Patent Document

CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims benefit of U.S. Provisional Patent Application No. 60/341,020, filed on Dec. 12, 2001, and entitled “Integrated Crystal Mounting and Alignment System for High-throughput Biological Crystallography”, which is hereby incorporated by reference in its entirety. 
     
    
     STATEMENT REGARDING FEDERAL FUNDING  
       [0002] This invention was made with U.S. Government support under Contract Number DE-AC03-76SF00098 between the U.S. Department of Energy and The Regents of the University of California for the management and operation of the Lawrence Berkeley National Laboratory. The U.S. Government has certain rights in this invention. 
     
    
     
       REFERENCE TO A COMPUTER PROGRAM  
         [0003]    Not applicable.  
         BACKGROUND OF THE INVENTION  
         [0004]    1. Field of the Invention  
           [0005]    The present invention generally relates to the transportation, robotic crystal mounting and alignment, manipulation, mounting, alignment of crystal samples in a variety of experimental environments. The present invention more particularly relates to the mounting, alignment, and exposure of samples to synchrotron radiation for high-speed x-ray crystallography.  
           [0006]    2. Description of the Relevant Art  
           [0007]    Overview of X-ray Crystallographic Systems  
           [0008]    Aspects of the present invention facilitate the transportation, as well as the remote and unattended mounting and alignment of frozen crystals—of e.g. biological materials, such as proteins, lipids, or deoxyribonucleic acids (DNA)—for x-ray diffraction analysis. A major challenge in the x-ray diffraction analysis system design is the necessity of storing the samples in liquid nitrogen before mounting and following removal, as well as maintaining the samples at near liquid nitrogen temperature throughout the mounting, alignment, and x-ray diffraction analysis data acquisition process. Additionally, the precision and stability of the crystal sample location alignment must be very high, with absolute sample position maintained within a few microns while rotating in one axis while being exposed to an incident x-ray beam through as much as a full 360°.  
           [0009]    Traditional x-ray diffraction analysis crystal sample handling procedures are operator-intensive, requiring continuous manual operator intervention at the measurement station. The ability to perform these tasks remotely and automatically significantly increases crystal mounting and measurement throughput, as well as reliability for large-scale protein crystallography characterization. An increase in throughput multiplies the number of samples that may be analyzed in a given time period, thus decreasing the time per sample, thereby lowering the cost associated with synchrotron-based x-ray crystallography.  
           [0010]    Synchrotron-Based X-ray Crystallography  
           [0011]    One embodiment of this invention is in the area of cryogenic protein crystallography at synchrotron sources, although the robotic mounting and alignment system can be adapted for other laboratory x-ray sources. Potential uses include high-volume protein characterization experiments. The level of application of this invention could range from a small experimental program processing only a few samples per day to large projects screening and analyzing many thousands of samples per year.  
           [0012]    Synchrotron-based x-ray crystallography is one application of this invention. Synchrotrons are capable of producing intense monochromatic pseudo-coherent photons of precisely controllable energies. The property of high intensity (otherwise known as high brightness) of the synchrotron x-ray beams means that acquisition of crystal lattice diffraction patterns can be done very rapidly, whereas other, lower intensity beams may require several times longer for a diffraction pattern to be acquired. The high brightness of the synchrotron radiation, combined with the narrow energy bandwidth achievable using a monochromator, can lead to exceedingly high-resolution x-ray diffraction patterns.  
           [0013]    The x-ray diffraction patterns can subsequently be analyzed to infer the relative spatial positions of the atoms constituting the crystal lattice structure. The overall x-ray diffraction analysis of crystals is known as x-ray crystallography. The information contained in the crystal structure can lead to important insights about the function of the molecule and into molecular-chemical interactions. Such insights can lead to targeted, and thus faster, pharmaceutical development and improved pharmaceuticals: a field known as ‘structure based pharmaceutical design’.  
           [0014]    Biological Crystallography Mounting Techniques  
           [0015]    Currently, most x-ray crystallography work is done using synchrotron x-ray sources. These x-ray sources are extremely expensive to operate, which means that time is precious. However, since synchrotron x-ray crystallography is still a recent phenomenon, most sample mounting is done manually, which is both slow and imprecise. Furthermore, since crystallography must be done on crystalline material, the sample must be maintained in a frozen state. Typically, this is ensured by keeping the sample at near liquid nitrogen temperatures.  
           [0016]    The requirement that the sample be maintained at liquid nitrogen temperature, however, requires that technicians and scientists can only mount the samples using cumbersome techniques of indirectly handling the sample. Thus, people cannot be allowed to inadvertently heat the sample, and reciprocally, the sample handling tools, and sample handling fixtures, cannot freeze the fingers of the people who do the mounting. To meet this requirement, clumsy tools resembling forceps or pliers are used. These tools are somewhat cumbersome, further adding time and difficulty in mounting and handling the sample.  
           [0017]    As more time is required to manually mount the sample, more heat is transferred from the ambient atmosphere, raising the crystal sample temperature. Some biological crystal samples, frozen at a critical point in a chemical reaction with another compound, continue their reactions at temperatures as low at 100° K., only about 22° K. above that of liquid nitrogen. This stringent maximum temperature requirement for some samples implies that the sample must be actively cooled during the entire mounting process, which adds still further time and complexity to the mounting process. It is preferable that the sample crystals be cooled to a temperature not in excess of 150° K., more preferably not in excess of 130° K., yet more preferably not in excess of 110° K., still more preferably not in excess of 100° K., yet still more preferably not in excess of 90° K., and most preferably not in excess of 80° K.  
           [0018]    The largest time-related issue with manual operator mounting of synchrotron x-ray crystallography samples is that the humans must enter the x-ray irradiation area (‘the hutch’) to mount and dismount the crystal samples. This action involves in turn a sequence of safety interlocking steps to protect the personnel from a harmful and potentially lethal dosage of x-rays used to irradiate the crystal sample to generate the diffraction patterns. Typically, one or more heavy lead-lined doors must be opened and closed, additional beam shutters inserted, and interlocking safety devices must be carefully verified for safe operation, prior to human access to the sample.  
           [0019]    The result is that manual mounting of a synchrotron x-ray crystallography sample is slow. As a result of being slow, manual mounting is very expensive as measured in synchrotron beam time.  
