embodiments, these methods can further comprise forming a plurality of preamplification products by subjecting an initial analyte comprising a plurality of polynucleotides to at least one cycle of PCR to form a detection mixture comprising a plurality of preamplification products. The detection mixture of preamplification products can be then used for further amplification using microplate  20  and high-density sequence detection system  10 . In some embodiments, preamplification comprises the use of isothermal methods. 
     In some embodiments, a two-step multiplex amplification reaction can be performed wherein the first step truncates a standard multiplex amplification round to boost a copy number of the DNA target by about 100-1000 or more fold. Following the first step, the resulting product can be divided into optimized secondary single amplification reactions, each containing one or more of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur, for example, using an aqueous target or using a solid phase archived nucleic acid. See, for example, U.S. Pat. No. 6,605,452, Marmaro. 
     In some embodiments, preamplification methods can employ in vitro transcription (IVT) comprising amplifying at least one sequence in a collection of nucleic acids sequences. The processes can comprise synthesizing a nucleic acid by hybridizing a primer complex to the sequence and extending the primer to form a first strand complementary to the sequence and a second strand complementary to the first strand. The primer complex can comprise a primer complementary to the sequence and a promoter region in anti-sense orientation with respect to the sequence. Copies of anti-sense RNA can be transcribed off the second strand. The promoter region, which can be single or double stranded, can be capable of inducing transcription from an operably linked DNA sequence in the presence of ribonucleotides and a RNA polymerase under suitable conditions. Suitable promoter regions may be prokaryote viruses, such as from T3 or T7 bacteriophage. In some embodiments, the primer can be a single stranded nucleotide of sufficient length to act as a template for synthesis of extension products under suitable conditions and can be poly (T) or a collection of degenerate sequences. In some embodiments, the methods involve the incorporation of an RNA polymerase promoter into selected cDNA molecule by priming cDNA synthesis with a primer complex comprising a synthetic oligonucleotide containing the promoter. Following synthesis of double-stranded cDNA, a polymerase generally specific for the promoter can be added, and anti-sense RNA can be transcribed from the cDNA template. The progressive synthesis of multiple RNA molecules from a single cDNA template results in amplified, anti-sense RNA (aRNA) that serves as starting material for cloning procedures by using random primers. The amplification, which will typically be at least about 20-40, typically to 50 to 100 or 250-fold, but can be 500 to 1000-fold or more, can be achieved from nanogram quantities or less of cDNA. 
     In some embodiments, a two stage preamplification method can be used to preamplify assay  1000  in one vessel by IVT and, for example, this preamplification stage can be 100× sample. In the second stage, the preamplified product can be divided into aliquots and preamplified by PCR and, for example, this preamplification stage can be 16,000× sample or more. Although the above preamplification methods can be used in microplate  20 , these are only examples and are non-limiting. 
     In some embodiments, the preamplification can be a multiplex preamplification, wherein the analyte sample can be divided into a plurality of aliquots. Each aliquot can then be subjected to preamplification using a plurality of primer sets for DNA targets. In some embodiments, the primer sets in at least some of the plurality of aliquots differ from the primer sets in the remaining aliquots. Each resulting preamplification product detection mixture can then be dispersed into at least some of the plurality of wells  26  of microplate  20  comprising an assay  1000  having corresponding primer sets and detection probes for further amplification and detection according to the methods described herein. In some embodiments, the primer sets of assay  1000  in each of the plurality of wells  26  can correspond to the primer sets used in making the preamplification product detection mixture. The resulting assay  1000  in each of the plurality of wells  26  thus can comprise a preamplification product and primer sets and detection probes for amplification for DNA targets, which, if present in the analyte sample, have been preamplified. 
     Since a plurality of different sequences can be amplified simultaneously in a single reaction, the multiplex preamplification can be used in a variety of contexts to effectively increase the concentration or quantity of a sample available for downstream analysis and/or assays. In some embodiments, because of the increased concentration or quantity of target DNA, significantly more analyses can be performed with multiplex amplified samples than can be performed with the original sample. In many embodiments, multiplex amplification further permits the ability to perform analyses that require more sample or a higher concentration of sample than was originally available. In such embodiments, multiplex amplification enables downstream analysis for assays that could not have been possible with the original sample due to its limited quantity. In some embodiments, the plurality of aliquots can comprise 16 aliquots with each of the 16 aliquots comprising about 1536 primer sets. In such embodiments, a sample comprising a whole genome for a species, for example a human genome, can be preamplified. In some embodiments, the plurality of aliquots can be greater than 16 aliquots. In some embodiments, the number of primer sets can be greater than 1536 primer sets. In some embodiments, the plurality of aliquots can be less than 16 aliquots and the number of primer sets can be greater than 1536 primer sets. For examples of such embodiments, see PCT Publication No. WO 2004/051218 to Andersen and Ruff. 
     Multiplex Methods 
     In some embodiments, multiplex methods are provided wherein assay  1000  comprises a first universal primer that binds to a complement of a first target, a second universal primer that binds to a complement of a second target, a first detection probe comprising a sequence that binds to the sequence comprised by the first target, and a second detection probe comprising a sequence that binds to a sequence comprised by the second target. In some embodiments, at least some of the plurality of wells  26  of microplate  20  comprise a solution operable to perform multiplex PCR. The first and second detection probes can comprise different labels, for example, different fluorophores such as, in non-limiting example, VIC and FAM. Sequences of the first and second detection probes can differ by as little as one nucleotide, two nucleotides, three nucleotides, four nucleotides, or greater, provided that hybridization occurs under conditions that allow each detection probe to hybridize specifically to its corresponding detection probe. 
     In some embodiments, multiplex PCR can be used for relative quantification, where one primer set and detection probe amplifies the target DNA and another primer set and detection probe amplifies an endogenous reference. In some embodiments, the present teaching provide for analysis of at least four DNA targets in each of the plurality of wells  26  and/or analysis of a plurality of DNA targets and a reference in each of the plurality of wells  26 . 
     Kits 
     In some embodiments, kits can be provided comprising materials suitable for carrying out polynucleotide amplification. In some embodiments, such kits can comprise microplate  20  and at least a master mix, such as described above herein. 
     In some embodiments, such kits can comprise solutions packaged for preamplification of targets for downstream or subsequent analysis including by multiplex PCR. In some embodiments, the kits can comprise a plurality of primer sets. In some embodiments, the kits can further comprise a set of amplification primers suitable for pre-amplifying a sample of target DNA disposed in at least some of the plurality of wells  26 . In some embodiments, the primers comprised in each of the plurality of wells  26  can, independently of one another, be the same or a different set of primers. 
