Abstract:
A nanovolume microcapillary crystallization system allows nanoliter-volume screening of crystallization conditions in a crystal card that allows crystals to either be removed for traditional cryoprotection or in situ X-ray diffraction studies on protein crystals that grow within. The system integrates formulation of crystallization cocktails with preparation of the crystallization experiments. The system allows the researcher to select either gradient screening in crystallization experiments for efficient exploration of crystallization phase space or a combination of sparse matrix with gradient screening to execute one comprehensive hybrid crystallization trial.

Description:
CROSS-REFERENCE TO A RELATED APPLICATION 
       [0001]    This application claims the benefit of U.S. Provisional Patent Application No. 61/061,536, filed Jun. 13, 2008, which is incorporated herein by reference. 
     
    
     STATEMENT OF GOVERNMENT LICENSE RIGHTS 
       [0002]    This subject matter was made, at least in part, with Government support as provided for by the terms of NIGMS U54 GM074961, awarded by the National Institute of General Medical Sciences. The Government has certain rights in the subject matter. 
     
    
     BACKGROUND 
       [0003]    The field of structural biology is generating technologies that increase throughput and efficiency each year. Such advances have inspired progression from gene to three-dimensional structure in three days. In an effort to improve efficiency, it is desirable to minimize the volume of protein required such that sufficient material for crystallization screening and optimization can be obtained from cell-free synthesis. With the “three day” structure goal in mind, it is desirable to develop several technologies to increase efficiency in the gene to structure pipeline. 
       SUMMARY 
       [0004]    This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
         [0005]    One aspect of the discussed subject matter includes a system for protein crystallization, which comprises a pumping system and pieces of software configured to execute on the protein crystallization system to control the pumping system. The system further includes one or more crystal cards coupled to the pumping system, each configured to house a mixer and a microfluidic capillary that is coupled to the mixer to facilitate storage and inspection of protein crystallization. 
         [0006]    Another aspect of the subject matter includes a method for gradient screening, which comprises regulating aqueous streams by independently controlling each aqueous stream with a pumping system exercised by pieces of software. The method further comprises mapping out crystallization phase space of a protein to illustrate transition from precipitation, to microcrystals, to single crystals in a protein crystallization experiment. 
         [0007]    A further aspect of the subject matter includes a method for hybrid screening, which comprises pre-forming precipitant plugs and pre-forming plug spacers, each separating two precipitant plugs from each other. The method further comprises forming gradients by merging precipitant plugs, plug spacers, and a protein stream. The method further includes mapping out crystallization phase space of a protein to illustrate transition from precipitation, to microcrystals, to single crystals in a protein crystallization experiment. 
         [0008]    A further aspect of the subject matter includes a method which comprises receiving a crystal card with capillaries, coating capillaries with a reagent to reduce the surface energy, and removing the reagent. 
         [0009]    In another aspect, the subject matter includes a crystal card, which comprises a substrate configured to house a mixer circuit and an inspection circuit. The crystal card further includes a layer bonded to the substrate and configured to peel from the substrate. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0010]    The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: 
           [0011]      FIG. 1  is a block diagram illustrating an exemplary nanovolume microcapillary crystallization system; 
           [0012]      FIG. 2  is a block diagram illustrating an exemplary pumping system of the nanovolume microcapillary crystallization system; 
           [0013]      FIG. 3A  is a pictorial diagram illustrating an exemplary user interface for configuring the pumping system; 
           [0014]      FIG. 3B  is a pictorial diagram illustrating an exemplary user interface for priming fluids to a crystal card of the system; 
           [0015]      FIG. 3C  is a pictorial diagram illustrating an exemplary user interface for specifying the production of nanoplugs in the crystal card wherein the nanoplugs are of equal size and equal content according to one embodiment of the subject matter; 
           [0016]      FIG. 3D  is a pictorial diagram illustrating an exemplary user interface for specifying the production of nanoplugs in the crystal card wherein the nanoplugs have varying concentrations of protein and precipitant according to one embodiment of the subject matter; 
           [0017]      FIG. 3E  is a pictorial diagram illustrating an exemplary user interface for specifying the production of nanoplugs in the crystal card wherein the nanoplugs have varying size and concentration for multiple precipitants according to another embodiment of the subject matter; 
           [0018]      FIG. 4A  is a pictorial diagram illustrating a top isometric view of one embodiment of a crystal card;  FIG. 4B  is a pictorial diagram illustrating a bottom isometric view of one embodiment of a crystal card;  FIG. 4C  is a pictorial diagram illustrating a top view of one embodiment of a crystal card;  FIG. 4D  is a pictorial diagram illustrating a side view of one embodiment of a crystal card;  FIG. 4E  is a pictorial diagram illustrating a bottom view of one embodiment of a crystal card; 
           [0019]      FIG. 5A  is a pictorial diagram illustrating a top isometric view of another embodiment of a crystal card;  FIG. 5B  is a pictorial diagram illustrating a bottom isometric view of another embodiment of a crystal card;  FIG. 5C  is a pictorial diagram illustrating a top view of another embodiment of a crystal card;  FIG. 5D  is a pictorial diagram illustrating a side view of another embodiment of a crystal card; 
           [0020]      FIG. 5E  is a pictorial diagram illustrating a bottom view of another embodiment of a crystal card; 
           [0021]      FIG. 6A  is a pictorial diagram illustrating a top isometric view of a third embodiment of a crystal card;  FIG. 6B  is a pictorial diagram illustrating a bottom isometric view of a third embodiment of a crystal card;  FIG. 6C  is a pictorial diagram illustrating a top view of a third embodiment of a crystal card;  FIG. 6D  is a pictorial diagram illustrating a side view of a third embodiment of a crystal card;  FIG. 6E  is a pictorial diagram illustrating a bottom view of a third embodiment of a crystal card; 
           [0022]      FIG. 7A  is a pictorial diagram illustrating a top isometric view of a fourth embodiment of a crystal card;  FIG. 7B  is a pictorial diagram illustrating a bottom isometric view of a fourth embodiment of a crystal card;  FIG. 7C  is a pictorial diagram illustrating a top view of a fourth embodiment of a crystal card;  FIG. 7D  is a pictorial diagram illustrating a side view of a fourth embodiment of a crystal card; 
           [0023]      FIG. 7E  is a pictorial diagram illustrating a bottom view of a fourth embodiment of a crystal card; 
           [0024]      FIG. 8A  is a pictorial diagram illustrating a top isometric view of a fifth embodiment of a crystal card;  FIG. 8B  is a pictorial diagram illustrating a bottom isometric view of a fifth embodiment of a crystal card;  FIG. 8C  is a pictorial diagram illustrating a top view of an embodiment of a crystal card;  FIG. 8D  is a pictorial diagram illustrating a side view of a fifth embodiment of a crystal card;  FIG. 8E  is a pictorial diagram illustrating a bottom view of a fifth embodiment of a crystal card; 
           [0025]      FIG. 9A  is a pictorial diagram illustrating a top isometric view of a sixth embodiment of a crystal card;  FIG. 9B  is a pictorial diagram illustrating a bottom isometric view of a sixth embodiment of a crystal card;  FIG. 9C  is a pictorial diagram illustrating an exploded isometric view of a sixth embodiment of a crystal card; 
           [0026]      FIG. 9D  is a pictorial diagram illustrating a top view of a sixth embodiment of a crystal card;  FIG. 9E  is a pictorial diagram illustrating a side view of a sixth embodiment of a crystal card;  FIG. 9F  is a pictorial diagram illustrating a bottom view of a sixth embodiment of a crystal card; 
           [0027]      FIG. 10  is a pictorial diagram illustrating one embodiment of a three-plus-one mixer of one embodiment of a crystal card; 
           [0028]      FIG. 11  is a pictorial diagram illustrating another embodiment of a three-plus-one mixer of one embodiment of a crystal card; 
           [0029]      FIG. 12  is a pictorial diagram illustrating a third embodiment of a three-plus-one mixer of one embodiment of a crystal card; 
           [0030]      FIG. 13  is a pictorial diagram illustrating a fourth embodiment of a three-plus-one mixer of one embodiment of a crystal card; 
           [0031]      FIG. 14  is a pictorial diagram illustrating a cross section through one embodiment of a crystal card; and 
           [0032]      FIGS. 15A-15V  are process diagrams illustrating an exemplary method for crystallizing molecules using a nanovolume microcapillary crystallization system. 
       