           [0020]    Biological Crystallography Sample Transportation  
           [0021]    Currently, there are relatively few synchrotron x-ray sources available for x-ray crystallography. Therefore, scientists wishing to use synchrotron x-ray sources face the dilemma of transporting the crystal samples to the synchrotron while simultaneously maintaining the crystal&#39;s cryogenically frozen state.  
           [0022]    A particularly fruitful use of synchrotron x-ray crystallography is in detection of chemical interactions within a specific biological sample. These interactions are evanescent in nature, sometimes reacting in the one-nanosecond time scale. Additionally, typical biological processes can follow a number of biochemical pathways that are time dependent. Some of these biochemical pathways proceed even at temperatures as low as 100° K. Thus, for a scientist to determine the crystalline structure of an intermediate state biochemical interaction, the biological sample must be frozen to a temperature low enough to inhibit further reaction, typically close to the liquid nitrogen temperature of about 77° K. under normal laboratory conditions.  
           [0023]    A Relevant Patent  
           [0024]    Abbott Laboratories is the named assignee of U.S. Pat. No. 6,404,849 B1 (the &#39;849 patent), entitled “Automated Sample Handling for X-Ray Crystallography”. The &#39;849 patent discloses algorithms for centering a crystal at a reference position relative to home position sensors, as well as the hardware for screwing a threaded sample holding device on and off a positioning device. The &#39;849 patent uses a multi-axis robot to move crystals from a sample rack to a positioning device.  
         SUMMARY OF THE INVENTION  
         [0025]    One aspect of the present invention is directed toward the transportation and manipulation of samples of cryogenically frozen biological particles, preferably protein crystals, mounted on standardized base-pin configured sample assemblies.  
           [0026]    The integrated crystal mounting and alignment system for high-throughput biological crystallography which transports and manipulates the sample assemblies comprises eight major components:  
           [0027]    1) a sample repository having a storage Dewar filled with liquid nitrogen, capable of keeping many samples cryogenically frozen, with a sample repository stage able to addressably move the sample assemblies to a point where a particular sample assembly can be extracted;  
           [0028]    2) a system for shipping, storing and handling of the sample assemblies at cryogenic temperatures, preferably liquid nitrogen temperatures;  
           [0029]    3) a computer-controlled sampling system sequencing a particular sample assembly through the steps of: a) selecting the particular sample assembly from the cryogenic sample repository, b) removing the selected sample assembly from the sample repository, c) transferring the sample assembly to a three axis positioner mounted on a goniometer head, d) centering the sample in the x-ray beam, e) exposing the sample (held by the mounted sample assembly) to x-ray radiation to produce a crystallographic image at a sequence of rotational exposure angles while simultaneously maintaining the sample&#39;s cryogenic temperatures, and f) replacing the sample assembly back in the cryogenic sample repository;  
           [0030]    4) a sample gripper capable of firmly grasping a sample assembly, while keeping the sample at cryogenic temperature;  
           [0031]    5) a gripper stage, using the sample gripper to: remove the sample assembly from cryogenic sample repository, transport the sample to a sample positioner, and replace the sample assembly in the sample repository, while at all times maintaining the temperature of the sample at or below 78° K.;  
           [0032]    6) a sample gripper defroster capable of keeping the sample gripper free of frost buildup during cycles of sample assembly mounting and dismounting (or unmounting) in ambient humid air;  
           [0033]    7) a sample positioner consisting of a precision three-axis positioner mounted on a precision goniometer; and  
           [0034]    8) an optical alignment system that provides feedback to the sample positioner for precise alignment of the sample to a predefined point in space within the x-ray beam during sample rotation. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0035]    [0035]FIG. 1 is a cross sectional view of a sample assembly with a crystal sample mounted.  
         [0036]    [0036]FIG. 2A is a top view of a sample cassette with one sample assembly.  
         [0037]    [0037]FIG. 2B is a cross sectional view through section  2 - 2  of FIG. 2A of a sample cassette with one sample assembly.  
         [0038]    [0038]FIG. 2C is an exploded view of the cross sectional view of FIG. 2B, with a sample cassette with one sample assembly.  
         [0039]    [0039]FIG. 3A is a top view of a sample cassette cover.  
         [0040]    [0040]FIG. 3B is a cross sectional view through section  3 - 3  of FIG. 3A of a sample cassette cover.  
         [0041]    [0041]FIG. 3C is a bottom view of a sample cassette cover showing liquid nitrogen venting features.  
         [0042]    [0042]FIG. 4A is a cross sectional view of a sample cassette assembly comprised of an assembled sample cassette with one sample assembly, and protected by a sample cassette cover.  
         [0043]    [0043]FIG. 4B is an exploded cross sectional view of a sample cassette assembly comprised of a sample cassette, one sample assembly, and a sample cassette cover, before assembly.  
         [0044]    [0044]FIG. 5A is a cross sectional view of a sample cassette carrier with six assembled sample cassettes present and the top slot vacant.  
         [0045]    [0045]FIG. 5B is a bottom view of the sample cassette carrier and all assembled sample cassettes absent.  
         [0046]    [0046]FIG. 6 is a cross sectional view of a cryogenic shipping container with sample cassette carrier with six sample cassette assemblies present, and the top slot vacant.  
         [0047]    [0047]FIG. 7A is a top view of a cassette deck with three sample cassettes present and one sample cassette absent.  
         [0048]    [0048]FIG. 7B is a sectional view of the cassette deck of FIG. 7A with three sample cassettes present and one sample cassette absent.  
         [0049]    [0049]FIG. 8 is a cross sectional view of a sample gripper.  
         [0050]    [0050]FIG. 9A is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the system has just grasped a sample assembly in the sample repository.  
         [0051]    [0051]FIG. 9B is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the system has retracted a pneumatic stage, known as SmallMove, causing the sample gripper to move vertically upwards.  
         [0052]    [0052]FIG. 9C is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the system has retracted the transverse vertical stage, known as UpDown, causing the sample gripper to move further vertically upwards.  
         [0053]    [0053]FIG. 9D is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the system has rotated the pneumatic 90° rotary stage, known as Rotary, causing the sample gripper to rotate toward the sample positioner.  
         [0054]    [0054]FIG. 9E is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the horizontal stage has moved the sample gripper in a long horizontal translation, causing the sample gripper to move to a predetermined distance toward the sample positioner.  