     In some embodiments, the kit can comprise at least one primer and at least one detection probe disposed in at least some of the plurality of wells  26 . In some embodiments, the kit can comprise a forward primer, a reverse primer, and at least one FAM labeled MGB quenched PCR detection probe disposed in at least some of the plurality of wells  26 . In some embodiments, the kit can comprise at least one detection probe, at least one primer, and a polymerase. In some embodiments, the kit can comprise at least one forward primer, at least one reverse primer, at least one labeled MGB quenched detection probe, at least one labeled MGB quenched detection probe used as a endogenous control, and a polymerase disposed in at least some of the plurality of wells  26 . In some embodiments, a ROX labeled detection probe can be used as a passive internal reference. Some embodiments comprise other detection probes to be used as a passive internal reference. In some embodiments, the kit can comprise reagents for preamplification. In some embodiments, reaction vessels, separate from microplate  20 , can contain any of the above reagents in a dried form, which can be coated to or directed to the bottom of at least some of the plurality of wells  26 . In some embodiments, the user can add a universal master mix, water, and a sample of target DNA to each of the plurality of wells  26  before analysis. 
     In some embodiments, a kit comprises a container containing assay reagents and a separate data storage medium that contains data about the assay reagents. The assay reagents can be adapted to perform an allelic discrimination or expression analysis reaction when mixed with at least one target polynucleotide. The other reagents can be, for example, components conventionally used for PCR and can comprise non-reactive components. In some embodiments, the assay reagents container can have a machine-readable label that provides information about the contents of the container. 
     In some embodiments, the data stored on the data storage medium can comprise computer-readable code that can be used to adjust, calibrate, direct, set, run, or otherwise control an apparatus, for example, high-density sequence detection system  10 . In some embodiments, the data stored on the date storage medium can be used to control high-density sequence detection system  10  to automatically perform PCR or RT-PCR of assay  1000 . See, for example, U.S. Patent Application Publication No. 2004/0072195. 
     Data Analysis 
     In some embodiments, as seen in  FIG. 58 , a plurality of microplates  20  having assay  1000  filled thereon can be analyzed as described herein with high-density sequence detection system  10  to generate data. In some embodiments, this data can be stored in a gene expression analysis system database  736 . Software can then be used to generate gene expression analysis information  738 . 
     In some embodiments, a gene expression analysis system can utilize computer software that organizes analysis sessions into studies and stores them in database  738 . An analysis session can comprise the results of running microplate  20  in high-density sequence detection system  10 . To analyze session data, one can load an existing study that contains analysis session data or create a new study and attach analysis session data to it. Studies can be opened and reexamined an unlimited number of times to reanalyze the analysis session data or to add other analysis sessions to the analysis. 
     In some embodiments, gene expression analysis system database  736  stores the analyzed data for each microplate  20  run on high-density sequence detection system  10  as an analysis session in database  736 . The software can identify each analysis session by marking indicia  64  of the associated microplate  20  and the date on which it was created. Once analysis sessions have been assigned to a study, various functions can be performed. These functions comprise, but are not limited to, designating replicates, removing outliers, filtering data out of a particular view or report, correction of preamplification values via stored values, and computation of gene expression values. 
     In some embodiments, real time PCR is adapted to perform quantitative real time PCR (qRT-PCR). In some embodiments, two different methods of analyzing data from qRT-PCR experiments can be used: absolute quantification and relative quantification. In some embodiments, absolute quantification can determine an input copy number of the target DNA of interest This can be accomplished, for example, by relating a signal from a detection probe to a standard curve. In some embodiments, relative quantification can describe the change in expression of the target DNA relative to a reference or a group of references such as, for an example, an untreated control, an endogenous control, a passive internal reference, an universal reference RNA, or a sample at time zero in a time course study. When determining absolute quantification, the expression of the target DNA can be compared across many samples, for example, from different individuals, from different tissues, from multiple replicates, and/or serial dilution of standards in one or more matrices. In some embodiments of the present teachings, qRT-PCR can be performed using relative quantification and the use of standard curve is not required. Relative quantification can compare the changes in steady state target DNA levels of two or more genes to each other with one of the genes acting as an endogenous reference, which may be used to normalize a signal from a sample gene. In some embodiments, in order to compare between experiments, resulting fold differences from the normalization of sample to the reference can be expressed relative to a calibrator sample. In some embodiments, the calibrator sample is included in each assay  1000 . The gene expression analysis system can determine the amount of target DNA, normalized to a reference, by determining 
       Δ C   T   =C   Tq   −C   Tendo    
     where C T  is the threshold cycle for detection of a fluorophore in real time PCR; C Tq  is the threshold cycle for detection of a fluorophore for a target DNA in assay  1000 ; and C Tendo  is the threshold cycle for detection of a fluorophore for an endogenous reference or a passive internal reference in assay  1000 . 
     In some embodiments, a gene expression analysis system can determine the amount of target DNA, normalized to a reference and relative to a calibrator, by determining: 
       ΔΔ C   T   =ΔC   T,q   −ΔC   T,cb    
     where C T,q  is the threshold cycle for detection of a fluorophore for the target DNA in assay  1000 ; C T,cb  is the threshold cycle for detection of a fluorophore for a calibrator sample; ΔC T,q  is a difference in threshold cycles for the target DNA and an endogenous reference; and ΔC T,cb  is a difference in threshold cycles for the calibrator sample and the endogenous reference. If ΔΔC T  is determined, the relative quantity of the target DNA can be determined using a relationship of relative quantity of the target DNA can be equal to 2 −ΔΔCT . In some embodiments, ΔΔC T  can be about zero. In some embodiments, ΔΔC T  can be less than ±1. In some embodiments, the above calculations can be adapted for use in multiplex PCR (See, for example, Livak et al. Applied Biosystems User Bulletin #2, updated October 2001 and Livak and Schmittgen,  Methods  (25) 402-408 (2001). 
     Triple Delta Analysis 
     In some embodiments, assay  1000  can be preamplified, as discussed herein, in order to increase the amount of target DNA prior to distribution into the plurality of wells  26  of microplate  20 . In some embodiments, assay  1000  can be collected, for example, via a needle biopsy that typically yields a small amount of sample. Distributing this sample across a large number of wells can result in variances in sample distribution that can affect the veracity of subsequent gene expression computations. In such situations, assay  1000  can be preamplified using, for example, a pooled primer set to increase the number of copies of all target DNA simultaneously. 
     In some embodiments, preamplification processes can be non-biased, such that all target DNA are amplified similarly and to about the same power. In such embodiments, each target DNA can be amplified reproducibly from one input sample to the next input sample. For example, if target DNA X is initially present in sample A at 100 target molecules, then after 10 cycles of PCR amplification (1000-fold), 100,000 target molecules should be present. Continuing with the example, if target DNA X is initially present in sample B at 500 target molecules, then after 10 cycles of PCR amplification (1000-fold), 500,000 target molecules should be present. In this example, the ratio of target DNA X in samples A/B remains constant before and after the amplification procedure. 
