Abstract:
A system and method provides for chemical and/or biochemical analysis using a microarray interrogated by a resonantly scanned beam of light.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This patent application claims priority benefit from U.S. Provisional Patent Application Ser. No. 60/687,292, entitled METHOD AND APPARATUS FOR SCANNED BEAM CHIP ASSAY, invented by Minhua Liang et al., filed Jun. 3, 2005. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present disclosure relates to methods and apparatuses for conducting light-addressed assays of microarrays, and more particularly to methods and apparatuses for scanned beam interrogation in microarray-based assay systems.  
       BACKGROUND  
       [0003]     Various technical papers and other publications document methods and apparatuses for conducting optical assays. These include, “Surface Plasmon Resonance Sensors: Review,” by Jiri Homola et al.; “Sensors and Actuators B 54” (1999), Elsevier; “Present and Future of Surface Plasmon Resonance Biosensors,” by Jiri Homola; “Anal Bioanal Chem” (2003); and “Biomedical Photonics Handbook,” Tuan Vo-Dinh, editor-in-chief, CRC Press (2003); all incorporated by reference herein.  
         [0004]     The present disclosure provides improvements over the prior art.  
       OVERVIEW  
       [0005]     According to an illustrative embodiment, a beam of light is scanned across a microarray surface, and the characteristics of light reflected, refracted, and/or emitted from the surface are measured. The microarray surface includes one or more chemical reagents bound to a surface, referred to as a surface reagent. A test fluid is allowed to flow across the microarray surface and one or more components, solutes, etc. are captured by the surface reagent. The captured solutes, which may be tagged or untagged, cause the surface of the microarray to interact with incident light in a characteristic manner. A detector receives light reflected or emitted from near the surface of the microarray. A controller analyzes the received light and determines one or more characteristics thereof. The controller then correlates the one or more characteristics to the chemical or biological attribute of the test fluid. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0006]      FIG. 1  is a diagram of a prism coupler-based surface plasmon resonance system according to an embodiment.  
         [0007]      FIG. 2  is a diagram of a grating coupler-based surface plasmon resonance system according to an embodiment.  
         [0008]      FIG. 3A  is a cross-sectional diagram of a microarray system responsive to plural patterned surface reagents for selectively attracting the entities from a fluid according to an embodiment.  
         [0009]      FIG. 3B  is a diagram of an interrogation beam scan pattern across a field of view and across a microarray supported within the field of view according to an embodiment.  
         [0010]      FIG. 4  illustrates the attraction of tag and non-tagged entities to a surface reagent according to an embodiment.  
         [0011]      FIG. 5  is a block diagram of the scanned beam interrogation system according to an embodiment.  
         [0012]      FIG. 6  is a block diagram according to an alternative embodiment that uses a non-imaging detector.  
         [0013]      FIG. 7  is a block diagram according to an alternative embodiment that uses a converging input beam.  
         [0014]      FIG. 8  is a block diagram according to a multiple wavelength excitation and detection according to an embodiment.  
         [0015]      FIG. 9  is a block diagram of a variant of the embodiment represented by  FIG. 8  that uses a non-imaging detector according to an embodiment.  
         [0016]      FIG. 10  is a block diagram of a variant of  FIG. 8  using converging input beams according to an embodiment.  
         [0017]      FIG. 11  illustrates a laboratory apparatus for carrying out an embodiment.  
         [0018]      FIG. 12  is a view of a beam scanner assembly according to an embodiment.  
         [0019]      FIG. 13  is a view of a test cell module according to an embodiment.  
         [0020]      FIG. 14  is a view of a focal plane camera showing its relationship to a collection lens according to an embodiment.  
         [0021]      FIG. 15  is a plot of intensity vs. time for a single pixel change in intensity arising from a change in refractive index of the dielectric according to an embodiment.  
         [0022]      FIG. 16  is a plot corresponding to  FIG. 21  where 64×64 pixels were averaged to provide the change in intensity according to an embodiment.  
         [0023]      FIG. 17  is a flow chart showing a process for calibrating system response according to an embodiment. 