         [0055]    [0055]FIG. 9F is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the SmallMove stage has moved the sample gripper a short horizontal translation to place the sample assembly in contact with the mounting post of the sample positioner.  
         [0056]    [0056]FIG. 9G is a partial front view of the integrated robotic crystal mounting and alignment system showing most of the major subsystems, where the horizontal stage has moved the sample gripper in a long horizontal translation, causing the sample gripper to move to a predetermined distance away from the sample positioner, leaving a sample assembly on the sample positioner mounting post.  
         [0057]    [0057]FIG. 10A is a partial front view of the sample positioner, including a tilt plate disposed between a goniometer and the X′ Y′ compound stage.  
         [0058]    [0058]FIG. 10B is a partial front view of the sample positioner, including another tilt plate disposed between the X′ Y′ compound stage and the Z′ stage. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0059]    Definitions  
         [0060]    “Biological crystal” means a crystallized frozen biological material, preferably a cryogenically frozen biological material at near liquid nitrogen temperatures.  
         [0061]    “Biological material” means either a collection of independent molecules, or a material having a non-covalently bound assembly of molecules derived from a living source. Examples include, but are not limited to, complexes of proteins, lipoprotein particles comprised of lipoproteins and lipids; viral particles assembled from coat proteins and glycoproteins; immune complexes assembled,from antibodies and their cognate antigens, deoxyribonucleic acids (DNA), ribonucleic acids (RNA), polysaccharides, etc.  
         [0062]    “Computer” means any device capable of performing the steps developed in this invention to result in an optimal waterflood injection, including but not limited to: a microprocessor, a digital state machine, a field programmable gate array (FGPA), a digital signal processor, a collocated integrated memory system with microprocessor and analog or digital output device, a distributed memory system with microprocessor and analog or digital output device connected with digital or analog signal protocols.  
         [0063]    “Cryogenic” means a temperature at or below that of liquid nitrogen at standard atmospheric pressure, −195.79° C. or 77.36° K.  
         [0064]    “Degree of freedom” means any one of the ways that a mechanical system can change spatial configuration, examples include, but are not limited to rotation, translation, and combinations of rotations and/or translations in one or more axes.  
         [0065]    “Gage vacuum” means a relative pressure lower than ambient atmospheric pressure.  
         [0066]    “Ferromagnetic” means a material capable of exhibiting alignment of atomic or molecular magnetic domains. Such a material is capable of magnetization, and is subjected to forces in the presence of a magnetic field.  
         [0067]    “Goniometer” typically means a device for measuring angles. Herein, it is used somewhat differently in that it rotates a surface to particular angles as instructed by a computer device using internal reference angular measurements.  
         [0068]    “Dewar” means any container for liquefied gases. These containers are traditionally typically double-walled with an evacuated interior having low thermal emissivity surfaces to reduce heat transfer from the container interior to the ambient atmosphere.  
         [0069]    Overview  
         [0070]    The present invention is directed toward the transportation and manipulation of samples of cryogenically frozen biological materials, preferably protein crystals mounted on sample assemblies comprised of a standardized base-pin configuration.  
         [0071]    When the hardware components are computer operated as a fully integrated crystal mounting and alignment system for high-throughput biological crystallography (herein referred to as the “system”), remote sample mounting and alignment of individual samples to a predetermined position in space (for subsequent X-ray illumination) can be achieved in less than 30 seconds with minimal remote operator involvement. In some instances, samples have been remotely mounted and aligned in a cycle of 20 seconds. Since no human operator physical presence is required in the room where active x-ray irradiation is taking place, there is no prolonged sequence of doors or interlocks to safely admit an operator, further increasing the speed of sample cycling.  
         [0072]    The sample assembly used in this invention is comprised of a sample base to which a thin sample tube is attached. The end of the sample tube not attached to the sample base has a sample loop, which contains the sample of frozen biological material to be examined. Typical samples range from 5-200 μm in size. The pin has a loop, which contains a cryogenically frozen sample.  
         [0073]    The system for shipping, storing and handling the samples is comprised of a standardized sample cassette containing positions for a plurality of sample assemblies, preferably 16 sample assemblies each in the present design. The outer dimension (diameter) of these sample cassettes is constrained by the maximum inner diameter of a standard cryogenic transport container. Presently, a preferred standard cryogenic transport container will accommodate seven of these disks representing a maximum of 112 sample assemblies. Improvements on this design would allow even more sample assemblies in a higher packing density arrangement.  
         [0074]    The computer-controlled high-throughput sample mounting and alignment system has been designed as a device optimized for reliable removal of the samples from the liquid nitrogen sample repository system and mounting on a sample positioner. The sample positioner, as further described below, positions the sample after it has been removed from the sample repository to a predetermined position in space through which an x-ray beam will subsequently pass, illuminating the sample and thereby generating crystallographic diffraction patterns. The major subsystems of the sample mounting and alignment system are: the sample positioner, a coordinated set of translational and rotating stages for positioning and orienting the sample gripper (known as the gripper stage), a repository system, a gripper defroster, an optical sample alignment system, and an x-ray camera. This system positions a sample crystal at a precise point in space for x-ray imaging analysis, or crystallography.  
         [0075]    The sample repository system is principally a liquid nitrogen filled storage Dewar removably placed atop a position-addressable repository stage. It accommodates up to four sample cassettes on a cassette deck corresponding to 64 samples per load. The individual sample assemblies are held fixed in the storage Dewar by a machinable magnetic material that attracts and retains the ferromagnetic material in the sample assembly. The storage Dewar is mounted on a two axis movable platform that provides alignment of any one of the 64 samples on the cassette deck  700  (FIG. 7A) beneath the sample gripper (described below). By using a larger Dewar, more sample assemblies can be tested in one loading. Loads of 300 or more sample assemblies may be used.  
         [0076]    In the preferred embodiment, the repository stage has one rotational degree of freedom, and one linear degree of freedom. By using a rotary stage atop a linear stage, valuable real estate is conserved, as the stage never has to move more than half of the diameter of the (preferably axisymmetric) storage Dewar.  
         [0077]    The storage Dewar itself has external position referencing features that precisely and removably locate the storage Dewar atop the rotary and linear stages. The storage Dewar also has an internal referencing system that is aligned with the external position reference features. A removable cassette deck is placed through alignment pins in the internal referencing system. In this fashion, by placing the storage Dewar on the repository stage, individual sample assemblies mounted on the cassette deck can be moved to a specific location required for sample pickup and subsequent mounting. Samples can likewise be moved from one position to another (empty) position either on the same or different sample cassette.  