     In some embodiments, a minor proportion of all target DNA can have an observed preamplification efficiency of less than 100%. In such embodiments, if the amplification bias is reproducible and consistent from one input sample to another, then the ability to accurately compute comparative relative quantitation between any two samples containing different relative amounts of target can be maintained. Continuing the example from above and assuming 50% reproducible amplification efficiency, if target DNA X is initially present in sample A at 100 target molecules, then after 10 cycles of PCR amplification (50% of 1000-fold), 50,000 target molecules should be present. Further continuing the example, if target X is initially present in sample B at 500 target molecules, then after 10 cycles of PCR amplification (50% of 1000-fold), 250,000 target molecules should be present. In this example, the ratio of template X in samples A/B remains constant before and after the amplification procedure and is the same ratio as the 100% efficiency scenario. 
     In some embodiments, an unbiased amplification of each target DNA (x, y, z, etc.) can be determined by calculating the difference in C T  value of the target DNA (x,y,z, etc.) from the C T  value of a selected endogenous reference, and such calculation is referred to as the ΔC T  value for each given target DNA, as described above. In some embodiments, a reference for a bias calculation can be non-preamplified, amplified target DNA and an experimental sample can be a preamplified amplified target DNA. In some embodiments, the standard sample and experimental sample can originate from the same sample, for example, same tissue, same individual, and/or same species. In some embodiments, comparison of ΔC T  values between the non-preamplified amplified target DNA and preamplified amplified target DNA can provide a measure for the bias of the preamplification process between the endogenous reference and the target DNA (x, y, z, etc.). 
     In some embodiments, the difference between the two ΔC T  values (ΔΔC T ) can be zero and as such there is no bias from preamplification. This is illustrated below with reference to  FIG. 213 . In some embodiments, the gene expression analysis system can be calibrated for potential differences in preamplification efficiency that can arise from a variety of sources, such as the effects of multiple primer sets in the same reaction. In some embodiments, calibration can be performed by computing a reference number that reflects preamplification bias. Reference number similarity for a given target DNA across different samples is indicative that the preamplification reaction ΔC T S can be used to achieve reliable gene expression computations. 
     In some embodiments of the present teaching, a gene expression analysis system can compute these reference numbers by collecting a sample (designated as Sample A and S A ) and processing it with one or more protocols. A first protocol comprises running individual PCR gene expression reactions for each target DNA (T x ) relative to an endogenous reference (endo), such as, for example, 18s or GAPDH. These reactions can yield cycle threshold values for each target DNA relative to the endogenous control; as computed by: 
       Δ C   T not preamplified   T   x   S   A   =C   T not preamplified   T   x   S   A   −C   T notpreamplified endo 
     A second protocol can comprise running a single PCR preamplification step on assay  1000  with, for example, a pooled primer set. In some embodiments, the pooled primer set can contain primers for each target DNA. Subsequently, the preamplified product can be distributed among plurality of wells  26  of microplate  20 . PCR gene-expression reactions can be run for each preamplified target DNA (T x ) relative to an endogenous reference (endo). These reactions can yield cycle threshold values for each preamplified target DNA relative to the endogenous control, as computed by: 
       Δ C   T preamplified   T   x   S   A   =C   T preamplified   T   x   S   A   −C   T preamplified endo 
     A difference between these ΔC T not preamplified  T x S A  and ΔC T preamplified  T x S A  can be computed by: 
       ΔΔ C   T   T   x   S   A   =ΔC   T not preamplified   T   x   S   A   −ΔC   T preamplified   T   x   S   A    
     In some embodiments, a value for ΔΔC T T x S A  can be zero or close to zero, which can indicate that there is no bias in the preamplification of target DNA T x . In some embodiments, a negative ΔΔC T T x S A  value can indicate the preamplification process was less than 100% efficient for a given target DNA (T x ). For example, when using an IVT process, a percentage of target DNA with a ΔΔC T  of +/−1 C T  of zero can be 50%. In another example, when using a multiplex preamplification process, a percentage of target DNA with a ΔΔCT of +/−1 C T  of zero can be 90 
     In some embodiments, an amplification efficiency can be less than 100% for a particular target DNA, therefore ΔΔC T  is less than zero for the particular target DNA. An example can be an evaluation of ΔΔC T  values for a group of target DNA from a 1536-plex for the multiplex preamplification process including four different human sample input sources: liver, lung, brain and an universal reference tissue composite. In this example, most ΔΔC T  values are near zero, however, some of the target DNA have a negative ΔΔC T  value but these negative values are reproducible from one sample input source to another. In some embodiments, a gene expression analysis system can determine if a bias exists for target DNA analyzed for different sample inputs. 
     In some embodiments of the present teachings, a gene expression analysis system can use ΔΔC T  values computed for the same target DNA but in different samples (Sample A (S A ) and Sample B (S B )) in order to determine the accuracy of subsequent relative expression computations. This results in the equation, 
       ΔΔΔ C   T   T   x   =ΔΔC   T   T   x   S   A   −ΔΔC   T   T   x   S   B    
     In some embodiments a value for ΔΔΔC T T x  can be zero or reasonably close to zero which can indicate that the preamplified ΔC T  values for T x  (ΔC T preamplified T x S A  and ΔC T preamplified T x S B ) can be used for relative gene expression computation between different samples via a standard relative gene expression calculation. 
     In some embodiments, a standard relative gene expression calculation can determine the amount of the target DNA. In some embodiments, a standard relative gene expression calculation employs a comparative C T . In some embodiments, the above methods can be practiced during experimental design and once the conditions have been optimized so that the ΔΔΔC T T x  is reasonably close to zero, subsequent experiments only require the computation of the ΔC T  value for the preamplified reactions. In some embodiments, ΔΔC T T x S A  values can be stored in a database or other storage medium. In such embodiments, these values can then be used to convert ΔΔC T preamplified T x S A  values to ΔΔC T not preamplified T x S A  values. In such embodiments, the ΔΔC T preamplified T x S y  values can be mapped back to a common domain. In some embodiments, a not preamplified domain can be calculated using other gene expression instrument platforms such as, for example, a microarray. In some embodiments, the ΔΔC T T x S A  values need not be stored for all different sample source inputs (S A ) if it can be illustrated that the ΔΔC T preamplified T x  is reasonably consistent over different sample source inputs. 