    
    
     DETAILED DESCRIPTION 
       [0033]    Various embodiments of the subject matter describe a nanovolume microcapillary crystallization system which comprises a pump, software configured to control the pump, and a crystal card that houses a mixer circuit and an inspection circuit. The crystal cards are suitably manufactured using materials that include one or more properties selected from a group consisting of X-ray transmission, optical clarity, moldability, chemical resistance and surface energy. The crystal cards house macromolecular crystals in various phases enabling either extraction of crystals from the crystal card or in situ X-ray diffraction. The crystals are promoted inside the crystal cards by formation of nanoplugs by the nanovolume microcapillary crystallization system. Nanoplugs are formed by combining streams of aqueous solutions with an immiscible and biologically inert carrier fluid, such as fluorocarbon solution. Streams of aqueous solutions, such as those composed of a target molecule, buffer, and precipitant solutions, are combined at the mixer circuit to form nanoplug crystallization experiments. The nanoplugs are incubated and monitored for crystallization. Nanoplug crystallization experiments can be suitably used to shed light on scientific questions regarding protein crystal nucleation and growth and to generate crystals for novel structure solution. 
         [0034]    The nanovolume microcapillary crystallization system facilitates two screening styles: gradient mode and hybrid mode. As used herein, the term gradient mode includes any suitable screening method that provides various crystallization phases of molecules. The gradient mode allows a crystallographer to finely scan a crystal card to reveal crystallization phase space of a particular molecule. Because each stream of aqueous solution used in the nanovolume microcapillary crystallization system can be independently controlled using the pump via the software, concentration gradients of desired granularity over a series of nanoplugs are suitably formed by changing the flow rates of the individual streams. As a precipitant stream decreases in flow rate, the nanovolume microcapillary crystallization system increases a flow rate of a buffer stream such that that the sum of the flow rates remains constant. Using the gradient mode, crystallization phase space of a particular molecule, such as a protein, can be mapped out to show a transition from precipitation, to microcrystals, to single crystals. 
         [0035]    As an enhancement to gradient mode, hybrid mode combines gradients with sparse matrix screening on one crystal card. Sparse matrix screening of molecule crystals in nanoplugs can be achieved by generating a pre-formed cartridge of different crystallizing agents. As used herein, the term hybrid mode includes hybrid screening, including any suitable screening method that includes pre-formed cartridges. The hybrid mode extends the concept of sparse matrix screening by pre-forming precipitant nanoplugs, separated by a nanoplug spacer (gas bubble), and forming a concentration gradient as they are merged with a molecule stream. Similar to the gradient mode, the hybrid mode generates gradients by coordinating flow rate change between the pre-formed precipitant nanoplugs and the buffer stream. By performing sparse matrix and gradient screening together on one crystal card, the hybrid mode is able to sample a large area of the crystallization phase space, generating 20-40 experiments from each pre-formed precipitant nanoplug. 
         [0036]    As used hereinabove and hereinbelow, the term “nanoplug” refers to a nanoliter-volume sized drop, such as a 10-20 mL aqueous drop, that fills a microfluidic channel of the system. Each nanoplug comprises a distinct microcrystallization experiment. As used hereinabove and hereinbelow, the term “mixer circuit” means the inclusion of a circuit having three aqueous channels and one carrier fluid channel that come together at a point upstream of the inspection circuit of a crystal card. Additional configurations are possible, such as mixers having four or five aqueous channels and one carrier fluid channel. The aqueous channels come together and suitably intersect the carrier fluid channel at a 90 degree angle. As used herein, the term “macro-micro interface” means the inclusion of a coupling between the syringes and a crystal card. In some embodiments, the syringes are connected to the mixer circuit or the inspection circuit via tubing, such as Teflon® (PTFE) tubing. In other embodiments, the tubing is connected to the mixer circuit or the inspection circuit using connectors configured to fluidically connect the inlets and outlets of the circuits with the tubing. As used hereinabove and hereinbelow, the term “inspection circuit” refers to a capillary or channel where fluids come together and form aqueous nanoplugs that flow inside the capillary. The inspection circuit can also be used to inspect the nanoplugs for crystal formation. Further, the inspection circuit can also be used to store crystals formed by the methods of the subject matter, and is therefore also referred to herein as a storage capillary. As used herein, the term “main channel” refers to the area of the inspection circuit that locates downstream of the mixer where the aqueous solutions and carrier fluid combine to form aqueous nanoplugs. As used hereinabove and hereinbelow, the term “molecule” includes small molecules, such as organic compounds and/or chemicals, and macromolecules. The term “biological molecule” refers to a molecule that is derived from, modeled on, or corresponds to a molecule from a biological source. The term also includes molecules synthesized or produced in vitro, such as by cell-free synthesis, and/or in vivo, such as recombinant proteins, mutant proteins, and artificial proteins, natural and artificial nucleic acid molecules, and other biological molecules that do not occur in nature. As used herein, the term “macromolecule” includes biopolymers such as nucleic acids, proteins, carbohydrates and lipids. For simplicity, the terms “protein” and “protein solution” are used herein to encompass other types of molecules in addition to proteins. 
         [0037]    A nanovolume microcapillary crystallization system  100  useful for molecule crystallization is shown in  FIG. 1 . A prepared sample  102  comprising a molecule for which the crystal structure is desired, for example a prepared protein sample, is provided in an aqueous solution  104 . Additional aqueous solutions are also provided; for example, a buffer solution and a precipitant solution. The buffer solution may comprise the buffer used to prepare the biological sample  102 . A carrier fluid is also provided. The carrier fluid is immiscible with the aqueous solutions. Suitable examples of carrier fluids include fluorinated oils; for example, FC-40 (3M Corp., St. Paul, Minn.). The aqueous solutions and carrier fluid  104  are provided to one or more syringes  106  which are connected to one or more pumps  108 . The pump  108  is controlled by software executed on a nanoplug-forming computer  110 . The software executed on the nanoplug-forming computer  110  regulates the flow of the aqueous solutions and carrier fluid  104  in a crystal card  112 . The flow of the aqueous solutions and carrier fluid in the crystal card  112  are observed through a magnifying device such as a microscope  114 . 
         [0038]    A pumping system  200  useful for regulating the flow of various fluids through a crystal card is shown in  FIG. 2 . Pump  1   202  controls syringe  1   204  and syringe  2   208 . Syringe  1   204  is loaded with an aqueous solution such as buffer  206 . Syringe  2  is filled with an aqueous solution such as a precipitant reagent  210 . Pump  2   212  controls syringe  3   214  and syringe  4   218 . Syringe  3   214  is filled with an immiscible fluid such as a carrier fluid  216 . Syringe  4   218  is filled with an aqueous solution containing a molecule, such as a protein of interest  220 . Suitable pumps include Harvard Twin 33 syringe pumps (Harvard Apparatus, Holliston, Mass.). In some embodiments, the syringe pumps have been modified by the manufacturer to provide better accuracy. Suitable syringes include Hamilton syringes, such as an 1800 series Hamilton Gas Tight syringe. Suitable syringe volumes range from 10 ul to 100 ul. The pumping system  200  is controlled by software executed on a nanoplug-forming computer  110 . 
         [0039]    Suitable software is provided for controlling the pumping system  108 .  FIGS. 3A-3E  illustrate representative user interfaces of the software of the system showing various modes that control the pumping system  200 .  FIG. 3A  shows a representative user interface  300  of a configuration mode of the software.  FIG. 3B  shows a representative user interface  302  of a prime mode of the software.  FIG. 3C  shows a representative user interface  304  of a constant mode of the software.  FIG. 3D  shows a representative user interface  306  of a gradient mode of the software.  FIG. 3E  shows a representative user interface  308  of a hybrid mode of the software of the system. 