         [0078]    The sample gripper contains a liquid nitrogen cooled split collet mechanism for grasping the sample assembly with sufficient force to overcome the magnetic attraction between the sample assembly base and the sample cassette base with its magnetic material, as well as to break free of any ice binding between them. The sample gripper is designed to encapsulate the sample tube with the sample crystal-containing sample loop within a liquid nitrogen temperature environment during the brief period when the gripper stage uses the sample gripper to transfer the sample assembly to the sample positioner.  
         [0079]    The sample gripper contains a low thermal capacity (preferably very thin stainless steel) outer shroud, which provides a sheath-flow of warm dry gas to prevent icing and frost formation during exposure to ambient temperature, moisture-laden air. After the sample assembly is mounted, the sample is maintained in a cryostream of dry, near liquid nitrogen temperature gaseous nitrogen during subsequent crystallographic measurements. Sufficient cryostream flow is provided to maintain the sample at liquid nitrogen temperatures despite beam heating induced by the probing synchrotron-produced x-ray heat load and closely juxtaposed ambient atmosphere.  
         [0080]    Removal of the sample assemblies from the sample positioner and replacement in the sample repository is accomplished using the same sample gripper. After removal, the mounting post upon which the sample assembly was just previously mounted could accumulate frost, interfering with the magnetic retention of the next sample assembly. If this frost becomes problematic, the mounting post could be actively heated.  
         [0081]    The sample gripper is periodically warmed to remove any ice accumulation. The gripper defroster provides either heated dry nitrogen gas or heated dry air to the outer shroud of the gripper through a pattern of small holes. Due to the low thermal mass of the outer shroud of the sample gripper (recall that it is preferably very thin stainless steel), defrost typically occurs in 6 seconds or less. The upper part of the sample gripper is furthermore heated, allowing the sample gripper to be left in the liquid nitrogen Dewar indefinitely to keep it cold, but at the same time not damaging the pneumatic actuator mounted on top operating at near room temperature, which cannot be exposed to cryogenic temperatures.  
         [0082]    The gripper stage moves the sample gripper through the mechanical motions used for removal and placement of the sample assembly between the sample positioning stage and the sample repository. The gripper stage comprises two orthogonal pneumatic translational motions (XY) (which could also be motor-controlled) upon which is mounted a third rotational (θ) motion mechanism. A relatively low force pneumatic actuator, mounted on the rotational motion mechanism, provides further independent vertical movement. The low force pneumatic actuator prevents damage from the sample gripper impacting misplaced sample assemblies or frost.  
         [0083]    The gripper stage motions are preferably based in part on pneumatic actuators to achieve precise and rapid motions in the liquid nitrogen cooled environment. The three-degree of freedom, (XYθ) positioner of the gripper stage has been designed to move the sample gripper with the required positioning precision within the restricted allowable physical envelope of the experimental setup. Electrically powered motors and actuators could potentially replace some or all of these pneumatic actuators, however, pneumatic actuators have proven to be simpler, cheaper, and easier to control and maintain in this application.  
         [0084]    The sample positioner is a three-degree of freedom stage, mounted on a precision goniometer to provide single axis rotation. The three-degree of freedom stage has been custom designed to position a sample assembly mounted on a mounting post at a precise position in the synchrotron x-ray beam, and, in coordination with the goniometer, to rotate the sample about that point. The three degree of freedom stage consists of two orthogonal motor-controlled translational motions (XY) upon which is mounted a third Z axis motion mechanism. By coordinating the motions of the XY stage and the goniometer, a mounted sample can be rotated about a defined point in space despite initial eccentric mounting. The overall hysteresis of the three degree of freedom stage is about 1.5 microns with a stability of about 1 micron. The system has been shown to be superior to existing commercial versions with respect to position precision and stability.  
         [0085]    The optical alignment system that provides feedback for precise alignment of the sample is based upon a high-resolution zooming camera that is optically aligned to a point in space that will be subsequently illuminated by the synchrotron x-ray beam. A software reference pixel location for the center of the x-ray beam is initially established. Visual images of the mounted crystal sample are then compared with this reference. Any necessary positional corrections are calculated and executed by the sample positioner to position the sample over the software reference position.  
         [0086]    The software and user interface designs have been structured to accommodate alternative centering feedback data based, for instance, on the measured x-ray intensities or data quality as indicated by the x-ray data collection imaging device.  
         [0087]    It should be noted that the system described herein could be used for positioning cryogenically stored samples to a particular point in space for many alternative types of measurements. These measurements may be done by observing the sample at a single prescribed point in space, or by observing the sample at a variety of rotational angles as it is rotated by the goniometer. Depending on the relative time durations of the sampling measurement, the sample may be statically paused at each measurement, or may be continually rotated. An example of a continual rotation measurement could be that of short pulse-width laser illumination using short pulses having microsecond, nanosecond, or picosecond pulse-widths. With such short pulse-widths, even a moving sample appears as if it were stationary.  
         [0088]    Data collection may be used on any experimental device outputting information that can be used as a measurement. Examples include, but are not limited to: x-ray crystallography, photonics-based tomography, photonics-based diffraction, surface spectroscopy, fluorescence correlation spectroscopy, photon arrival time interval distribution analysis, fluorescence resonance energy transfer methods, mass spectrometry, and evanescent wave methods, scanning probe microscopy, taccimetry, profilometry, and atomic force microscopy.  
         [0089]    Sample Assembly  
         [0090]    Referring now to FIG. 1, a sample assembly  100  is comprised of a sample  110  captured in a sample loop  120 . The sample  110  is preferably frozen at cryogenic temperatures, such as that of liquid nitrogen at standard atmospheric pressure, −195.79° C. or 77.36° K. The sample loop  120  is in turn attached by adhesive  130 , preferably alpha cyanoacrylate, to sample tube  140 . The sample tube  140  is attached to sample base  160  by insertion into hole  150 . The sample base  160  has an upper land  180  for retention, and a lower recess  170  for mounting.  
         [0091]    Sample base  160  is ferromagnetic, preferably easily free-machining ANSI  1118  steel with zinc electroplating after machining for corrosion resistance. The sample tube  140  is preferably stainless steel or other low thermal conductivity material, preferably already containing an attached 50 to 1000 μm nylon sample loop  120 .  