     
    
     DETAILED DESCRIPTION  
       [0024]     The art often refers to phenomena involved in prior assay systems as surface plasmon resonance (SPR). There may be some debate within the scientific community as to the precise physics involved in these and similar systems. In the interest of clarity, this disclosure generally uses the term surface plasmon resonance as a generic term to refer to such systems and phenomena, even though other physics may be at work. It will be understood that the teaching herein extends to alternative systems.  
         [0025]      FIG. 1  shows a prism coupler-based surface plasmon resonance system.  FIG. 2  shows a grating coupler-based surface plasmon resonance system. In each case, and interrogation beam  102  impinges upon a metal layer  104  of a microarray  101 . Metal layer  104  may be formed from gold or silver for example. As is known to the art, the relative tendency of the impinging beam to be guided along the layer in a manner approximating total internal reflection vs. reflecting off the layer depends upon the ratio of the index of refraction of the metal layer to the dielectric layer  304 . In turn, the index of refraction of the dielectric (surface reagent) layer  304  depends upon the concentration of test entities held by the surface reagent. Referring to  FIG. 1 , the strength of the response beam  106  (according to certain interpretations of the physics) depends on the ratio of the index of refraction of the metal layer  104  to the index of refraction of the dielectric (surface reagent) layer  304 , and in turn on the concentration of test entities held by the dielectric (surface reagent) layer  304 . The structure of the surface reagent layer  304  will be explained more fully below. Referring to  FIG. 2 , diffraction orders  106   a ,  106   b , and  106   c , respectively the zeroeth, −1, and +1 orders, are selectively reflected from the grating surface corresponding to the concentration of test entities on the surface. While only three refraction orders are shown, additional orders may be present.  
         [0026]      FIG. 3A  is a simplified partial cross-sectional diagram of a microarray  101  and test fluid  108 . The microarray  101  includes a substrate  302  and a metal layer  104  that has a surface reagent layer  304  comprising two types of surface reagents  306  and  308  for selectively attracting respective entities  312  and  314  from a fluid. In one exemplary embodiment the metal layer is approximately 50 nm thick gold. The interrogation beam  102  is transmitted through the transparent dielectric substrate  302  and impinges on the top side of gold layer  104 . A layer of one or more surface reagents  306 ,  308  coats the bottom surface of metal layer  104  to form the surface reagent layer  304 .  
         [0027]     According to some embodiments, the surface reagent molecules may include an anchoring end configured to bond to the surface of the microarray, a conductive link that may for example comprise an alternating single-, double-bond carbon chain coupled to the anchoring end, and a reactive end coupled to the conductive link. The reactive end may be selected to provide preferential coupling with a test entity.  
         [0028]     The test fluid  108  flows past the surface reagent layer  304  as indicated by the arrow  310 . In this example, the test fluid  108  includes two types of test entities  312  and  314 , which may for example be proteins, DNA strands, other molecules, etc. In the example of  FIG. 3A , two types of surface reagents  306  and  308  are shown patterned onto the surface of metal layer  104  to form the surface reagent layer  304 . The surface reagent molecules identified as  306   a ,  306   b ,  306   c , and  306   d  are selected to attract a first type of entity  312 . As shown in  FIG. 3A , surface reagent molecules  306   a ,  306   b , and  306   d  are filled by respective entities  312  and surface reagent molecule  306   c  is empty. Similarly, surface reagent molecules identified as  308   a - d  are selected to attract test entities  314  from test fluid  108 . Surface reagent molecules  308   a  and  308   d  are shown filled with test entities  314  and surface reagent molecules are shown empty. According to some embodiments, surface reagents may be selected to fill proportionally to the concentration of respective test entities in the test fluid  108 . Accordingly, the ratio of filled sites of surface reagent  306  to filled sites of surface reagent  308  (optionally when modified by one or more conversion factors), may be proportional to the relative concentrations of test entities  312  and  314  in the test fluid  108 . Conversion factors may include compensation for relative attraction (e.g. electro-negativity ratios), diffusivity, geometric factors, temperature, flow rate, Reynolds number, miscibility, etc. that may change the particular relationship between concentration and proportion of filled surface reagent sites.  