         [0040]    Referring now to the crystal cards of the subject matter disclosed herein, a representative example of one embodiment of a crystal card is shown in  FIGS. 4A-4E . The crystal card  400  is configured to be about the same size as a standard microscope slide, being about 76.20 mm long and about 25.40 mm wide (or about 3 inches long by about 1 inch wide). The crystal card  400  is about 1.0 to 1.5 mm thick. The crystal card is manufactured of transparent polycarbonate by injection molding (Siloam Biosciences, Inc.). 
         [0041]    Referring now to the embodiment shown in  FIGS. 4A and 4B , the crystal card  400  has an upper surface  402  and a lower surface  414  that is parallel to the upper surface  402 . The crystal card  400  further comprises a substrate configured to house a mixer circuit  404  and a storage and inspection circuit  406 . The mixer circuit  404  is comprised of four microfluidic channels  421 ,  422 ,  424 , and  426 . See  FIG. 4C . Channels  421 ,  422 , and  424  come together and intersect channel  426  at a 90 degree angle. Each channel comprises an inlet  410 . See  FIG. 4E . The inspection circuit  406  comprises a long microfluidic capillary channel that locates just downstream of the mixer  404  and ends at an outlet  412 . The length of the microfluidic capillary  406  is about 67 cm. The microfluidic capillary channel  406  is also referred to as an inspection circuit, in which crystals produced in the card may be stored in the channel  406  until subjected to in situ X-ray diffraction analysis or extracted for cryocooling. The microfluidic channels  421 ,  422 ,  424 ,  426  and the capillary channel  406  are substantially square in cross-section and have an inner diameter of about 200 micrometers (μm)×200 μm. However, other configurations of the channels are possible. 
         [0042]    Referring now to  FIG. 4D , the crystal card  400  further comprises a layer  420  that is thermally bonded to the substrate and configured to peel from the substrate. The peelable layer  420  is thermally bonded to the substrate surface  414 . In other embodiments, the peelable layer  420  may be chemically bonded to the substrate. The peelable layer  420  is about 0.10 to 0.14 mm thick. The peelable layer  420  is suitably configured such that removal of the peelable layer  420  exposes the interior space of the inspection circuit channel  406 . The crystal card  400  further comprises a macro-micro interface that connects the syringes to the crystal card. In one embodiment, the macro-micro interface includes sections of rigid plastic tubing  430  (for example, tubing made of PEEK™ polymer) that are connected at one end to the inlets  410  and outlet  412 , and are connected at the other end to slip fit connectors  432  made of flexible silicone tubing. The slip fit connectors  432  are configured to accept Teflon® tubing (PTFE) (not shown). The other end of the tubing is connected to a syringe of the system. The Teflon® tubing has an inner diameter of 360 um and an outer diameter of 760 um (ID/OD 360/760), whereas the connecter  432  has an inner diameter of 760 um, thereby forming a gas and liquid tight seal when the Teflon® tubing is inserted into the connecter  432 . 
         [0043]    In operation, the channel  421  is connected to tubing that is filled with an aqueous solution, such as a buffer that is used in the protein solution of interest. Channel  422  is connected to tubing that is filled with a precipitant solution. As used herein, it is understood that the term precipitant is interchangeable with the term crystallant. Channel  424  is connected to tubing that is filled with a solution containing a target molecule of interest. In one embodiment, the target biological molecule is a protein. Channel  426  is connected to tubing that is filled with a carrier fluid. Suitable examples of carrier fluids include fluorinated oils or fluorocarbons, such as FC-40, although others are possible. The carrier fluid is immiscible with the aqueous fluids and preferentially wets the walls of the inspection circuit microchannel, thereby separating segments of the combined aqueous solution into nanoplugs that span the width of the channel. In one embodiment, the aqueous nanoplugs are about 10-20 mL in volume. 
         [0044]    Referring now to  FIGS. 5A-5E , a representative example of another embodiment of a crystal card of the subject matter is shown. Similar elements between different figures have similar reference numbers, wherein the first digit increases by one and corresponds to the figure number. For the sake of brevity, elements that are similar between the different Figures will not be described further. In the embodiment shown in  FIGS. 5A-5E , the inlet  510  is located in a shallow cylindrical depression  508  located in a top surface  502  of the crystal card  500 . The cylindrical depression  508  is configured for attaching a connector (not shown) that connects tubing to the inlets  510  and outlet  512 . The dimensions of the crystal card  500  are shown in  FIG. 5E . The crystal card  500  is 76.2 mm long and 25.4 mm wide. The inlets  510  are spaced 4.5 mm apart. The parallel channels of the inspection circuit  506  are 2.0 mm apart. However, as will be appreciated by a person skilled in the art, other suitable configurations are possible. 
         [0045]    Referring now to  FIGS. 6A-6E , a representative example of a third embodiment of a crystal card is shown. For the sake of brevity, similar elements that are described in previous figures are not described here. In the embodiment shown in  FIGS. 6A-6E , the inlets  610  and outlet  612  are positioned below a cylindrical projection  608  that is connected to and extends outwardly from the surface  602 . The projection  608  is configured for attaching a connector (not shown) that connects tubing to the inlets  610  and outlet  612 . Digressing, the crystal cards illustrated in the embodiments shown in  FIGS. 4-6  are manufactured from transparent polycarbonate plastic by injection molding (Siloam Biosciences, Inc.). 
         [0046]    Returning now to  FIGS. 7-9 , representative embodiments of a second type of crystal card will be described.  FIGS. 7A-7E  illustrate a representative example of another embodiment of a crystal card of the subject matter. For the sake of brevity, similar elements that are described in previous figures are not described here. In the embodiment shown in  FIGS. 7A-7E , the top surface  702  of the crystal card  700  further comprises two rows of ports  708 . The ports are configured to receive a plastic connector (not shown) that is suitable for connecting tubing to the inlets  710  and outlets  712  located beneath the port  708 . The surface  702  comprises 28 ports  708 . However, different numbers of ports are possible depending on the design of the crystal card  700 . The port  708  extends about 2.5 mm above the surface  702  of the crystal card  700 . A hole is suitably drilled in the bottom center portion of the port  708  such that it aligns with and is in fluidic connection with the inlets  710  and outlets  712 . The center of the ports are spaced about 4.5 mm apart. The hole drilled in the bottom of the port  708  is about 0.2 mm (200 um) in diameter. It will be understood that not every port is connected to the circuit channels such that only desired ports to connect tubing to the inlets  710  and the outlets  712  need be drilled. In other embodiments, a laser is used to drill holes through the peelable layer  720  before it is bonded to the bottom surface  714 . The laser-drilled holes are configured to be in fluidic connection with the inlets  710  and the outlets  712 . Tubing is connected to the laser-drilled holes using a specially designed crystal card holder (not shown). 
         [0047]    Referring still to  FIGS. 7A-7E , the crystal card further comprises two separate asymmetrical microfluidic channel circuits  706 A,  706 B. In  706 A, the inspection circuit is about 270 mm long. In  706 B, the inspection circuit is about 306 mm long. In both circuits  706 A and  706 , the outlet  712  is located on the opposite side of the circuit from the inlets  710  and the mixer circuits  704 A,  704 B. The embodiment shown in  FIGS. 7A-7E  comprises two separate configurations of the mixer circuit  704 A,  704 B. As shown in more detail in  FIG. 10 , the mixer circuit  704 A comprises a short neck region approximately 0.20 mm long between the aqueous channels and the carrier fluid channel. As shown in more detail in  FIG. 11 , the mixer circuit  704 B lacks a neck region between the aqueous channels and the carrier fluid channel. The mixer circuit  704 A was found to be suitable for aqueous nanoplug formation in a crystal card. 
         [0048]    Referring now to  FIGS. 8A-8E , a representative example of another embodiment of a crystal card of the subject matter is shown. For the sake of brevity, elements that are similar to previously described elements are not further described here. The crystal card  800  comprises two separate symmetrical microfluidic channel circuits  806 . In this embodiment, the outlet  812  is located on the same side of the circuit  806  as the mixer  804  and the inlets  810 . 