         [0092]    Sample Cassette  
         [0093]    Referring now to FIGS. 2A, 2B and  2 C, a sample cassette  200  holding one sample assembly  100  is shown. The sample cassette  200  has a cassette base  210  into which a keyed shaft  220  is affixed on one end  225 . A magnet  250 , preferably a machinable magnet of polymeric matrix material, is inserted into the cassette base  210 , preferably in a recess  255  in the cassette base  210 . The machinable magnet  250  has one opening  230  aligned with each corresponding cassette base opening  265 . Sample assemblies  100  are then able to be loosely inserted into cassette base  210  through openings  265  and be retained by the upper surface  260  of magnet  250 , with cooling liquid nitrogen able to flow through each magnet  250  opening  230  and cassette base  210  opening  265  to directly contact, and hence, cool the sample assembly  100  through direct contact with lower recess  170  (shown in FIG. 1).  
         [0094]    In this embodiment of the sample cassette  200 , 16 sample assemblies can be accommodated in a regular pattern forming two concentric circles. However, any other regularized or nonregularized pattern (not shown in FIG. 2B) could be used as well.  
         [0095]    Keyed shaft  220  is present to align a cassette cover  300  (described below) so that it does not damage the samples  110  when they are covered for transport or storage. Many other functionally equivalent methods, using multiple unkeyed shafts in a pattern, or external jigs, would also achieve the result that cassette cover  300  placement onto the sample cassette  200  would not damage any of the samples  110 . The attachment of keyed shaft  220  is preferably by a threaded screw into a threaded recess in the keyed shaft, however, many other mechanically equivalent attachment methods would also work, including, but not limited to press fit, shrink fit, adhesive attachment, welding, or threading.  
         [0096]    Sample Cassette Cover  
         [0097]    Referring now to FIGS. 3A, 3B and  3 C, a sample cassette cover  300  is a cylindrical shape with a keyed opening  310  passing through a threaded section  330 . A cylindrical neck  340  portion protrudes above the main top surface  320 , and is centered on the sample cassette cover  300  center  305 . An indexing feature  350  serves as clearance for an alignment feature to be discussed below.  
         [0098]    In the sample cassette cover  300  bottom surface  355 , pluralities of sample assembly recesses  360  provide protection to samples assemblies  100  (shown in FIG. 1) placed in them. In this embodiment, an outer vent ring  370  and an inner vent ring  380  vent the sample assembly recesses  360 ; these details are not shown with hidden lines in FIG. 3A to minimize confusion of hidden lines. The outer vent ring  370  and an inner vent ring  380  are respectively ported to the exterior of the sample cassette cover  300  with pluralities of outer vent ports  375  and inner vent ports  385 . This venting arrangement allows for the flow of liquid nitrogen to each of the sample assembly recesses  360 , ensuring that sample assemblies  100  (shown in FIG. 1) as well as the cassette cover  300  are amply cooled by liquid nitrogen. It also allows the venting of nitrogen gas, which might otherwise build up inside the sample assembly recesses  360 .  
         [0099]    Sample Cassette Assembly  
         [0100]    Referring now to FIG. 4A, a sample cassette assembly  400  is shown comprised of a sample cassette cover  300  assembled in place over a sample assembly  100 , and retained by sample cassette  200 . Now referring to both FIGS. 4A and 4B, sample assembly  100  is magnetically retained on the upper surface  260  of magnet  250 .  
         [0101]    During installation, the sample cassette cover  300  first encounters keyed shaft  220 . The sample cassette cover  300  must first be rotationally aligned relative to sample cassette  200  so that the keyed shaft  220  may translate into the keyed opening  310 . Since the keyed shaft  220  is taller than the installed sample assembly  100 , the sample  110  cannot be damaged by the sample cassette cover  300  during normal installation of the sample cassette cover  300  as sample assembly recesses  360  in the sample cassette cover  300  slides over each of the sample assemblies  100 .  
         [0102]    The sample cassette cover  300  has one sample assembly recess  360  for each matching cassette base  210  opening  265 . The depth of the sample assembly recesses  360  exceeds the retained height of the sample assembly  100 . Additionally, the sample assembly recesses  360  have smaller diameters than the width of the sample assembly  100 , so that the bottom surface  355  of the sample cassette cover  300  positively retains the sample base  160  by contacting upper land  180 .  
         [0103]    When assembled as shown in FIG. 4A, the sample assemblies  100  are positively sandwiched between the sample cassette cover  300  and the sample cassette  200 . Now referring additionally to FIG. 3C, the outer vent ring  370  and the inner vent ring  380  allow for filling of the sample assembly recesses  360  with liquid nitrogen through outer vent ports  375  and inner vent ports  385 .  
         [0104]    In one embodiment of the invention, the assembled cassette cover  300  and sample cassette  200  are inverted so that sample assembly recesses  360  form liquid nitrogen repositories, thus keeping the biological crystal sample  110  immersed in liquid nitrogen and maintaining the sample at a cryogenic temperature until all of the liquid nitrogen has boiled away. In this embodiment, several minutes of room temperature exposure can be tolerated by the sample with minimal temperature rise when moving samples from shipping container to cryogenic sample repository.  
         [0105]    Additional mechanical components (not shown) clip and retain the sample cassette cover  300  to the sample cassette  200 , although many other methods of positively retaining the parts together exist, and are readily designed by those skilled in the mechanical design arts.  
         [0106]    Sample Cassette Carrier  
         [0107]    Refer now to FIGS. 5A and 5B, where a cassette carrier  500  is depicted. A top hook  510  is press fit into a low thermal conductivity sleeve  515 , preferably comprised of fiberglass or other low thermal conductivity low temperature plastic, which has inserted into it a stainless steel rod  520 . The stainless steel rod  520  is welded to a tab  525  formed by two narrow slots  530  on either side. The tab  525  is part of a sheet  580  that encompasses, and is attached to, about half the diameter of a plurality of shelves  550 . The method of attachment could be any that survives repeated thermal shocks from room temperature to liquid nitrogen temperature. In this embodiment, three screws  590  are used to attach each shelf  550  to the sheet  580 .  
         [0108]    The distances between the shelves  550  form a set of shelf openings, or landings  535 . For purposes of illustration, the top-most landing  535  is vacant, without a sample cassette assembly  400 . The other six shelve openings in the diagram each show sample cassette assemblies  400  present.  