     In some embodiments, after microarray sample-to-sample differences are in a ΔΔC T  format, then real-time PCR data can be directly compared to data from other platforms. In some embodiments, a ΔΔΔC T  calculation can be a validation tool to confirm that relative quantitation data can be compared from one amplification/detection process to another. In some embodiments, ΔΔΔC T  calculation can be a validation tool to confirm that relative quantitation data can be compared from one sample input source to another sample input source, for example, comparing a sample from liver to a sample from brain in the same individual. In some embodiments, ΔΔΔC T  calculation can be a validation tool to confirm that relative quantitation data can be compared from one high-density sequence detector system  10  to another high-density sequence detection system  10 . In some embodiments, ΔΔΔC T  calculation can be a validation tool to confirm that relative quantitation data can be compared from one platform to another, for example, data from real time PCR to data from a hybridization array is especially valuable for cross-platform validation. In some embodiments, real time PCR and hybridization array data can be directly compared. In some embodiments, the TaqMan ΔΔC T  can be compared to a microarray output converted to the ΔΔC T  format. In such embodiments, the resultant ΔΔΔC T , if within +/−1 C T  of zero, can determine a high-degree of confidence that the actual fold difference observed within each of the two platforms is correlative. 
     Assay Controls 
     In some embodiments, high-density sequence detection system  10  measures the relative quantities of target DNA using the C T  value from a PCR growth curve, as described herein. The measured C T  value for target DNA for a given assay may vary depending on the system and/or microplate  20  in which the assay  1000  is measured. That is, such variation may arise from manufacturing differences in high-density sequence detection system  10  and/or thermal non-uniformity from variances in production of microplate  20 . 
     In some embodiments, normalization may be the adjusting of a set of raw measurements. For example, a variable storing target DNA levels, quantities may be represented in copy numbers, according to some transformation function in order to make such data compatible between different samples. For example, adjusting copy numbers for a target DNA quantity will produce measurements normalized against a quantity of total RNA and therefore such data can be expressed in specific meaningful and/or compatible units. Without relevant normalization, raw measurements may not carry information that is easily interpretable. 
     In some embodiments, several of the plurality of wells  26  of microplate  20  can be allocated for controls. In some embodiments, the control comprises a template. The template can be, for example, a synthetic oligonucleotide or plasmid, genomic DNA, or other natural DNA or RNA. In some embodiments, the template can contain analogs of naturally occurring nucleotides with modifications to the base, sugar, or phosphate backbone, such as PNAs. 
     In some embodiments, exogenous templates can be used as controls and such templates can be introduced into assay  1000  in one of the following ways: 
     (i) the template at a known concentration can be introduced into a reverse transcription reaction along with the sample; 
     (ii) the template at a known concentration can be introduced into a preamplification reaction along with the sample; 
     (iii) the template at a known concentration can be introduced into assay  1000  along with the sample; or 
     (iv) the template at a known concentration can be spotted onto at least one of a plurality of wells  26 . 
     In some embodiments, the exogenous template can be spotted and dried into at least some of the plurality of wells  26  at a known and defined concentration and the C T  value measured from those of the plurality of wells  26  comprising the control. This C T  value can be used to correct for high-density sequence detection system  10 , microplate  20 , and sample filling/pipetting variations. In these embodiments, assay  1000  can be used to fill at least some of the plurality of wells  26 , but assay  1000  would not contain any exogenous template that would be amplified. In some embodiments, the template can be filled into at least some of the plurality of wells  26  at a known and defined concentration and the C T  value can be measured from the plurality of wells  26  comprising the control to correct for variations from sample filling and pipetting. Templates can also be detected in some of the plurality of wells  26  as an internal control. In such embodiments, the detection probe for the template would produce a different signal than the detection probe for the target DNA. In some embodiments that include a preamplification method to amplify targets prior to PCR, the template can also be designed such that it can be preamplified. Thus, if the template is introduced to assay  1000  prior to preamplification and subsequently measured on microplate  20 , its C T  value could be used to correct for variations in the efficiency of sample preamplification as well as filling/pipetting errors. 
     In some embodiments, the plurality of wells  26  used for controls on microplate  20  can be allocated to contain at least one fluorescent dye that can be spotted and dried down into microplate  20  and hydrolyzed at the time of sample filling. Such plurality of wells  26  can be used to improve calibration of detection system  300  for optical aberrations. In some embodiments, a dye can be used at known concentration and the signals therefrom can be used to optimize the detection sensitivity of high-density sequence detection system  10  (such as the exposure time of the CCD in a detection system  300 ). In some embodiments, the plurality of wells  26  comprising a series dilution of control wells can be used for such calibrations and optimizations. In some embodiments, some of the plurality of wells  26  can be used as controls for identification of the position of the plurality of wells  26 . In some embodiments, at least some of the plurality of wells  26  on microplate  20  can comprise a passive internal reference dye (PIR), such as for example, ROX. The signal from the PIR can be used to locate the plurality of wells  26  by detection system  300 . In some embodiments, prior to beginning PCR, background signals from quenching dyes can be used to determine the locations of the plurality of wells  26  by detection system  300 . In some embodiments, controls can be used to determine filling errors. That is, signals from the PIR can be used to determine if sample filling errors have occurred by looking for an absent or an abnormally high or low signal in the PIR detection image or channel. These signals can indicate an empty well, or an overfilled or under filled well, respectively. In some embodiments, controls can be used to determine spotting errors. The background signals from quenching dyes can be used to determine if spotting errors occurred by looking for an absent or an abnormally high or low signal in the quenching detection image or channel. 
     In some embodiments, controls can be used as quality control for spotting reagents onto microplate  20 . Controls can be measured (by imaging or scanning) for the weak background fluorescence of the dried down reagents to determine if the plurality of wells  26  were spotted correctly and/or in the correct orientation. In some embodiments, one or more fluorescent, infrared, ultraviolet, or visible dyes are introduced into the reagents prior to spotting. When dried down, the fluorescent dyes can be measured to determine if spotting was performed correctly. In some embodiments, the addition of extra dyes to the spotting reagents can be useful for spotting reagents that do not have an inherent fluorescent signal, such as for example the use of reagents comprising SYBR® detection probes. In such embodiments, these additional dyes could also be used as internal controls for identifying filling and pipetting errors. 
     In some embodiments, the plurality of wells  26  without detection probes or primers and/or the plurality of wells  26  that are completely empty or filled with buffer or other solution not containing dye can be used for background correction. The plurality of wells  26  comprising controls without templates (no template controls (NTC)) can also be used for background correction and/or for confirming lack of contamination of the plurality of wells  26  by other samples. In some embodiments, the plurality of wells  26  comprising controls without assay  1000  can be used to confirm lack of contamination during spotting. In some embodiments, the plurality of wells  26  containing varying amounts of a single or multiple dyes can be used to determine if high-density sequence detection system  10  is capable of detecting signals within the expected dynamic range independent of assay performance. In some embodiments, the plurality of wells  26  containing varying amounts of a single or multiple dyes can be used to correct for optical crosstalk or other means of signal correction or normalization. Examples include serial dilutions, multiple titration points, dye ladders, as well as replicates and combinations thereof. In some embodiments, pin hole arrays are used for optical calibration. The controls described above, individually or in combinations thereof, can be incorporated into a single microplate  20  to be used to verify high-density sequence detection system  10  performance in the field at the time of installation or during manufacture. 