         [0049]    Referring now to  FIGS. 9A-9E , a representative example of another embodiment of a crystal card of the subject matter is shown. For the sake of brevity, elements that are similar to previously described elements are not further described here. A crystal card  900  comprises a single microfluidic circuit comprising one mixer circuit  904  and a long inspection circuit  906 . The inspection circuit  906  is about 665 mm long.  FIG. 9C  illustrates an exploded view of the crystal card  900 . Piece  930  comprising ports  908  is bonded to piece  940  comprising the microfluidic circuit channels. The peelable layer  920  is thermally bonded to the bottom surface  914  of piece  940 . However, in other embodiments, the peelable layer  920  may be chemically bonded to the substrate surface  914 . The peelable layer  920  is suitably configured such that removal of the peelable layer  920  exposes the interior space of the inspection circuit channel  906 . Digressing, the crystal cards illustrated in the embodiments shown in  FIGS. 7-9  are manufactured from transparent cyclic olefin copolymer (COC) or comparable plastic (ThinXXS Microtechnology AG, Germany). 
         [0050]    Returning now to  FIGS. 10-13 , representative examples of mixer circuits will now be described.  FIG. 10  shows a representative example of one embodiment of a mixer circuit that corresponds to the mixer  704 A shown in  FIG. 7 . The mixer circuit  1000  comprises three aqueous channels  1021 ,  1022  and  1024 . The aqueous channels are separated from the carrier fluid channel  1026  by a neck region  1007 . The channels are oriented such that the three channels  1021 ,  1022 ,  1024  containing aqueous solutions come together and intersect the channel  1026  containing the carrier fluid at a 90 degree angle. The mixer  1000  further comprises a portion of an inspection circuit  1006 . Referring still to  FIG. 10 , the dimensions of the mixer  1000  will now be described. The neck region  1007  is about 0.2 mm long. Channel  1021  is about 0.2 mm in diameter. Channels  1022 ,  1024  are about 0.141 mm in diameter. Channels  1006 ,  1026  are about 0.2 mm in diameter. However, other suitable dimensions for a mixer circuit are possible. 
         [0051]      FIG. 11  shows a representative example of another embodiment of a mixer circuit that corresponds to the mixer circuit  704 B shown in  FIG. 7 . The mixer circuit  1100  comprises three aqueous channels  1121 ,  1122  and  1124 . The aqueous solution channels connect directly to the carrier fluid channel  1106  in the absence of a neck region. The mixer circuit feeds into the inspection circuit  1126 . The channels are oriented such that the three channels containing aqueous solutions come together and intersect the channel containing the carrier fluid at a 90 degree angle. The diameter of channel  1121  is about 0.2 mm. The diameter of channels  1122 ,  1124  is about 0.141 mm. The diameter of the junction region between the aqueous channels and the carrier fluid channel  1126  is about 0.285 mm. However, other suitable dimensions for a mixer are possible. 
         [0052]    Referring now to  FIG. 12 , another view of the mixer circuit  704 A described in  FIG. 7  is shown. The mixer circuit  1200  comprises three aqueous channels  1221 ,  1222  and  1224  that are connected by a short neck region to the carrier fluid channel  1226 . The channels are oriented such that the three channels containing aqueous solutions come together and intersect the channel containing the carrier fluid at a 90 degree angle. Each channel has an inlet  1210 . Downstream of the mixer circuit  1204 , the solutions feed into a portion of an inspection circuit  1206 . Referring still to  FIG. 12 , the dimensions of the mixer circuit  1200  will now be described. The inlets  1210  are located about 4.4 mm from the channels  1206 ,  1226 . The aqueous channels  1221 ,  1222 ,  1224  make a right angle turn about 2.9 mm from the inlet. The right angle turn has an inner radius R0.300 and an outer radius R0.500. The portion of channels  1221 ,  1222 ,  1224  that are disposed in a plane parallel to channel  1206  are about 1.300 mm from channel  1206 . The aqueous channels  1221 ,  1222 ,  1224  make a 45 degree turn before connecting with each other upstream of the neck region. The inner diameter of channel  1206  is about 0.200 mm (200 um). The parallel portions of channel  1206  are about 1.2 mm apart. However, other suitable dimensions are possible. 
         [0053]      FIG. 13  shows a representative example of another embodiment of a mixer that corresponds to the mixer circuits  804  and  904  shown in  FIGS. 8 and 9 . The mixer circuit  1300  comprises aqueous channels  1321 ,  1322 , and  1324 . The aqueous channels are separated from the carrier fluid channel  1306  and the inspection circuit  1326  by a short neck region. The diameter of the neck region is about 0.200 mm. However, other suitable dimensions are possible. The channels are oriented such that the three channels containing aqueous solutions come together and intersect the channel containing the carrier fluid at a 90 degree angle. Downstream of the mixer circuit, the solutions flow into the inspection circuit  1326 . 
         [0054]      FIG. 14  shows a representative example of a cross-section through a crystal card similar to the embodiment illustrated in  FIG. 9 . The crystal card  1400  is comprised of three layers  1420 ,  1430  and  1440 . Layer  1430  comprises the ports as shown in  FIGS. 7-9 . Layer  1430  is about 0.4 mm thick. Layer  1440  comprises the microfluidic channel circuit and is about 1.5 mm thick at the edge. Layer  1420  comprises the peelable layer attached to the bottom surface of the crystal card  1400 , and is about 0.14 mm thick. 
         [0055]      FIGS. 15A-15V  illustrate a method  5000  for crystallizing molecules using a nanovolume microcapillary crystallization system. From a start block, the method  5000  proceeds to a set of method steps  5002 , defined between a continuation terminal (“Terminal A”) and an exit terminal (“Terminal B”). The set of method steps  5002  describes the preparation of a crystal card and the connection of the crystal card to a pump. 
         [0056]    From Terminal A ( FIG. 15B ), the method  5000  proceeds to a set of method steps  5008  where the crystal card is manufactured from a suitable material, such as polydimethylsiloxane (PDMS) or plastic by injection molding. The method then returns to a point of invocation. The method  5000  next proceeds to a set of method steps  5010  defined by a continuation terminal (“Terminal A 2 ”). The set of method steps  5010  treats the microcapillary surface of the crystal card to reduce the surface energy. 
         [0057]    From Terminal A 2  ( FIG. 15C ), the method  5000  proceeds to decision block  5014  where a test is performed to determine whether the crystal card is manufactured from plastic. If the answer to the test is NO, the method proceeds to another continuation terminal (“Terminal A 4 ”). If the answer to the test at decision block  5014  is YES, the method proceeds to another decision block  5016  where another test is performed to determine whether the plastic is polycarbonate. If the answer to the test at decision block  5016  is NO, the method  5000  proceeds to another continuation terminal (“Terminal A 5 ”). If the answer to the test at decision block  5016  is YES, the method  5000  proceeds to another continuation terminal (“Terminal A 6 ”). 
         [0058]    From Terminal A 4  ( FIG. 15D ), the method  5000  proceeds to block  5018  where the method treats the crystal card as manufactured from PDMS. The method proceeds to block  5020  where the microcapillary surface is treated with a perfluorinated silane solution for 2 hours at room temperature. The method then proceeds to block  5022  where the perfluorinated silane solution is removed by vacuum. At block  5024 , the microcapillary surface of the crystal card is dried using a gas such as air under pressure at 5-10 psi for 1 hour. The method then returns to the point from which the steps of Terminal A 2  were invoked, and proceeds to another continuation terminal (“Terminal A 3 ”). See block  5012 . 
         [0059]    From Terminal A 5  ( FIG. 15E ), the method  5000  proceeds to block  5026  where the method treats the crystal card as made of a plastic comprising cyclic olefin copolymer (COC) or comparable plastic. At block  5028 , the microcapillary surface is treated with a reagent to reduce the surface energy (hydrophobicity) of the plastic for 2 hours at room temperature. Suitable reagents for reducing the surface energy include fluorinated copolymer solutions, but other reagents are possible. Suitable fluorinated copolymer solutions include a two percent fluorinated copolymer solution in a fluorosolvent, such as Cytonix PFC 502AFA (Cytonix Corp., Beltsville, Md.). Cytonix PFC 502AFA is manufactured to adhere to polycarbonate and reduce the surface energy to 6-10 dyne/cm. To apply the fluorinated copolymer solution, the crystal card is filled from the outlet with the Cytonix PFC 502AFA solution. At block  5030 , the fluorinated copolymer solution is removed by vacuum. At block  5032 , the microcapillary surface is dried using a gas such as air under pressure of 5-10 psi for 1 hour. The method  5000  then proceeds to block  5034  where the crystal card is heated to 60° C. for 1 hour. The method then returns to the point of invocation of the steps of Terminal A 2 . See block  5012  at Terminal A 3 . 