         [0109]    In FIG. 5B, each shelf  550  has an inner  551  and outer  552  radius concentric about a center point  555 , which is the same center point for a mounted sample cassette assembly  400 . During insertion of the sample cassette assembly  400 , cylindrical neck  340  (shown in FIGS. 3A and 3B) of the sample cassette cover  300  slides into obround slot  560 , where retaining spring  570 , secured by fastener  575 , retains the sample cassette cover  300 . Retaining spring  570  deflects partially into retaining spring recess  565 . When fully inserted, cassette cover  300  center  305  is roughly concentric with center point  555 .  
         [0110]    Cryogenic Transport Container  
         [0111]    Before shipping, the cryogenic transport container  600  of FIG. 6 is initially precooled with liquid nitrogen for shipping per the manufacturer&#39;s directions. Some of these precooling steps can take as long as four hours to complete.  
         [0112]    Referring now to FIG. 6, cryogenic transportation container  600  has a removable insulation plug  610 , which inserts into a corresponding cylindrical bore  630  in bulk insulation  620 . When cylindrical bore  630  is initially empty except for nitrogen gas and liquid, the cassette carrier  500 , having one or more sample cassette assemblies  400 , is first inserted. Subsequently, the removable insulation plug  610  is installed. Further liquid nitrogen, if needed, is added per the manufacturer&#39;s directions. Depending on the manufacturer of cryogenic transportation container  600 , ambient temperature exposure, and the detailed construction of the container, cryogenic temperatures below −150° C. can be maintained during shipment for up to 200 hours.  
         [0113]    Cassette Deck  
         [0114]    Refer now to FIGS. 7A and 7B. The cassette deck  700  is shown. The cassette deck  700  has a center reference hole  710 , three mounting holes  760 , and a larger diameter keying hole  750  to uniquely orient the cassette deck  700  with respect to hardware incorporated into the sample repository (not shown). Orientation pins  720  are preferably press fit into the cassette deck  700 . The orientation pins  720  provide a unique orientation of sample cassettes  200  which are positioned between the two other outer positioning pins  740 . This pattern is replicated in four quadrants. In one quadrant there is just an outline of the area  745  that is normally occupied by a sample cassette  200 .  
         [0115]    By using this arrangement, in conjunction with the sample cassette  200  design, all sample assemblies  100  are uniquely positioned with respect to the cassette deck  700 . The unique positioning allows for unattended sample assembly  100  mounting and demounting using a sample gripper described below.  
         [0116]    Sample Gripper  
         [0117]    The sample gripper  800  is shown in FIG. 8. An upper actuator flange  865  is connected to a lower actuator flange  805 . The actual mechanism of the actuator is not shown, as these are readily commercially available as either solenoidal electrical or pneumatic force/displacement devices. The preferred actuator is pneumatic. The lower actuator flange  805  has been modified so that in conjunction with gripper flange  815 , a port  810  is formed. The port  810  attaches to a plenum  812 . The plenum  812  allows a continuous gas connection with a series of cylindrical openings  820 , which at their apex, connect to small openings  825 . Input gas can be attached to port  810 , fill plenum  812 , pass through a plurality of cylindrical opening  820 , and emit at small openings  825  into an outer shroud area  832 . The outer shroud area  832  is formed by an inner tube  830  and outer tube  835 , which are both attached to gripper flange  815 . The outer tube  835  necks down to a removable close fitting shroud tube  840 . The inner tube  830  has a very low thermal mass, low heat capacity material, and is preferably both very thin walled, and made of stainless steel. At the lower end of the inner tube  830 , is attached a collet sleeve  845 . The collet sleeve  845  is preferably silver soldered (not shown) to the stainless steel inner tube  830 . The inner tube  830  must be sufficiently thick so as to keep from bucking under axial compressive forces generated by the collet sleeve  845 .  
         [0118]    The split collet  850  has an actuation movement relative to the collet sleeve  845 , closing the split collet  850  about a sample assembly  100  located within its grasp. When split collet  850  is retracted upwards, the collet sleeve  845  causes compressive closure of the collet actuation surface  846 , with consequent high force retention of the sample assembly  100  located within the split collet  850  in a collet recess  847 . The split collet  850  is pulled upward by collet adapter  854 , which connects the split collet  850  to the collet tube  855 . The collet tube  855  is in turn connected to the actuator adapter  860 . The actuator adapter  860  connects collet tube  855  to the actuator tube  870 . Thus a vertical motion of the actuator tube  870  causes the same vertical motion of the actuator adaptor  860 , collet tube  855 , collet adapter  854 , and in turn the split collet  850 .  
         [0119]    The temperature of the split collet  850  is measured by a temperature sensing element  852  located at the bottom of a temperature sensing hole  851 . Wires (not shown to minimize drawing clutter) ascend upward through the temperature sensing hole  851 , through a matching hole in collet adapter  854 , and exit the sample gripper  800  through the center bore  875  of actuator tube  870 .  
         [0120]    To cool the sample gripper  800  down to temperatures appropriate for sample assembly  100  pickup (e.g. liquid nitrogen temperature), the collet sleeve  845  end of the sample gripper  800  is immersed in liquid nitrogen. At this time, there is no sample assembly  100  present. A small gage vacuum of 3-4 inches of mercury is drawn on port  810 , which is communicated through the small openings  825  to the outer shroud area  832 . Vent port  833  in inner tube  830  allows the vacuum to pull liquid nitrogen up and around split collet  850 . Since the collet is split, liquid nitrogen fills the interior  853  of the split collet  850 . The temperature-sensing element  852  is used to register when the split collet  850  temperature has cooled sufficiently for sample assembly  100  pickup.  
         [0121]    When the sample gripper  800  is moving the sample assembly  100 , room temperature dry nitrogen gas is fed through port  810  into the outer shroud area  832  to preclude frost buildup on inner tube  830  or split collet  850 . The frost buildup is prevented by the simple expedient of keeping moisture away from any of the cold surfaces of the inner tube  830  or split collet  850 , by the flow of the dry nitrogen gas, preferably in laminar flow.  
         [0122]    The sample gripper  800  is drawn showing most details, with the center section abbreviated by a cut  890 .  