         [0029]     While the microarray  101  is held in a field-of-view (FOV), the interrogation beam  102  is scanned across the microarray in a scan path  110 . A portion of the incident interrogation beam  102  may be reflected, scattered, emitted, etc. as a response beam  106 . According to some theories, the tendency for reagent molecules  306 ,  308  to resonate at the interrogation wavelength may be modified by the presence or absence of captured test entities. The relative resonance of the reagent molecules affects the transmission, reflection, etc. of the response beam  106 . According to some theories, whether surface reagent sites  306 ,  308  are filled or unfilled determines that index of refraction of the system, and accordingly determines the strength of the response beam  106 . Thus, the intensity of the response beam  106  may be proportional to the proportion of filled sites within the traversed portion of the microarray.  
         [0030]     In addition to or alternatively to using the intensity of the response beam  106  to measure the state of the reagent sites  306 ,  308 , other electromagnetic response characteristics may be measured to provide a measure of surface concentrations. For example interrogation beam wavelength sensitivity, wavelength shift between the interrogation and response beams, polarization state, etc. may be detected and correlated to surface concentrations.  
         [0031]     As described more fully below, a microarray substrate  302  may be loaded with one or a plurality of types of surface reagents  306 ,  308 . Alternatively, the concentration, relative activity, etc. of a particular surface reagent may be modified across the surface reagent layer  304 . Such varying surface reagents may be patterned across the surface to provide a broader spectrum of detection, greater dynamic range, greater sensitivity, greater specificity, etc. Interfaces between particular surface reagents  306 ,  308  may be selected to provide preferential coupling to different parts of particular or related molecules. Such an approach may be used to provide specificity to isomers, bring reactants into a desired proximity and/or orientation, etc.  
         [0032]      FIG. 3B  illustrates a scan path  110  across a microarray  101  held in a field-of-view  316 . In the illustrative embodiment, the scan path may be a two-dimensional pattern sequentially scanned by the interrogation beam. The 2D pattern may be scanned in a periodically repeating manner to provide successive frames of response from the microarray. According to some embodiments, the frames may be repeated at a video rate such as 60 Hz to provide dynamic detection of changes in surface concentration of test entities. In embodiments where the microarray is patterned with cells  320  of varying surface reagent properties (several of whose positions are indicated), beam location may be correlated to a set of test criteria associated with the surface reagent property in the location.  
         [0033]     The microarray  101  is shown bearing an indicia  318  that may be scanned by the interrogation beam. According to some embodiments, the indicia may be a linear or 2D bar code symbol that provides orientation, calibration, and/or other characterization data for the microarray. According to some embodiments, a single-width, variable placement symbology such as BC  412 , etc. may be used.  
         [0034]     The symbol may directly encode orientation, calibration or characterization data. Alternatively the symbol may encode a serial number, lot code, etc. that may be used as an access code to lookup relevant orientation, calibration or characterization data.  
         [0035]     The scan pattern or path  110  is illustrated as a pattern corresponding to a resonant bi-directional horizontal scan superimposed over a smooth vertical ramp. Accordingly, raster pinch may be seen at the lateral extremes of the scan pattern. The microarray  101  is illustrated as occupying a portion of the field of view  316  with over-scan regions at each lateral extreme as well as optional over-scan regions at each vertical extreme. Provision for such over-scan regions may be desirable to reduce the degree of raster pinch and hence deviation from parallel horizontal scan lines. Additionally or alternatively, interpolation, correction scanning, etc. may be included to provide a respective virtual or real scan pattern that more nearly approaches straight horizontal scan lines.  
         [0036]     One or more calibration patches  322  may be included. The calibration patches may be configured to maintain a constant beam response such as, for example, by excluding surface reagents or providing non-reactive surface reagents therein. As will be explained later, the calibration patches may be used to calibrate system response such as, for example, variations in illuminator power output. Such calibration may be used to reduce noise and provide more accurate tracking of variations in the system and therefore more accurate determination of test patch response.  
         [0037]     The scan pattern  110  provides sequential addressing of the surface of the microarray  101 . Thus a sequentially addressed, non-imaging detector may be used to detect the response beam  106 , with spatial information being provided by the time sequential pattern of the scanned beam.  