         [0060]    From Terminal A 6  ( FIG. 15F ), the method  5000  proceeds to block  5036  where the crystal card is pre-chilled on ice. At block  5038 , the microcapillary surface is treated with a fluorinated copolymer solution such as Cytonix PFC 502AFA for 2 hours on ice. The polycarbonate crystal card inlets may be prone to cracking if incubated with the 502AFA solution at higher temperatures. The method then proceeds to continuation terminal A 5  where it skips to block  5030  and performs the steps in blocks  5030 ,  5032 , and  5034 . The method then returns to a point at which the steps of Terminal A 2  were invoked. See Terminal A 3  at block  5012 . The set of method steps at block  5012  couples the crystal card to the pump. 
         [0061]    From Terminal A 3  ( FIG. 15G ), the method  5000  proceeds to block  5040  where syringe  1  is filled with a buffer or aqueous solution. At block  5042 , syringe  2  is filled with a precipitant solution. At block  5044 , syringe  3  is filled with a carrier fluid. A representative example of a suitable carrier fluid includes a fluorinated carbon solution. Suitable examples of a fluorocarbon fluid include FC-40. FC-40 has a high surface tension with the detergents used in solubilizing membrane proteins. The surface tension enables nanoplug formation and crystallization. In a representative embodiment, the carrier fluid is a fluorinated oil which is immiscible with aqueous fluids. The carrier fluid surrounds and separates the aqueous nanoplugs as they are formed, moving them forward through the crystal card during the method. At block  5046 , syringe  4  is filled with a protein solution containing the protein of interest in a suitable buffer. At block  5048 , suitable tubing such as Teflon® tubing is attached to the needle of each syringe. At block  5050 , syringes  1  and  2  are attached to pump  1 , and syringes  3  and  4  are attached to pump  2 . At block  5052 , the tubing is connected to the crystal card via a macro-micro interface. Suitable connections for the macro-micro interface are described above. The method then proceeds to exit Terminal B. 
         [0062]    From Terminal B, the method  5000  proceeds to a set of method steps  5004 , defined between a continuation terminal (“Terminal C”) and an exit terminal (“Terminal D”). The set of method steps  5004  receives instructions to regulate fluid flow through the crystal card to obtain crystals. From Terminal C ( FIG. 15H ), the method  5000  proceeds to a set of method steps  5054 , defined by a continuation terminal (“Terminal C 1 ”). The set of method steps  5054  configures the pump. 
         [0063]    From Terminal C 1  ( FIG. 15I ), the method  5000  proceeds to block  5060  where the method receives instructions on the type of syringe pump model to be controlled by the system. Suitable pumps include Harvard Apparatus Twin Syringe Pump Model 33 (Harvard Apparatus, Holliston, Mass.), which has been modified by the manufacturer to provide better accuracy. As illustrated by  FIG. 2 , each syringe pump controls two syringes. At block  5062 , the method receives instructions on the serial communication port of a computer used to control the pump system. The communication ports are configured such that each syringe pump receives instructions at the same time, thereby preventing time delays and allowing the solutions to flow through the crystal card simultaneously. The method proceeds to block  5064  where the method receives instructions on the volume of each syringe connected to the pumps. At block  5066 , the method determines the diameter of each syringe connected to the pumps. The method then proceeds to return to a point at which the steps of the Terminal C 1  were invoked. 
         [0064]    From block  5054 , the method  5000  proceeds to a set of method steps  5056  defined by a continuation terminal (“Terminal C 2 ”). The set of method steps primes fluids to the mixer circuit of the crystal card. From Terminal C 2  ( FIG. 15J ), the method  5000  proceeds to block  5068  where the method receives instructions on which syringe will be used to dispense fluids into the mixer of the crystal card. At block  5070 , the method receives instructions on the flow rate from each syringe. At block  5072 , the method receives instructions on the volume of fluid to be dispensed by the syringe. At block  5074 , the method dispenses or aspirates fluid from a fluidic channel upstream of the mixer circuit. The method then continues to another continuation terminal (“Terminal C 4 ”). 
         [0065]    From Terminal C 4  ( FIG. 15K ), the method  5000  proceeds to decision block  5076  where a test is performed to determine whether the syringe is dispensing an aqueous fluid. If the answer to the test at decision block  5076  is NO, the method proceeds to another continuation terminal (“Terminal C 5 ). If the answer to the test at decision block  5076  is YES, the method proceeds to block  5078  where the method receives instructions to stop the aqueous fluid at the mixer circuit and before the fluid enters the inspection circuit. The method then continues to Terminal C 2  and repeats the above identified process steps for the next syringe. From Terminal C 5  ( FIG. 15K ), the method  5000  proceeds to block  5080  where the method receives instructions to stop the carrier fluid downstream of the mixer circuit and slightly inside the inspection circuit. The method then proceeds to return to a point from which the steps of Terminal C 2  were invoked. 
         [0066]    Digressing, an illustrative process for priming aqueous solutions and the carrier fluid to the mixer of the crystal card will now be described in detail. First, the empty crystal card mixer circuit is positioned on the microscope stage for observation during priming. The method receives instructions to dispense a solution, for example buffer, from syringe  1  to the mixer. The buffer is dispensed into the fluid channel connected to syringe  1  until the user observes that the solution has reached the region of the mixer just upstream of the junction between the fluidic channels. The method then receives instructions to stop dispensing the solution. Solution may be removed from the channel by instructing the method to aspirate the reagent. It is suitable to refrain aqueous solutions from entering the inspection circuit of the crystal card. The method is repeated for each of the three fluid channels connected to syringes dispensing aqueous solutions; for example, syringe  4  (protein solution) and syringe  2  (precipitant solution). The carrier fluid is then dispensed into the fourth fluid channel connected to syringe  3 . The carrier fluid is dispensed into the fourth fluid channel until the fluid travels through the mixer junction and just slightly enters the inspection circuit (fifth channel) of the crystal card. The method then receives instructions to stop dispensing the carrier fluid. 
         [0067]    Returning to block  5056 , the method  5000  proceeds to a set of method steps  5058  defined by a continuation terminal (“Terminal C 3 ”). The set of method steps receives instructions to produce aqueous nanoplugs in the inspection circuit of the crystal card. From Terminal C 3  ( FIG. 15L ), the method  5000  receives instructions on which nanoplug formation protocol will be performed at block  5082 . The method then proceeds to decision block  5084  where a test is performed to determine whether the instruction received was to perform the constant mode. If the answer to the test at block  5084  is NO, the method proceeds to another continuation terminal (“Terminal C 6 ”). If the answer to the test at decision block  5084  is YES, the method proceeds to block  5086  where the method receives instructions on the flow rate for each syringe. The method then proceeds to block  5088  where the method receives instructions on the total volume of fluid to pass through the mixer circuit. At block  5090 , the method produces aqueous nanoplugs inside the inspection circuit of the crystal card wherein each nanoplug is suitably of equal size and has the similar concentration of protein and precipitant. The method then proceeds to return to a point of invocation. From block  5058 , the method proceeds to exit terminal D. 