         [0123]    Integrated Crystal Mounting and Alignment System for High-throughput Biological Crystallography  
         [0124]    Referring now to FIG. 9A, we see the integrated crystal mounting and alignment system for high-throughput biological crystallography  900  as viewed down an axis parallel to the incoming synchrotron x-ray beam  901 . A frame  902  connects the various subsystems, and will not be fully described other than to say that it must be sufficiently stable and stiff to keep most components accurately positioned to within about 1 μm.  
         [0125]    The subsystems include a repository stage, a gripper stage, a sample positioner, a cryostream unit, a video alignment subsystem, and a collimation and beam blocking subsystem. These subsystems are more fully described sequentially below.  
         [0126]    The repository stage is comprised of the Y 1 , linear stage  904 , which is mounted on the frame  902 . Atop the Y 1 , linear stage  904  is attached rotary stage  906 , which rotates about a vertical axis of revolution. Storage Dewar  908  removably attaches to the rotary stage  906  at a repeatable position and orientation using standard mechanical and precision engineering fixturing techniques that are well known in these arts. The storage Dewar  908  is nominally filled with enough liquid nitrogen  910  to amply cover any sample assemblies  100  that may be present in any sample cassettes  200 . The cassette deck  700  is mounted on position referencing components not described here, which allows sample cassettes  200  to be addressably positioned relative to the frame  902  with high accuracy.  
         [0127]    The gripper stage moves the sample gripper  800  relative to the frame  902 . It comprises a horizontal stage  926 , an UpDown stage  924 , and a rotary stage  920 . A long travel Y 2  linear stage, known as the horizontal stage  926 , moves horizontally (along the plane of the paper) a vertical stage mounted transversely thereon, known as UpDown  924 . The UpDown stage  924  comprises a platform  922  that serves as a mounting base for a 90° rotary stage  920 , known as Rotary, preferably a pneumatic 90° rotary stage. The 90° rotary stage  920  top mounts a small travel, lighter actuation force pneumatic stage called SmallMove  918 , which serves as a mount for the sample gripper  800 . Rotary stage  920  rotate the sample gripper  800  between a downward position (as shown in FIG. 9C) and a horizontal position (as shown in FIG. 9D).  
         [0128]    The sample positioner is comprised of a high precision rotary stage known as a goniometer  928 , which is mounted on the frame  902 . The goniometer  928  rotates an angle θ (theta) along an axis typically parallel to the horizontal plane. Upon the goniometer  928  is mounted a. A Z′ stage  932  with a magnetic mounting post  934  mounts onto the X′Y′ stage  930 . At the particular goniometer  928  angle θ depicted in FIGS.  9 A- 9 G, the X′Y′ stage  930  moves in and out of the plane of the paper (the X′ axis), and up and down in the plane of the paper (the Y′ axis). These motions will rotate with continued rotations of the goniometer  928  in a typical kinematic rotating frame of reference. The mounting post  934  is mounted upon, and moved by, compound X′Y′ stage  930 . The mounting post  934  is spring preloaded (to prevent hard sample assembly  100  mountings), and rides on two sets of three bearings, each set of which forms an equilateral triangle.  
         [0129]    In an alternate embodiment of the system, the sample positioner is further comprised of a tilt plate  929  (as depicted in FIG. 10A) disposed between the high precision rotary stage known as the goniometer  928 , and the compound X′Y′ stage  930 , to which the Z′ stage  932  is attached as before. The effect of the tilt plate  929  is to rotate the Z′ stage  932  eccentrically with respect to the axis of rotation of the goniometer  928  so that the positioner rotates the sample  110  about an axis non-orthogonal with the compound X′Y′ stage  930  and the Z′ stage  932 . The tilt plate  929  preferably forms a tilt angle of at least 15°, more preferably of at least 10°, yet more preferably of at least 5°, still more preferably of at least 2°, and most preferably of at least 1°. The alignment and centering operations described below may be used either directly by ignoring the effect of the tilt plate  929 , or by including the angle of the tilt plate  929  in the alignment and centering algorithms. Regardless of the eccentricity induced by the tilt plate  929 , the properly centered sample  110  will maintain location within the x-ray synchrotron beam  901  when the beam is operational, and the same spatial position when the x-ray synchrotron beam  901  is non-operational, as further described below. Rotations of the sample  110  will normally require operation of the compound X′Y′ stage  930  and the Z′ stage  932  for correct positioning.  
         [0130]    In yet another embodiment (shown in FIG. 10B) another tilt plate  931  may be disposed between the compound X′Y′ stage  930  and the Z′ stage  932  to effect the eccentricity described above. In this further embodiment, the other tilt plate  931  would preferably form a tilt angle of at least 15°, more preferably of at least 10°, yet more preferably of at least 5°, still more preferably of at least 2°, and most preferably of at least 1°.  
         [0131]    In operation, a sample assembly  100  (already removed here, and thus not shown) is retained by the mounting post  934  by magnetic attraction. The mounting post  934  could readily be heated to prevent frost formation, but heating has not yet proven necessary. The coordinated motions of the rotation addressable goniometer  928 , the compound X′Y′ stage  930  and the Z′ stage  932  allow a sample to be rotated in space about a predetermined point, preferably the incoming synchrotron x-ray beam  901 .  
         [0132]    A commercially available cryostream unit  936  emits a stream of near liquid nitrogen temperature nitrogen gas to cool the sample  110  when the sample assembly  100  is mounted on the mounting post  934 . The cryostream unit  936  is actuated along axis  938  so as to: 1) prevent interference with the sample gripper  800  when mounting or unmounting sample assemblies  100  (not shown), 2) not optically occlude the camera  948  optical aperture, and 3) not interfere with the projection of the incoming synchrotron x-ray beam  901 , regardless of whether or not the x-ray beam  901  is operational.  
         [0133]    The video alignment subsystem is comprised of a commercially available backlighter  956  on a vertically extendible backlighter stage  958 . During alignment, the backlighter stage  958  raises the backlighter  956  so that the sample  100  (not shown, but mounted on mounting post  934 ) is back-lit when viewed by the high resolution macroscopic zooming video camera  948 . During x-ray irradiation, the backlighter  956  is retracted so that it is out of the direct x-ray beam  901 .  