         [0038]      FIG. 4  illustrates the attraction of tagged and non-tagged entities to a surface reagent. As with the example of  FIG. 3 , substrate  302  supports a metal layer  104  which holds surface reagents  304 . As the test fluid  108  flows past, the surface reagent molecules attract respectively tagged and untagged entities  402  and  312 . As shown, tagged entities  402  are bound or otherwise associated with a tagging entity  404 . In the example of  FIG. 4  and elsewhere herein, tagging entities  404  may be photoluminescent entities adapted to absorb a first wavelength of light and responsively emit a second wavelength of light. As will be shown below, one aspect of some embodiments is the ability to simultaneously or in rapid succession determine the presence or absence of test entities  312 / 410  on surface reagent  304  and the presence or absence of tags  404 .  
         [0039]     According to some embodiments, photoluminescence may be used as a proxy for SPR interrogation. That is, the photoluminescence of the fluorescent tags may be used to determine the presence/absence/concentration of entities to which the tags are affixed. Similarly, in addition to responses to an incident light beam, chemiluminescence may be determined.  
         [0040]      FIG. 5  is a block diagram of a scanned beam interrogation system according to an embodiment. A laser scanner module  502  includes a laser driver  504  that drives a first laser  506 . A beam of light  508  emitted by the first laser  506  is shaped by beam shaping optics  510  and impinges upon a scan mirror  512 . According to some embodiments, scan mirror  512  may be a MEMS scanner that is driven by a MEMS driver  514  also present in the laser scanner module  502 . According to exemplary embodiment the laser  506  emits coherent light at 658 nm wavelength at a power range of 0 to 25 milliwatts.  
         [0041]     The scanner module  502  is aligned with the test cell module  516  such that the scanned light beam  518  (shown in three illustrative positions) is collimated by a lens  520 . A linear polarizer  522  polarizes the light to couple with the microarray (described below) in TM (or P) mode. Sequentially scanned parallel beams of light  102  enter a coupling prism  524  and impinge upon the surface of the microarray  526  as described and shown above. The test fluid space  528  has provision for flow of a test fluid from an input  530  to an output  532 . According to some embodiments a plurality of inputs and outputs are used. According to an illustrative embodiment, the microarray includes a plurality of test patches  320   a ,  320   b , and  320   c , each of which may be loaded with a different particular surface reagent. As described above, impinging beams  102  are reflected or absorbed by regions of the metal/dielectric corresponding to the test cells on the microarray according to the presence or absence of test entities in surface reagent sites. In surface plasmon resonance mode, impinging light rays  102  are reflected (or not reflected) as reflected rays  106  through the coupling prism  524  to a collecting lens  536 . Similarly, photoluminescent emission from test cells  320   a ,  320   b , and  320   c  may also output at least a portion of its energy to collecting lens  536 .  
         [0042]     Test patches  320  may be reduced to a single patch, may be arranged in a one-dimensional pattern such as a series of stripes, may be arranged in a 2D rectilinear or “checkerboard” pattern, or in other patterns. The microarray may be aligned with its test patches facing toward the light beams  102  as shown, or alternatively may be turned to face in the opposite direction such that the light beams first pass through a transparent substrate. In the latter case, the substrate may be made separate or integral with the coupling prism  524 . A range of test patch sizes may be used according to the requirements of a given application. For example, a single-patch microarray may be arranged with the single patch imaged across the entire field of view of the scanner module  502  and detector/controller module  538 . This may be useful, for example, when adsorption or reaction rate data is desired to be collected (for which some embodiments may be used). Alternatively test patches corresponding to a single pixel may be used. Patch sizes of 1×1 (pixel), 2×2, 4×4, 8×8, 16×16, 32×32, and 64×64 have been simulated from data taken from a single, multiple pixel (covering substantially the entire field-of-view) cell microarray. Additionally, a square aspect ratio need not be maintained.  
         [0043]     The collection optic  536  is configured to focus the response beams  106  onto the focal plane array  540 . In this case, it may be appropriate to align the focal plane detector  540  parallel to the collection optic and to select focal distances such that the distance from the collection optic  536  to points on the microarray  526  corresponding to the test patches  320  falls within the depth-of-field of the system. According to some embodiments, the focal length of the collection optic  536  may be varied dynamically to keep the current location of the interrogation beam  102  in focus. According to some embodiments, a scattering screen, photoluminescent (PL) screen, etc. may be formed on the output face of the coupling prism  524  such that the collection optic  536  focuses the scattered image from the coupling prism output face onto the focal plane detector  540 . While the collection optic  536  is simplified to show a single lens, it may include multiple elements including elements such as lenses, mirrors, and diffractive elements.  