         [0068]    From Terminal C 6  ( FIG. 15M ), the method  5000  proceeds to decision block  5092 , where a test is performed to determine whether the method was instructed to perform gradient mode. If the answer to the test in block  5092  is NO, the method proceeds to another continuation terminal (“Terminal C 7 ”). If the answer to the test in decision block  5092  is YES, the method proceeds to block  5094  where the method receives instructions on the maximum flow rate for the syringes with variable flow. In one embodiment, the variable flow syringes contain the buffer and precipitant. In another embodiment, syringes  1  and  2  are the variable flow syringes. However, the method can designate any syringe to be a variable flow syringe. In one embodiment, the combined flow rate of the variable flow syringes equals the maximum flow rate. For example, in one embodiment, the method provides instructions for the flow rate of syringe  1  to equal 2 μl/min, whereas the method provides instructions for the flow rate of syringe  2  to equal 0 (zero) μl/min. In this embodiment, the maximum flow rate equals 2 ul/min (2+0 μl/min). The method then proceeds to block  5096  where the method receives instructions on the constant flow rate for the syringe controlling the carrier fluid. In one embodiment, syringe  3  controls the carrier fluid. In one embodiment, the carrier fluid flow rate equals the total flow rate of the aqueous solutions (buffer, precipitant, and protein solutions). In another embodiment, the flow rate for the carrier fluid may be selected to be slower or faster than the total flow rate of the aqueous fluids. Slower carrier fluid rates generate larger aqueous nanoplugs with smaller segments comprising carrier fluid between nanoplugs. Faster carrier fluid rates generate smaller aqueous nanoplugs with larger carrier fluid segments between the nanoplugs. The method then proceeds to block  5098  where the method receives instructions on the constant flow rate for the syringe controlling the protein solution. In one embodiment, syringe  4  controls the carrier fluid. In one embodiment, the protein flow rate equals the sum of the flow rate of the other aqueous solutions (buffer and precipitant). Changing the flow rate of the protein solution changes the ratio of protein-to-crystallization conditions in each nanoplug. The method then proceeds to block  6000  where the method receives instructions on the total aqueous volume to be dispensed during a single iteration or cycle of the method. The method then proceeds to another continuation terminal (“Terminal C 8 ”). 
         [0069]    From Terminal C 8  ( FIG. 15N ), the method  5000  proceeds to block  6002  where the method receives instructions on the volume of each aqueous nanoplug that will be dispensed into the inspection circuit. At block  6004 , the method receives instructions on the total number of iterations or cycles to be performed (i.e., the number of times the gradient screening steps are repeated). In one embodiment, if the method receives instructions to run zero iterations, the pumps will stop when the total aqueous volume selected at block  6000  is dispensed. In another embodiment, if the method receives instructions to run one or more iterations, the pumps will stop when the process steps described above have been repeated the desired number of times. At block  6006 , the method reciprocally varies the flow rate of the buffer and precipitant solutions such that the sum of the buffer and precipitant solution flow rates equals the maximum flow rate selected at block  5094 . For example, in one embodiment, at block  5094  the method provides instructions for the flow rate of syringe  1  to equal 2 μl/min and provides instructions for the flow rate of syringe  2  to equal 0 μl/min, such that the maximum flow rate equals 2 μl/min. When the method starts, the flow rate from syringe  1  will begin at 2 μl/min and ramp down to 0 μl/min, while the flow rate from syringe  2  will simultaneously ramp up from 0 μl/min to 2 μl/min. At block  6008 , the method produces a series of aqueous nanoplugs inside the inspection circuit wherein each drop is of equal size but varies in the concentrations of protein and precipitant in each drop. At block  6010 , the method terminates after the desired number of iterations or cycles has been performed. The method then returns to block  5058  where the method proceeds to exit terminal D. 
         [0070]    From Terminal C 7  ( FIG. 15O ), the method  5000  proceeds to decision block  6012  where a test is performed to determine whether the method was instructed to perform hybrid mode. If the answer to the test at block  6012  is NO, the method proceeds to another continuation terminal (“Terminal C 9 ”). If the answer to the test at block  6012  is YES, the method proceeds to another decision block  6014  where a test is performed to determine whether a precipitant cartridge has been prepared. If the answer to the test at decision block  6014  is NO, the method proceeds to another continuation terminal (“Terminal C 10 ”). If the answer to the test at block  6014  is YES, the method proceeds to another continuation terminal (“Terminal C 11 ”). 
         [0071]    From Terminal C 9  ( FIG. 15P ), the method  5000  proceeds to decision block  6016  where a test is performed to determine whether the method was instructed to perform the pulsatile mode. If the answer to the test at decision block  6016  is NO, the method returns to Terminal C 3  where the above identified steps are repeated. If the answer to the test at decision block  6016  is YES, the method proceeds to block  6018  where the method receives instructions on performing the pulsatile mode. The method then returns to block  5058 . From block  5058 , the method exits to Terminal D. 
         [0072]    From Terminal C 10  ( FIG. 15Q ), the method  5000  proceeds to block  6020  where a syringe is connected to tubing, such as Teflon® tubing, containing carrier fluid. The method then proceeds to block  6022  where the syringe is connected to a syringe pump. At block  6024 , the method receives instructions to enter a defined volume, for example, about 40 mL, and aspirates an air bubble of about 40 mL into the tubing. At block  6026 , the method aspirates a defined volume, for example, about 120 mL, of a precipitant solution into the tubing. At block  6028 , the method repeats the above two steps until a suitable number of precipitants are loaded into the tubing. For example, a suitable number of precipitants can range from 1-24 or more. At block  6030 , the method aspirates carrier fluid, about 1 μL, into the open tip of the tubing. At block  6032 , the tubing is connected to the precipitant inlet of the crystal card. The method then proceeds to continuation Terminal C 11 . 
         [0073]    At Terminal C 11  ( FIG. 15R ), the method  5000  proceeds to block  6034  where the method receives instructions on the starting flow rate of the buffer solution (syringe  1 ). At block  6036 , the method receives instructions on the change in the flow rate (step size) of the buffer solution. The step size is the change in the rate of flow that will be applied at each ramp up or down of the method. At block  6038 , the method receives instructions on the starting flow rate of the precipitant cartridge (syringe  2 ). At block  6040 , the method calculates the change in the flow rate (step size) of the precipitant solution. In one embodiment, the step size for the buffer equals the step size for the precipitant. At block  6042 , the method sums the buffer and precipitant flow rates to determine the total flow rate. At block  6044 , the method receives instructions on the starting flow rate for the carrier fluid (syringe  3 ). At block  6046 , the method receives instructions on the change in the flow rate (step size) of the carrier fluid. The method then proceeds to another continuation terminal (“Terminal C 12 ”). 
         [0074]    From Terminal C 12  ( FIG. 15S ), the method  5000  proceeds to block  6048  where the method receives instructions on the constant flow rate of the protein solution (syringe  4 ). The method then proceeds to block  6050  where the method receives instructions on the number of ramp up steps (rate of flow changes) for each precipitant. At block  6052 , the method sets the number of ramp down steps to equal the number of ramp up steps for each iteration or cycle of the method. At block  6054 , the method receives instructions on the number of iterations or cycles to be performed. In one embodiment, one iteration or cycle corresponds to a single precipitant loaded in the precipitant cartridge. At block  6056 , the method receives instructions on the duration of each ramp step. For example, in one embodiment, the duration of each ramp step is 1.5 seconds. At block  6058 , the method reciprocally varies the buffer and precipitant flow rates such that the sum equals the starting rates. The method then proceeds to another continuation terminal (“Terminal C 13 ”). 
         [0075]    From Terminal C 13  ( FIG. 15T ), the method  5000  proceeds to block  6060  where the method varies the flow rate of the carrier fluid. The method then proceeds to block  6062  where the method produces a series of nanoplugs inside the inspection circuit wherein each drop has equal amounts of protein and varying amounts of precipitant and buffer. In one embodiment, the method provides a varied amount of precipitant with a constant amount of protein for each cycle. Table 1 illustrates one embodiment of the method described above for the hybrid mode. The method then proceeds to Terminal D. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Values specified by the method in hybrid mode. 
               