     In some embodiments, a procedure for calibration of spectral sensitivity can employ a reference standard to apply a correction to a spectrum such that each of the plurality of wells  26  signal for each filter is normalized to a specific value. In some embodiments, the reference standard can comprise serial dilutions, multiple titration points or dye ladders, as well as replicates and combinations thereof. In some embodiments, the reference comprises multiple dyes (e.g., two, three, four, five, or more) in some of the plurality of wells  26  of microplate  20 . In some embodiments, the value should be identical across all instruments and time periods in order to preserve the calibration. In some embodiments, a reference can be fluorescent reference standard. In some embodiments, the reference can be used in normalizing a single high-density sequence system  10 . In some embodiments, the reference can be used to normalize a group of high-density sequence systems  10 . In some embodiments, the procedure normalizes thresholds and baselines over a group of high-density sequence detector systems  10  so that C T  values are similar across the group for the same assay  1000 . In some embodiments, the controls are templates. 
     In some embodiments, the templates are introduced into a mixture comprising a sample prior to reverse transcription and the resulting C T  values generated from the templates are used to correct for variations in the efficiency of the reverse transcription reaction relative to the expected C T  value. In some embodiments, templates are introduced into a mixture comprising a sample prior to preamplification and the resulting C T  values generated from the templates are used to correct for variations in efficiency of the preamplification reaction. In some embodiments, the templates are introduced into a mixture comprising the sample prior to amplification and the resulting C T  values generated from the templates are used to correct for variations in efficiency of amplification. In some embodiments, different templates are introduced into the mixture comprising a sample at the three different steps (i) reverse transcription, (ii) preamplification and (iii) amplification and the resulting C T  values generated from the templates are calculated for each of the three steps. In such embodiments, the resulting C T  value generated from the templates can be used to determine which of the three steps can be responsible for large deviations of C T  measurements from the expected values. Multiple exogenous templates with varying relative concentrations can be added to a sample mixture in any of the three steps or all of the steps. In some embodiments, a standard plot for absolute quantitation of a sample run on microplate  20  can be calculated. The standard plot can be used to normalize data attained from different microplates  20  or from different samples on the same microplate  20 . 
     In some embodiments, a control can comprise an endogenous template or a set of endogenous templates within a sample that can be used in a wide range of tissues. In some embodiments, the endogenous template can be selected so that the average signal produced during amplification is consistent from sample to sample. In some embodiments, the appropriately selected endogenous template can be used to normalize for variations in sample quantity in the plurality of wells  26 . In some embodiments, results from endogenous controls can be compared from results from exogenous control to distinguish variations in sample quantity and variations in assay performance. A dataset can be normalized by using a function of multiple endogenous templates as controls. For example, a regression of the mean expression values from multiple endogenous controls and can be chosen to be expressed across the entire expression range. Other examples of normalization using a function include functions of the mean signal across microplate  20 , median normalization, quantile normalization, and lowness normalization. In some embodiments, the endogenous controls are relatively invariantly expressed across standard experimental conditions or biological conditions, for example, a tumor, or non-tumor tissue. In some embodiments, the endogenous controls are relatively, invariantly expressed across different tissue types, for example, brain and lung. In some embodiments, a single endogenous control can be used for normalization. In some embodiments, multiple endogenous controls are used for normalization. 
     In some embodiments, microplate  20  comprising a calibrated dilution series of DNA targets and single exon assays can be run on high-density sequence detection system  10  and the data collected can be used to calibrate for absolute quantity or copy number estimations or as in comparison to other array platforms. In such embodiments, microplate  20  can comprise a combination of replicated bacterial DNA and human DNA. For example, microplate  20  can be spotted with 96 different primer sets and 64 replications of the ten-fold primer sets. The human sample can be split and then spiked with bacterial targets to make a set of four ten-fold dilutions. Microplate  20  comprising 96 primer sets with 64 replications can be filled with the set of four ten-fold dilutions and run in high-density sequence detection system  10  producing data for 16 replications of each dilution of the set. The data collected can be used for calculation of high-level performance parameters such as tabulating bad data, calibrating random error model, estimating systematic errors, and estimating starting copy number. 
     In some embodiments, controls can be used for spatial normalization that compensates between at least two channels of signal that is being collected by detections system  300 . The channels for which a signal can be being collected and imaged can be different band passes and the optical performance can change with wavelength and detection probe. In some embodiments, spatial normalization can be accomplished by calibration images of each of the at least two channels collected from a mixture of a pure detection probe spotting to the channel. In some embodiments, a control comprising a mixture of dyes can be spotted onto microplate. In such embodiments, the control comprising a mixture of dyes produces a high signal to noise ratio when detected in detection system  300  of high-density sequence detection system  10 . In such embodiments, spatial normalization correction can be calculated by the use of spatial trends of the measurements of the controls. The controls comprising a mixed dye can be placed in the grid throughout microplate  20 . In some embodiments, to correct all extracted normalization intensities for the spatial trends, a coarse image can be collected and normalized to a 1, 2D median smoothed inner plated under every feature collected is then divided into the image of the extracted normalized intensities. In some embodiments, spatial normalization allows for platform comparisons of data, removes specific instrument effects, or improves cross instrument and cross platform comparisons. In some embodiments, any of the controls discussed above can be adapted for genotyping applications. 
     Assay Selection and Polynucleotide Library 
     In some embodiments, a method is provided for supplying a user with assays useful in obtaining structural genomic information, such as the presence or absence of one or more SNPs, and functional genomic information, such as the expression or amount of expression of one or more genes. As such, in some embodiments, the assays can be configured to detect the presence or expression of genetic material in the sample. 
     In some embodiments, a method of compiling a library of polynucleotide data sets can be provided. In such embodiments, the data sets can correspond to polynucleotides that each function as a primer for producing a nucleic acid sequence that can be complementary to at least one target SNP, as a detection probe for rendering detectable the at least one target SNP, or as both. According to some embodiments, the method can comprise selecting for the library polynucleotide data sets that each correspond to a respective polynucleotide that contains a sequence that is complementary to a respective first allele in each of the at least one target, if, under a set of reaction conditions a number of parameters are met by each polynucleotide corresponding to the data sets in the library. 