         [0044]     According to some embodiments, the scan pattern of the scanner module  502  may be varied as a function of aspects of the microarray  526  and the test cell or cells  320  thereon. For example, fiducials, bar codes, etc.  318 , as shown in  FIG. 3B , may be formed across or around the periphery of the microarray for providing accurate alignment, calibration and/or characterization information about a particular microarray. Characterization traits that may be determined include the arrangement and type of test patches, the spacing of the test patches, the spectral response characteristics of the test patches, the coupling length of the test patches, the surface reagent characteristics of the test patches, etc. The detector and controller systems (described below) determine the characterization, calibration, and/or alignment of the microarray and may responsively drive the scanner module  502  to a modified scan pattern. Alternatively, the scan pattern may be maintained substantially constant while the sampling of the detector system (described below) is modified to allow for microarray characteristics. Alternatively, the characterization data may be used to select conversion data in the software program  548  described below, log inventory or use of a microarray, or act as input to provide other functions.  
         [0045]     As indicated by the example of  FIG. 5 , a scan mirror  512  sequentially scans output beam  518  and hence interrogation beam  102 . That is, the beam  102  is scanned onto test patch  320   a  at a first instant in time, and onto test patches  320   b  in  320   c  that other instants in time. As shown, the surface state of test patches  320   a  and  320   c  is such that substantially no response beam  106  is produced, while the surface state of test patch  320   b  cooperates with the incoming interrogation beam to create a response beam  106 . While this simplified response may be substantially the manner in which a given microarray responds under a set of conditions, it may also be the case that some amount of response beam is present from each test patch with the relative attenuation varying according to the (empty or full) state and proportion of the surface reagent molecules within each test patch.  
         [0046]     A detector/controller module  538  is aligned to receive light from the collecting lens  536 . Reflected beam  106  passes through collection lens  536  and impinges on a CCD, CMOS, or other focal plane detector  540 . A frame grabber  542  receives the image from the focal plane detector  540  and transfers it to memory  544 . A microprocessor or other central processing unit  546  controls memory  544  and frame grabber  542  and is configured to run a software program  548  that calculates the presence or absence and/or concentration of test entities in the test fluid  528 , decodes indicia on the microarray, and/or performs other functions. Controller  538  also includes control software  550  for controlling the laser driver  504  and the MEMS driver  514  within the laser scanner module  502 . Alternatively concentration, presence, or absence of entities may be calculated in a separate computer system.  
         [0047]      FIG. 6  illustrates an alternative detector system that uses a non-imaging detector  602  to capture the instantaneous light received from the test cells. As indicated above, the scan mirror  512  sequentially addresses the test patches  320   a ,  320   b , and  320   c  (here shown in an exemplary manner with the surface reagent facing the input beam). Thus according to some embodiments light from the test patches need not be imaged by focal plane detector. Rather, the timing of light received by the photodetector is correlated to the instantaneous mirror position to determine which test patch corresponds to the currently received light. A collection optic  536  may be used to increase the numerical aperture over which a scattered or reflect response beam  106  is collected. In some embodiments, the collection optic  536  may comprise a telecentric lens aligned to converge parallel response beams from across the microarray  526  onto the detector  602 .  
         [0048]      FIG. 7  is a block diagram according to an alternative embodiment wherein one or more lenses  520  is (are) operable to receive a sequentially scanned beam  518  and produce corresponding sequentially scanned converging beams  702 . In this way, a given test patch  320  on the microarray  526  receives light at a variety of input angles over successive instants in time. Light reflected at the respective instants in time  106  is imaged onto the focal plane detector  540  by collection optic  536 , or alternatively onto a non-imaging photodetector  602  (not shown). When using a non-imaging photodetector, the instantaneous beam position may be used to map the response from a variety of collection angles as described in conjunction with  FIG. 6 , above and the collected light may be collected over a relatively narrow aperture to determine the reflectance angle. The system of  FIG. 7  may be configured to determine the characteristics of the test patch as a function of impinging light angle. For example, as the index of refraction changes slightly, the angles of which detection rays  704  are reflected change. Thus, the intensity pattern of light moves across the surface of focal plane imager  540 . In addition to systems using a single lens  520 , a plurality of lenses, such as a microlens array for example, may be used. Such a system is operative to introduce a variety of input angles to a plurality of test patches. In such a case, it may be useful for the microlens array  520  to be made interchangeable to allow arrangements of lenses corresponding to a variety of arrangements of test patches  320  may be used.  