             
          
           
               
                 One 
                   
                   
                   
                 Carrier 
                   
                   
                   
                 Total 
               
               
                 cycle 
                   
                 Buffer 
                 Precipitant 
                 Fluid 
                 Protein 
                 Sum 
                 Duration 
                 time 
               
               
                 up 
                 Step 
                 (ul/min) 
                 (ul/min) 
                 (ul/min) 
                 (ul/min) 
                 Aqueous 
                 (sec) 
                 (sec) 
               
               
                   
               
               
                   
                 0 
                 0.2 
                 0.6 
                 2.2 
                 0.6 
                 1.4 
                 1.5 
                   
               
               
                 Ramp up 
                 1 
                 0.3 
                 0.5 
                 2.0 
                 0.6 
                 1.4 
                 1.5 
               
               
                   
                 2 
                 0.4 
                 0.4 
                 1.8 
                 0.6 
                 1.4 
                 1.5 
               
               
                   
                 3 
                 0.5 
                 0.3 
                 1.6 
                 0.6 
                 1.4 
                 1.5 
               
               
                   
                 4 
                 0.6 
                 0.2 
                 1.4 
                 0.6 
                 1.4 
                 1.5 
                 6.0 
               
               
                 Ramp down 
                 1 
                 0.5 
                 0.3 
                 1.6 
                 0.6 
                 1.4 
                 1.5 
               
               
                   
                 2 
                 0.4 
                 0.4 
                 1.8 
                 0.6 
                 1.4 
                 1.5 
               
               
                   
                 3 
                 0.3 
                 0.5 
                 2.0 
                 0.6 
                 1.4 
                 1.5 
               
               
                   
                 4 
                 0.2 
                 0.6 
                 2.2 
                 0.6 
                 1.4 
                 1.5 
                 6.0 
               
               
                 Step size 
                   
                 0.1 
                 0.1 
                 0.2 
                 stable 
               
               
                   
               
             
          
         