     In some embodiments, the method can comprise determining a background signal value by calculating a first normalized ratio of a fluorescence intensity of a respective polynucleotide that contains a sequence that is complementary to a first allele comprised in the at least one target nucleic acid sequence, reacted with first assay reactants in the absence of the target nucleic acid sequence, and under first conditions of fluorescence excitation, to a dye fluorescence intensity of a passive-reference dye under the first conditions. The method can comprise comparing a difference between a second normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with the first assay reactants in the presence of the target nucleic acid sequence, to the dye fluorescence intensity, and the background signal value. The method can comprise comparing a difference between a third normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with second assay reactants that contain a second allele comprised in the at least one target nucleic acid sequence to the dye fluorescence intensity, wherein the second allele differs from the first allele, and the background signal value. 
     In some embodiments, the method can comprise determining whether at least one individual from a population of individuals has a genotype identifiable under the first conditions that result from reacting the respective polynucleotide with the first assay reactants and in the presence of the target nucleic acid sequence, wherein the population comprises at least one individual that has the identifiable genotype and at least one individual that does not have the identifiable genotype. The method can comprise determining whether at least one individual from the population has an identifiable minor allele of the identifiable genotype, under the first conditions that result from reacting the respective polynucleotide with the first assay reactants in the presence of the target nucleic acid sequence. See U.S. Patent Application Publication No. 2003/0190652 to De La Vega et al. 
     Other Applications and Methods 
     In some embodiments, high-density sequence detection system  10  can be used for a variety of biological applications, or assays, other than PCR. In some embodiments, high-density sequence detection system  10  comprising optical illumination and detection system  300  can be used in imaging microplates that fit a SBS standard footprint from low density microplates, for example, 96, 384, or 1536 well microplates to high-density microplates, for example, 6144 or 31104 well microplate. In some embodiments, using lower density microplates high-density sequence detection system  10  can detect multiple, discrete events within a well, for example, for imaging fluorescently tagged antibodies binding to receptors on the surface of a cell for high-throughput cell-based screening. In some embodiments, high-density sequence detection system  10  is not limited to imaging only microplate  20  but can be used in the imaging of gels, blots, nitrocellulose membranes, and the like with features at high-density. 
     In some embodiments, high-density sequence detection system  10  can image microplates, nitrocellulose membranes, gels, films, blots, and the like. Detection can be, in some embodiments, for isotopic changes, chemiluminescent emissions, chemifluorescent emissions, fluorescent emissions, calorimetric changes, and time-lapse studies of any of the above detection methods. In some embodiments, high-density sequence detection system  10  can be used as a spectrophotometer or spectrofluorometer for samples contained in microplate  20 . For example, high-density sequence detection system can be used for methods for the measurement and/or analysis of absorbance (UV-Vis-NIR) by adding a detector to opposite side from excitation side of microplate  20 ; for methods for the measurement and/or analysis of fluorescence intensity; for methods for the measurement and/or analysis of fluorescence polarization by adding at least one polarizing filter to detection system  300 ; or for methods for the measurement and/or analysis of time resolved fluorescence. In some embodiments of high-density sequence detection system  10  can be modified to increase read out speed of CCD pixels. In some embodiments, high-density sequence detection system  10  can be used for methods for the measurement and/or analysis of luminescence. In some embodiments, high-density sequence detection system  10  can be used for time-limited chemiluminescent reactions and in such embodiments, high-density sequence detection system  10  can be modified to manipulate reagents in microplate  20  to begin the reactions. 
     Isothermal Amplification 
     According to some embodiments, high-density sequence detection system  10  can be used to perform various isothermal procedures in, for example, the areas of molecular diagnostics, genotyping, gene expression monitoring, and drug screening. Such isothermal procedures can include, for example, those useful in genetic, biochemical, and bioanalytic processes, such as processes for detecting a target DNA, processes for detecting a mutation, processes for detecting a polymorphism, processes for detecting a single base insertion or deletion, and for processes for identifying SNPs. In some embodiments, the high-density sequence detection system  10  can be used to perform isothermal amplification according to U.S. Pat. No. 6,692,917. 
     In some embodiments, processes for identifying SNPs can include, for example, assays for single-base discrimination and/or quantitative detection of DNA or RNA sequences, for example, SNPs and mutations (single base changes, insertions or deletions in DNA and RNA molecules), from samples containing genomic DNA, total RNA, cell lysates, purified DNA, purified RNA, or nucleic acid amplification products, for example, PCR or RT-PCR products. Other assays that can be carried out using high-density sequence detection system  10  of the present teachings include the processes and methods taught in U.S. Pat. No. 6,692,917. 
     In some embodiments, the assays can be performed using a high-density sequence detection system  10  wherein assay  1000  comprises reaction components, including, for example, the first oligonucleotide, the detection probe, or both the first oligonucleotide and the detection probe. In some embodiments, such components can be attached to microplate  20 , directly or through a spacer and/or linker molecule, including for example, a carbon chain, a polynucleotide, biotin, or a polyglycol. In some embodiments, the assays can be performed alone or in combination with nucleic acid amplification assays, including for example, standard or multiplex PCR. 
     Protein Assays 
     In some embodiments, high-density sequence detection system  10  can be used to detect the binding activity of primary antibody reagents as direct labeled conjugates or indirect conjugate forms, for example, conjugate enzymes or conjugate Quantum Dots (Qdots). Cells from a variety of sources can be used including in vitro tissue culture and peripheral blood leukocytes. In some embodiments, binding events can be detected or imaged from microplate  20 , or alternatively, on nitrocellulose membranes with high-density separation channels and/or bands, for example, using a Western blot technique. In some embodiments, when using a Western blot, one protein in a mixture of any number of proteins can be detected while also providing information about the size of the protein and such information can indicate how much protein has accumulated in cells. 
     Referring to an illustrative example, first proteins are separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates the proteins by size. Nitrocellulose membrane is placed on the gel and the protein bands are electrokinetically transported onto the nitrocellulose membrane. This results in a nitrocellulose membrane imprinted with the same protein bands as the gel. The nitrocellulose membrane is then incubated with a primary antibody made by inoculating a rabbit and diluting the antisera (from blood). The primary antibody sticks to the protein and forms an antibody-protein complex with the protein of interest. The nitrocellulose membrane is then incubated with a secondary antibody, an antibody enzyme conjugate. The secondary antibody is an antibody against the primary antibody and has the ability to stick to the primary antibody. The conjugate enzyme can comprise a molecular flare stuck onto the antibodies so they can be visualized. The enzyme is incubated in its specific reaction mix resulting in bands wherever there is a protein-primary antibody-secondary antibody-enzyme complex such as wherever the protein of interest is located. In some embodiments, high-density sequence detection system  10  can be used to detect a flash of light that is given off by the enzyme and, in some embodiments, detection system  300  of high-density sequence detection system  10  can be customized for the particular conjugated labels. 