         [0049]     Alternatively, the microarray  526  may be stepped across the FOV as indicated by arrow  704  to sample reflection angles from a plurality of test patches  320  or from a plurality of locations on a test patch  320 .  
         [0050]      FIG. 8  is a block diagram according to a multiple or flexible wavelength embodiment of the system. According to the illustrative embodiment, the laser scanner module  502  may include a plurality of laser drivers  504 , here shown as an array, drive a plurality of lasers  506   a ,  506   b ,  506   c ,  506   d , and  506   e . The plurality of lasers produce a plurality of colored laser beams that are combined into a single beam  508  by a beam combiner  802 . According to the illustrative embodiment, laser  506   a  corresponds to an ultraviolet (UV) wavelength, laser  506   b  corresponds to a blue (B) wavelength, laser  506   c  corresponds to a green (G) wavelength, laser  506   d  corresponds to a red (R) wavelength, and laser  506   e  corresponds to an infrared (IR) wavelength. The input beams are introduced to a wavelength or beam combiner  802  and the beam combiner  802  produces a composite beam  508  containing the respective wavelengths at their respective powers. By driving the laser array, light at selected wavelengths may be made to impinge on the test patches  320 . Alternatively or additionally, one or more wavelengths may be selected to produce photoluminescence of tags as described above. As illustrated, reflected, photoluminescently emitted light, and/or chemiluminescently emitted light is imaged by focal plane imager  540 , or alternatively by a non-imaging photodetector as described above.  
         [0051]     According to an alternative embodiment, one or more wavelength agile lasers, which may optionally be combined with additional channels of fixed wavelength or variable wavelength lasers, may be driven to produce particular wavelengths of light.  
         [0052]     According to some embodiments a composite beam  508  is scanned by the MEMS scanner  512  to form a sequentially scanned interrogation beam  102  that includes the plurality of wavelengths. Alternatively, the lasers  506  may be sequentially driven to provide an interrogation beam that contains one wavelength while interrogating a given test patch  320   a . A different laser may then be driven during interrogation of a second test patch  320   b , etc. Alternatively, a plurality of wavelengths less than the total number of channels may be used to interrogate a given test patch  320 . Accordingly, an analysis of variance between wavelength response of test patches may be conducted with confounding of higher order interactions selected to improve system throughput.  
         [0053]     Alternatively, the plural or flexible laser(s) of  FIG. 8  may be used to provide other variations in the input beam. For example, the polarizer  522  after lens  520  may be omitted and the laser beams may be polarized prior to combining. Thus, a variety of beam polarizations may be introduced to the microarray. This may be used for example for experimental analysis and/or to accommodate slight misalignments in the system to maintain (for example) TM polarization at the metal surface  104 .  
         [0054]      FIG. 9  is a block diagram according to a variant of an embodiment of  FIG. 8  wherein an analyzer  902  is used to detect rotation of polarization imparted by the polarizer  522 . Polarization rotation may be affected by the state of the test patches  320 . Such a system may be operable with a single wavelength laser scanner module  502  or a multiple wavelength system as illustrated. In multi-wavelength systems, the amount of polarization rotation as a function of wavelength may be used to provide an interrogation modality.  
         [0055]      FIG. 10  illustrates a variant wherein a scanner module  502  includes plural wavelengths or wavelength agile beams that are focused by optical element  520  to converge on a test patch  320  as converging interrogation beams  702 . As with the illustrative embodiment of  FIG. 7 , an array of optical elements  520  may be used to produce an array of converging beam test points coincident with the microarray  526 . It may be advantageous for certain applications to substitute one or more reflective or diffractive optical elements for the lens  520 . Reflective optical elements in particular are known to minimize chromatic distortion and may be especially useful with a multiple wavelength system.  