       
     
         [0076]    From Terminal D at block  5004 , the method  5000  proceeds to a set of method steps  5006 , defined between a continuation terminal (“Terminal E”) and an exit terminal (“Terminal F”). The set of method steps  5006  performs diffraction experiments on the crystals obtained from the crystal card. From Terminal E ( FIG. 15U ), the method  5000  proceeds to decision block  6064  where a test is performed to determine whether crystals were extracted from the inspection circuit of the crystal card prior to diffraction. If the answer to the test at block  6064  is NO, the method proceeds to another continuation terminal (“Terminal E 1 ”). If the answer to the test at block  6064  is YES, the method proceeds to block  6066  where a peelable layer is removed from the bottom surface of the crystal card. In one embodiment, the peelable layer is bonded to the plastic part of the crystal card that contains the microfluidic channels. The bond is designed to be strong enough to prevent fluid from leaking out of the microfluidic circuit but weak enough to be manually peeled off. In one embodiment, the bond is a thermal bond. In another embodiment, the bond is a chemical bond. Removal of the peelable layer exposes the interior of the microfluidic channels of the crystal card, allowing access to the aqueous nanoplugs. In another embodiment, the aqueous nanoplugs that contain crystals are retained in the microfluidic channels of the crystal card after the peelable layer is removed. At block  6068 , the crystal formed in the inspection circuit is extracted from the crystal card using a cryoloop. In one embodiment, the cryoloop is a nylon cryoloop. At block  6070 , the crystal is cryocooled, and diffraction data is obtained. The method then proceeds to exit Terminal F where the method terminates execution. 
         [0077]    From Terminal E 1  ( FIG. 15V ), the method  5000  proceeds to block  6072  where the crystal card containing crystals is mounted onto the goniometer of an X-ray source. At block  6074 , the method obtains diffraction data from crystals located in situ inside the inspection circuit. The method then proceeds to block  5006  and exit Terminal F. The method then terminates execution. 
         [0078]    The above described crystal extraction steps can be used in combination with the gradient screening of various embodiments of the subject matter to generate crystals of methionine-R-sulfoxide reductase. Crystals were removed from the crystal card using a cryoloop and then cryocooled for diffraction experiments. As an example, a 1.7 Å data set was collected at SBC-CAT beamline 19BM located at the Advanced Photon Source at Argonne National Laboratories and the structure was subsequently solved and refined. The final coordinates and structure factors were deposited to the Protein Data Bank (accession code 3CXK). 
         [0079]    The crystal card of various embodiments of the subject matter is also suitable for in situ diffraction. In situ diffraction allows the crystallographer to assess the quality of a crystal before being altered by the cryoprotection process. For robust crystals, it can allow complete diffraction data to be collected. The crystal card is sufficiently X-ray transparent to be mounted onto the goniometer of an X-ray source for diffraction data collection at room temperature. For example, a simple test was conducted to analyze the absorption of the X-Rays by the crystal card. The beam current in the ion chamber normalized to the APS ring current (I/I 0 ) was measured with and without the crystal card inserted at a wavelength of 0.979261 A (12.66099 keV). I/I 0  without the crystal card measured 1.91671 E-6 and I/I 0  with the crystal card measured 1.5511 E-6. This constitutes a 19% X-ray absorbance by the crystal card. Further, the crystal card can be translated along its X and Y axis to collect data from multiple crystals to be combined for a complete data set. To demonstrate this technique, a crystal card containing Lysozyme crystals was mounted on the goniometer head at NE-CAT beamline 24ID-C located at the Advanced Photon Source at Argonne National Laboratories. Data were collected at room temperature from three crystals in the crystal card. Crystallographic data are provided in Appendix A. 
         [0080]    Regarding structure determination, data sets were collected at the Advanced Photon Source: beamline 19BM at 100K for methionine-R-sulfoxide reductase and beamline 24-IDC at room temperature for lysozyme. Data were integrated and scaled with HKL2000. For the lysozyme structure, intensities were integrated separately for each of the three data sets using the mosflm package. The structures of lysozome and methionine-R-sulfoxide reductase were solved by molecular replacement using Molrep and PDB entries 1IEE and 3CEZ as the search models, respectively. Structures were refined with Refmac5 and model building was performed with Coot. 
         [0081]    While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 APPENDIX A 
               
             
             
               
                   
               
               
                 Crystallographic Data: 
               
             
          
           
               
                   
                   
                 methionine-R- 
               
               
                   
                 Lysozyme 
                 sulfoxide reductase 
               
               
                   
                   
               
             
          
           
               
                 Data Collection 
                   
                   
               
               
                 Unit Cell (Å) 
                 a = 79.18, 
                 a = 42.00, b = 45.17 
               
               
                   
                 c = 38.38 
                 c = 45.40, α = 88.4 
               
               
                   
                   
                 β = 83.7, γ = 69.1 
               
               
                 Space group 
                 P4 3 2 1 2 (No. 96) 
                 P1 (No. 1) 
               
               
                 Resolution (Å) 
                 50-1.90 
                 50-1.70 
               
               
                 Wavelength (Å) 
                 0.97950 
                 0.97932 
               
               
                 Total Reflections 
                 54,338 
                 118,181 
               
               
                 Unique Reflections 
                 10,151 
                 32,539 
               
               
                 I/(sigI)* 
                 11.4 (2.9)  
                 23.1 (2.2)  
               
               
                 R merge  (%)* 
                 13.7 (58.4) 
                  6.8 (42.3) 
               
               
                 Completeness (%)* 
                 98.8 (98.5) 
                 95.3 (87.4) 
               
               
                 Redundancy 
                 5.4 (5.0) 
                 3.6 (3.3) 
               
               
                 Wilson B factor (Å 2 ) 
                 24.1 
                 22.1 
               
               
                 Refinement 
               
               
                 Resolution (Å) 
                 50-1.90 
                 50-1.70 
               
               
                 Reflections (working/test) 
                 9,412/480   
                 30,828/1,650  
               
               
                 R working /R free  (%) 
                 19.6/23.0 
                 16.6/19.9 
               
               
                 Number of atoms (protein/water) 
                 1001/45  
                 2082/180  
               
               
                 r.m.s. deviation bond length (Å) 
                 0.016 
                 0.015 
               
               
                 r.m.s. deviation bond angle (degrees) 
                 1.607 
                 1.408 
               
               
                 Average B factor (Å 2 ) (All atoms) 
                 28.8 
                 28.3 
               
               
                 Average B factor (Å 2 ) (Protein) 
                 28.5 
                 27.5 
               
               
                 Average B factor (Å 2 ) (Water) 
                 35.9 
                 37.0 
               
               
                 Coordinate error (Å) Based on R free   
                 0.149 
                 0.095 
               
               
                 Ramachandran Analysis (%) 
               
               
                 Most Favored (chain A/B) 
                 89.4 
                 91.7/90.8 
               
               
                 Additionally Allowed (chain A/B) 
                 10.6 
                 7.3/8.3 
               
               
                   
               
               
                 *Parenthesis indicates values for the 2.00 Å to 1.90 Å resolution shell for lysozyme and 1.76 Å to 1.70 Å shell for methionine-R-sulfoxide reductase.