     By way of example in some embodiments, Green Fluorescent Protein (GFP) is extracted from  Aequorea Victoria . GFP is a small protein (about 27 Kd) and the DNA sequences coding for GFP can be manipulated by recombinant DNA technology to create gene fusions between GFP and any protein of interest. Such DNA constructs can then be introduced into living cells to express the GFP fluorescent tags on the protein of interest. The GFP fluorescent tag can be used to localize a protein of interest to a specific cell type and/or subcellular localization in living cells and organisms. In some embodiments, high-density sequence detection system  10  optics can be modified to enable 2-40× magnification of individual wells or a small number of wells, adding an x-y stage and adding z-axis autofocus. In some embodiments, high-density sequence detection system  10  can be used to perform GFP-based protein localization assays using microplate  20 . In some embodiments, for gene expression, the GFP DNA coding sequence can be placed behind a promoter and/or regulatory DNA sequence of interest, and introduced into cells and this can be used to perform promoter studies in living organisms. 
     In some embodiments, fluorescence resonance energy transfer (FRET) assays can be used to determine the exact time and place of colocalization. Energy transfers from the excited fluorophore to the nearby acceptor fluorophore. In some embodiments, donor and acceptor molecules are less than 10 nm apart and the emission spectra of the donor fluorophore overlap the excitation spectra of the acceptor fluorophore. The farther apart the molecules are, the weaker the transfer energy. Extremely low light levels require, in some embodiments, a highly sensitive cooled CCD with high quantum efficiency and fast readout rates. FRET images can be taken at different wavelengths. In some embodiments, high-density sequence detection system  10  can be modified to perform FRET assays in microplate  20 . High-density sequence detection system  10  optics can be modified to enable magnification (e.g., 2-40×) of individual wells or a small number of wells, adding an x-y stage, and adding z-axis autofocus. In some embodiments, high-density sequence detection system  10  can be used to perform FRET assays using microplate  20 . In some embodiments, high-density sequence detection system  10  can produce a series of time lapse images for FRET. 
     Assays Using QDots As Labels 
     Quantum dots (QDots) are fluorescent nanoparticles made of inorganic molecules, for example, CdSe and an emission wavelength of a QDot is determined by its physical size. In general, QDots have large stokes shifts, with excitation wavelengths on the order of 408 nm and emission wavelengths starting at around 520 nm and In some embodiments, Qdots can have greater photostability, greater spectral separation, and brighter emission relative to organic fluorescent dyes. It is possible to label, or conjugate QDots to molecules of interest for molecular biology assays, such as antibodies. Further, mixtures of QDots can be employed to provide multiplexing capability. Some embodiments include the use of beads coated with different QDot nanocrystals to detect gene expression levels. For example, 9 μm paramagnetic beads can be coated with mixtures of QDot nanocrystals. Unique spectral codes can be created using four different fluorescent colors of QDot nanocrystals coated onto the beads at defined ratios. Then an outer protective coat can be applied and cross-linked. In some embodiments, gene-specific oligonucleotide probes are conjugated to the bead surface and each gene-specific bead can be identified by its unique QDot nanocrystal spectral code. Gene-specific beads can be combined to form custom gene panels. In some embodiments, many beads of each different type are added to each well  26  with the different bead types having been coated with the spectral code corresponding to the different target DNA. 
     Referring to an illustrative example, total RNA is isolated from cells or tissue and the sample can then be labeled with biotin. Unbound biotin can be separated from the biotynilated-sample complex by washing, size exclusion, or any of a number of other well-known processes. The cleanly separated biotin labeled sample can then be added to the bead mixtures in microplate  20  and allowed to hybridize to the beads. A reporter can be created by attaching streptavidin to a fifth QDot nanocrystal label. Unattached streptavidin can be separated from the QDot labeled streptavidin in a manner similar to that used for separating the unbound biotin, as before. Cleanly separated streptavidin can then be added to the mix. This fifth QDot (the reporter) provides quantitative information on gene expression. The QDot nanocrystal-labeled streptavidin can bind to the biotinylated targets. To separate any unbound, non-specific biotin and streptavidin, another wash step, or size exclusions step, can be added to separate them from the biotin-streptavidin complexes (sample-biotin to bead-oligo-streptavidin complex). Alternatively, the beads can be allowed to settle to the bottom of wells  26  of microplate  20 , which is then imaged. For example, QDots have been linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. In some embodiments, each bead can be identified by reading its spectral code and can quantify the amount of target hybridized to each coded bead. In some embodiments, high-density sequence detection system  10  can be optimized for the excitations and emissions of QDots. In some embodiments, with the multiplexing capabilities afforded by spectral codes, a whole genome gene expression analysis can be completed on a microplate  20 . 
     Cellular Assays 
     In some embodiments, with the addition of humidity control and CO 2  to the existing temperature control-chamber, high-density sequence detection system  10  can accommodate live cell assays in microplate  20 . In some embodiments, high-density sequence detection system  10  is modified to comprise magnification (e.g., 2-40×) and an x-y stage. In some embodiments, throughput can be increased by imaging more than one well at a time, with lower resolution and/or lower magnification images. 
     In some embodiments, using a lower magnification and/or image resolution, high-density sequence detection system  10  can simultaneously read multiple wells in real time. This can be useful, for example, for optimizing assay conditions and determining dose response curves. In some embodiments using microplate  20 , more such assays can be run in shorter time leading to better optimizations and more accurate IC 50  value determinations. 
     In some embodiments, microplate  20  can be modified using coatings, activations, and the like to make it more amenable to a particular assay. For example, for growing and staining adherent cells, for example, high protein binding (affinity to molecules for hydrophobic and hydrophilic domains—high binding of antibodies), and for low binding capacity (affinity to molecules of hydrophobic domains). 
     In some embodiments, high-density sequence detection system  10  comprising microplate  20  can be used to analyze cell differentiation such as identifying morphological changes following membrane dye incorporation; analyze cell cycle employing the detection of G1, S and G2/M phases of a cell cycle; determine mitotic index by detection using antibodies to identify M-phase specific marker; identify cell adhesion by detecting attachment and morphology; or monitor colony formation by detecting the enumeration of one or more colonies. In some embodiments, high-density sequence detection system  10  comprising microplate  20  can be used to study slow ion channels by employing, for example, detection of ion flux fluorescent DiBAC4(3) reporter. In some embodiments, high-density sequence detection system  10  comprising microplate  20  can be used to study protein kinase by using standard antibody methods; study translocation by identifying movement of proteins between plasma membrane, cytoplasm, and the nucleus; study fluorescent proteins such as EGFP and Reef Coral Fluorescent Protein in multiplex assays; identify quantum dots using limited spectral overlap from distinct conjugates; or to study cell based screening such as data lactamase, adipogenesis, hybridoma, expression cloning and/or lectin binding. In some embodiments, high-density sequence detection system  10  comprising microplate  20  can be used to study G-protein coupled receptors. In such embodiments, the membrane proteins are encoded by about 20% of genes and most organisms and are critical for cellular communication, electrical and ion balances, structural integrity of cells and their adhesions, as well as other like functions. In some embodiments, high-density sequence detection system  10  can be used for the analysis of DNA/RNA/protein quantitation and purity; PicoGreen/Nanoorange and Bradford assays; analysis of ELISA and/or enzyme kinetics; analysis of drug dissolution profiles; analysis of caspase-3 and protease assays; analyzing Catch Point cAMP assays; analysis of IMAP kinase assays; analysis of intrinsic tryptophan fluorescence; analysis of membrane permeability assays; analysis of FluoroBlok cell migration assays; analysis of delfia assays; analysis of immunohistochemistry; analysis of tissue staining; analysis of hybridization arrays; or analysis of amino assay. 