         [0056]     Response beams  106  at various angles may simultaneously or sequentially include a plurality of wavelengths for detection by the detector  540  in the detector-controller module  538 . As with the example of  FIG. 7 , the test cell module  516  may include a mechanism for stepping the microarray  526  past the interrogation point to sequentially interrogate a plurality of test patches  320  or a plurality of spots on a test patch  320 .  
         [0057]      FIG. 11  illustrates a laboratory microarray interrogation apparatus according to an embodiment. The scanner module  502  is aligned to scan a beam through a lens  520  and polarizer  522  onto the test module  516 . Light is selectively reflected from the surface of the microarray according to principles described above. The selectively reflected light is collected by a collection lens  536  and imaged by a focal plane camera  540 . A computer monitor  502  displays an image of the microarray  1102  including a fiducial  1104  for determining alignment.  
         [0058]      FIG. 12  is a close-up view of the beam scanner assembly  502  aligned to scan a beam of light through a lens  520 .  
         [0059]      FIG. 13  is a close-up view of the test cell module according to an embodiment. The test cell module  516  includes a body  1302 , a coupling prism  524 , and fluid introduction and removal lines  1304 . Also shown is a polarizer  522  through which the interrogation beam is received according to some embodiments, and a collection lens  536  aligned to receive response beams from the microarray held by the body  1302  of the test cell module.  
         [0060]      FIG. 14  is a close-up view of the focal plane camera  540  showing its relationship to a collection lens  536  according to an embodiment.  
         [0061]      FIG. 15  is a plot of laboratory data taken with the system illustrated by  FIGS. 11-14 .  FIG. 15  shows the change in light intensity received by a single, particular pixel on the detector as a function of time. The time plotted corresponds to the addition of a dielectric index changing material through the flow lines to the test fluid.  
         [0062]      FIG. 16  is a plot of laboratory data corresponding to  FIG. 15 , but where the plotted data is the average of a 64 by 64 pixel region.  
         [0063]      FIG. 17  is a flow chart describing a method for calibrating system response that, according to an embodiment, may be performed by the software program  548  running in the controller, or optionally may be run by a computer used to record and/or analyze the results of a test. Laser gain curves, detector sensitivity, amplifier gain, and other system variables may change with time, temperature, power fluctuations, etc. Experiments have shown that laser gain may be the largest of such changes, especially when certain laser technologies such as second harmonic generation (SHG) lasers are used. Such changes may result in concentration-to-code relationships that are non-constant. Measurement of the system response to areas of known SPR may be used to compensate for such system variables.  
         [0064]     As described above and shown in  FIG. 3B , a microarray may include one or more calibration patches  322 . The calibration patches may for example include a non-reactive surface agent whose SPR response is not affected by concentrations in the test fluid. The calibration patches may be formed in one or more locations on the microarray. Alternatively or additionally, calibration patches may be formed in the field-of-view of the scanned beam outside the perimeter of the microarray such as in the overscan region. The relationships between calibration patches formed on the test cell module body and calibration patches on the microarray, between calibration patches formed on the test cell module body and test patches on the microarray, and/or between calibration patches formed on the microarray and test patches on the microarray may be used to provide system calibration. One or more of the test patches may provide a response corresponding to a zero response. Another test patch may provide a response corresponding to a full scale response such as a mirror surface for example.  
         [0065]     Referring to the flow chart  1701  of  FIG. 17 , the beam is scanned in a pattern and encounters a calibration patch and enters step  1702 . The response of the calibration patch is cached. Step  1702  may optionally include averaging a plurality of response pixels. After caching the response, the program enters step  1704 . According to some embodiments, step  1704  may involve simply overwriting a previously stored calibration value with the most recently cached value. According to other embodiments, step  1704  may involve applying a statistical algorithm such as updating the stored calibration value with an average or weighted average of the previously stored value and a recently determined value. Proceeding to step  1706 , the system determines whether a new beam position in the scan pattern corresponds to a calibration patch or to a test patch. If the beam position corresponds to a calibration patch, the program loops and returns to step  1702 . If the beam position corresponds to a test patch, the program proceeds to step  1708 .  