     Dielectric Spectroscopy of Molecular Biology Assays 
     In some embodiments of high-density sequence detection system  10 , an electrically conductive circuitry can be added to microplate  20  to transform a plurality of wells  26  into resonant cavities. In some embodiments, a terminal antenna can be placed in close proximity to a sample in each of the plurality of wells  26 , such as a coplanar waveguide device. Such circuitry can deliver electrical signals in the Hz-GHz frequency ranges, for example in the microwave ranges, to the samples. In some embodiments, an electrical connector can be added to microplate  20  in order to connect it to the generated and measured electrical signals from external sources, such as an Agilent vector network analyzer. Such a system can be used to measure changes in the dielectric properties of the samples contained in the plurality of wells  26  of microplate  20 . Examples of events that cause changes in dielectric properties, which can be detected or monitored by such a system, include monitoring cell growth and/or death, detecting DNA hybridization, detecting protein-protein and protein-small molecule interactions, detecting protein conformational changes, detecting ion channel flux in cells, and monitoring bulk properties such as pH, and salt concentration. 
     Monitoring Surface Plasmon Resonance in Real-time 
     In some embodiments of high-density sequence detection system  10 , microplate  20  can be modified to have an electrically conductive thin layer which can be, for example, gold, on bottom wall  36  of plurality of wells  26 . In some embodiments, surface plasmon resonance (SPR) can occur when polarized light incident at an angle for total internal reflection strikes the electrically conductive layer at the interface between media of different refractive index, for example, microplate material with high refractive index and the assay  1000  with low refractive index. In some embodiments, an evanescent wave of electric field intensity can be generated and interacts with (is absorbed by) free electron clouds in the gold layer. In some embodiments, this interaction can generate electron charge density waves called plasmons and can cause a reduction in the intensity of the reflected light. High-density sequence detection system  10  can be modified to illuminate microplate  20  with incident polarized light covering a range of incident angles. In some embodiments with further modifications, high-density sequence detection system  10  can measure reflected light at different angles of transmission from microplate  20 . In some embodiments, the resonance angle at which the intensity minimum occurs can be a function of the refractive index of the solution close to the gold layer, for example, a biological sample flowing over the gold layer in the plurality of the wells  26  of microplate  20 . In some embodiments, modified high-density sequence detection system  10  can be used to detect SPR analysis such as protein interactions, small molecule (drug candidates) interactions with their targets, membrane-bound receptor interactions, DNA and RNA hybridization, interactions between whole cells and viruses, recognition of cell surface carbohydrates and molecular interactions, such as binding and dissociation. 
     Determining Presence of Specific DNA Oligonucleotide Sequences Using Bioelectronic Detection 
     In some embodiments, high-density array of gold electrodes can be incorporated into microplate  20 . In some embodiments, capture probes and signal probes can be designed and manufactured for a specific target DNA. In some embodiments, capture probes can be coated onto the gold electrodes forming a monolayer on the gold surface. In some embodiments, signal probes can be tagged with ferrocenes. In some embodiments, the target DNA can be amplified by PCR and when added to the monolayers on the gold electrodes, specific target DNA can hybridize to the capture probe. An electrochemical signal can be generated when the amplicon hybridizes to the capture probe and the ferrocene-labeled signal probe, thereby bringing a reporter molecule, ferrocene, into contact with the monolayer on the gold electrode. In some embodiments, an AC voltammogram is obtained when the specific target DNA is detected in a sample, but no electronic signal is registered when the specific target DNA is absent from the sample. 
     Optical Planar Waveguides 
     In some embodiments, microplate  20  can comprise a high-density array of planar waveguides to selectively excite only fluorophores located at or near the surface of the waveguide. The waveguide can be constructed by depositing a high refractive index material onto a low refractive index material. In some embodiments, a parallel laser light beam is coupled into the waveguiding film by a diffractive grating which is etched into the substrate material of microplate  20 . In some embodiments, the light propagates within the waveguiding film and creates a strong evanescent field perpendicular to the direction of propagation into the adjacent medium, for example, one of plurality of wells  26  in microplate  20 . In some embodiments, the field strength of the evanescent wave can decay exponentially with distance, so only fluorophores at or near the surface are excited. In some embodiments, selective detection of DNA hybridization, immunoaffinity reactions, and membrane receptor based assays can be analyzed using microplate  20  comprising a high-density array of planar waveguides. 
     Microplate Applications for Localized Heating, Gradient Thermocycling 
     In some embodiments, microplate  20  can comprise heat generating electronics and such electronics can be associated with, or in proximity to, one or more of plurality of wells  26  in microplate  20 . In some embodiments, temperatures in a plurality of wells  26  or subsets thereof can be controlled to create a gradient thermocycler. In some embodiments, microplate  20  comprising heat generating electronics can be used, for example, to determine optimum assay parameters such as oligo melting point temperatures and/or can be used to improve synchronization of thermal cycling with detection system  300  in high-density sequence detection system  10 . In some embodiments, when detection system  300  is limited to reading only a portion of microplate  20  at a time, thermal cycling reactions can be started or stopped selectively by use of microplate  20  comprising heat generating electronics to correspond with optical detection. 
     Portals 
     In some embodiments, a web-based user interface can be provided that comprises a web-based gene exploration system operable to provide information to assist a user in selecting one or both of a stock assay and a custom assay. In some embodiments, the web-based gene exploration system can comprise a search function operable to identify genetic material based on a portion of known data. The search function can provide one or more parameters identifying gene structure or function for selection by the user. 
     In some embodiments, systems are provided comprising a web-based user interface configured for ordering stock assays and/or requesting custom designed assays. Such assays can then be delivered to the user. In some embodiments, such assays are configured to detect presence or expression of genetic material. Assays that detect the presence or expression of genetic material can comprise assays for detecting SNPs or for detecting expressed genes. In some embodiments, the web-based user interface can be configured to receive criteria related to the SNP or to the expressed transcript for which an assay is ordered. Such methods, kits, assays, web interfaces, and the like are disclosed in U.S. Patent Application Publication No. 2004/0018506 to Koehler et al.