         [0066]     As mentioned above, a microarray and/or test cell module body may include a plurality of test patches. According to some embodiments, the location and/or size of test patches may be determined by reading an optical indicia on the microarray or on the microarray package.  
         [0067]     Proceeding to step  1708 , the interrogation beam is scanned over a test patch and the response measured. After caching the response, the program proceeds to step  1710 . In step  1710 , the code value measured in step  1708  may be modified according to the stored calibration value. For example, the measured value may be modified by dividing by the difference between the stored calibration value and a nominal value, wherein the nominal value represents the value that would be returned from the calibration patch if all system components were responding at their intended levels.  
         [0068]     I.e.:
 
Corrected Value=Measured Value/(Calibration Value−Nominal Value)
 
         [0069]     In this way, if the response of the system is higher than nominal, for example because the laser has output a brighter beam than nominal, the calibration value will be larger than the nominal value and the denominator will be greater than one. Dividing the measured value by the denominator will reduce the measured value to a corrected value that corresponds to nominal system response. The corrected value is then output to an output file, a computer monitor, a data plotting program, etc.  
         [0070]     After performing step  1710 , the program loops to decision step  1706  and again determines whether the currently illuminated point on the microarray corresponds to a test patch or a calibration patch. The program proceeds according to the state.  
         [0071]     The inclusion of a plurality of calibration patches may be used to determine systematic response of the system vs. beam position and/or provide multiple updates per image frame to account for system response variations across a range of frequencies.  
         [0072]     Aspects of the beam scanner module, detector module, and/or controller and operation thereof may be better understood by reference to one or more of U.S. Pat. No. 6,140,979 by Gerhard et al.; U.S. Pat. No. 6,151,167 by Melville; U.S. Pat. No. 6,245,590 by Wine et al.; U.S. Pat. No. 6,362,912 by Lewis et al.; U.S. Pat. No. 6,433,907 by Lippert et al.; and U.S. Pat. No. 5,629,790 to Neukermans et al.; all hereby incorporated by reference. This disclosure may be further understood by reference to one or more of U.S. patent application Ser. No. 10/873,540 by Wiklof et al.; U.S. patent application Ser. No. 10/984,327 by Sprague et al.; U.S. patent application Ser. No. 10/630,062 by Wiklof et al.; U.S. patent application Ser. No. 10/118,861 by Bright et al.; U.S. patent application Ser. No. 11/316,683 by Skumik et al.; and U.S. patent application Ser. No. 11/316,326, by Straka et al., all hereby incorporated by reference.  
         [0073]     The preceding overview, brief description of the drawings, and detailed description describe exemplary embodiments according to the present invention in a manner intended to foster ease of understanding by the reader. Other structures, methods, and equivalents may be within the scope of the invention. For example, methods and physical embodiments may be combined or used singly.  
         [0074]     Additionally, while terms such as “laser scanner module” and “laser emitter” have been used to describe embodiments, it is possible to provide a beam scanner that produces light from sources other than lasers. Such sources may include, for example, photoluminescent sources, incandescent sources, arc emission sources, light emitting diodes, etc. Similarly, lasers may include a number of different technologies include laser diodes, second harmonic generation lasers, gas lasers, etc. Accordingly, except where noted, terms such as “beam scanner module” and “beam source” should be regarded as functional and structural equivalents of corresponding terms such as “laser scanner module” and “laser source”.  
         [0075]     Further, while embodiments described herein refer to a modularity comprising a scanner module, a test cell module, and a detector-controller module, other groupings of components should be viewed as structural and functional equivalents. For example, some or all components may be included as part of an overall assembly having no overt modularity per se. Alternatively, the controller may be included locally or remotely; or may be grouped with the scanner module, the detector module, or the test cell module without departing from the scope and spirit of the disclosure and claims contained herein.  
         [0076]     While the terms “light” and “light beam” are used, it should be recognized that “light” corresponds to a range of electromagnetic energies that extends beyond wavelengths that may be typically detected by human vision. Except where noted, “light” should be read to encompass a range of wavelengths broader than the visible spectrum.  
         [0077]     As such, the scope of the invention described herein shall be limited only by the claims.