         [0134]    Collimation and beam blocking is typically required to respectively form a parallel incoming x-ray beam of a controlled diameter, or stop the beam altogether. For collimation to work properly, a small aperture must be aligned with the incoming x-ray beam. Collimation and beam blocking of the x-ray beam is effected by using the collimator vertical actuator  940  to raise a piezoelectric actuator  942 , to which an x piezoelectric actuator  944  is attached, which moves a selection of collimators having various diameters and beam blocks  946  into the x-ray beam  901 . Note that the collimators and beam blocks  946  with their associated actuators, are in a non-interfering plane from the backlighter  956  so that each may operate independently without collision.  
         [0135]    To collimate or locally block the synchrotron x-ray beam  901 , the various diameter collimators and beam block  946  is moved up into the x-ray beam  901  by the collimator vertical actuator  940 , and is precisely positioned for optimal collimation by small, precise movements effected by piezoelectric actuators  942  and  944 .  
         [0136]    Application of the Invention to Mount a Sample Assembly  
         [0137]    Refer now to FIGS.  9 A- 9 F, which is a sequence of partial front views of the integrated crystal mounting and alignment system for high-throughput biological crystallography  900  with most of the major subsystems illustrated. The sequence of FIGS.  9 A- 9 F show some of the major steps involved in conveying a sample assembly  100  to the sample positioner mounting post  934  for alignment using camera  948  and subsequent data collection from crystallographic diffraction of the incoming synchrotron x-ray beam  901  by the sample  110  (not shown).  
         [0138]    Initially, the sample gripper  800  must be cooled sufficiently to safely grasp a sample assembly  100 . The sample gripper  800  is initially partially immersed in the liquid nitrogen  910  so that the temperature-sensing element  852  (shown in FIG. 8) is cooled to a temperature of at least −150° C. prior to continuing with the sample assembly pickup. This initial sample gripper  800  immersion is located away from any resident sample assemblies  100  present in the storage Dewar  908 . For this purpose the sample gripper  800  is immersed in the storage Dewar  908  until a set temperature is achieved. This initial cooling procedure typically requires over a minute. However, in normal operation, the cooling down procedure is only required once in a set of samples since the sample gripper  800  remains cold with repeated immersions in the liquid nitrogen  910 . When a sample assembly  100  pickup occurs, further additional cooling will take place as the sample gripper  800  is immersed in the storage Dewar  908 .  
         [0139]    FIGS.  9 A-G correspond to the integrated crystal mounting and alignment system  900  moving through a sequence of configurations as described more fully below. It is appreciated that there are many alternative sequences and minor variations that may be used to effect the same operations.  
         [0140]    In FIG. 9A, the system is in the process of using the sample gripper  800  to grasp a sample assembly  100  in the storage Dewar  908 . From there, it will move the sample assembly  100  to the sample positioner mounting post  934  for alignment and x-ray probing. At this step in the protocol, a sample assembly  100  has been grasped by sample gripper  800 . Prior to grasping the sample assembly  100 , the following setup steps have occurred: (1) the gripper  800  is released; (2) the heater  950 , collimator beam blocks  946 , the cryostream unit  936  are retracted so as to not interfere with other movements; (3) Rotary  920  is rotated down so that the sample gripper  800  assumes a vertical orientation; (4) UpDown  924  has been moved down; (5) SmallMove  918  has been downwardly extended; and (6) the gripper stage has moved the sample gripper  800  to a location in the storage Dewar  908 , and immersed the sample gripper  800  in the liquid nitrogen  910  until the sample gripper  800  has reached a temperature of −130° C. as measured by the temperature sensing element  852  (indicated but not shown in FIG. 8). Gripping is accomplished by moving the sample gripper  800  over a selected sample assembly  100  located in a predefined location pattern in the storage Dewar  908 . The sample gripper  800  is actuated, causing the split collet  850  to exert a pressure on the sample assembly  100 . The friction generated by this pressure is sufficient to overcome the frost buildup and/or magnetic attraction of the sample assembly  100  to the sample cassettes  200  in the storage Dewar  908 .  
         [0141]    Next, in FIG. 9B, the system has retracted the SmallMove  918  pneumatic stage, causing the sample gripper  800  to be moved vertically upwards. Depending on the liquid nitrogen  910  fill level in the storage Dewar  908 , the sample assembly  100  may have cleared the liquid nitrogen  910  surface, as depicted here. Not shown in the drawings is an automated liquid nitrogen fill apparatus, to keep the liquid nitrogen  910  fill level at a specified level. The storage Dewar  908  is typically nearly full, so that the sample gripper  800  is partially immersed even when SmallMove  918  is retracted to its highest vertical position.  
         [0142]    Next, in FIG. 9C, the system has actuated the transverse vertical stage, known as UpDown  924 , causing the platform  922  to move further vertically upwards, carrying the sample gripper  800  vertically upwards, so that the grasped sample, assembly  100  vertically clears the top of the storage Dewar  908 .  
         [0143]    Next, in FIG. 9D, the system has rotated the pneumatic 90° rotary stage  920 , known as Rotary, causing the sample gripper  800  and sample assembly  100  to rotate 90° clockwise and point toward the sample positioner mounting post  934 . Sample assembly  100  is essentially collinear with mounting post  934 .  
         [0144]    Next, in FIG. 9E, the sample gripper  800  has moved by the long horizontal travel linear stage, known as the Y 2  stage  926 , causing the sample gripper  800  to move to a predetermined distance toward the sample positioner mounting post  934 , in a vector parallel to the Z′ stage  932  motion (horizontally as indicated in FIG. 9E), with sample assembly  100  approaching the mounting post  934  in advance of sample gripper  800 .  
         [0145]    Next, in FIG. 9F, the SmallMove  918  pneumatic stage has been extended in a short horizontal translation to mount the sample assembly  100  gently in contact with the magnetically attractive mounting post  934  of the sample positioner. After contact with the mounting post  934  is accomplished, the gripper assembly  800  releases its grip on the sample assembly  100 . Now the cryostream unit  936  is actuated along line  938  to approach the sample  110  mounted on the sample assembly  100  mounted on the mounting post  934 . The cryostream unit  936  is then activated to cool the sample assembly  100  sample  110  (still cryogenically shielded in the gripper assembly  800 ) which is now roughly located at a spatial position where the x-ray beam  901  will subsequently irradiate.  
         [0146]    Next, in FIG. 9G, the sample gripper  800  has been moved a predetermined distance away from the sample positioner mounting post  934  by the long travel Y 2  linear stage, known as the horizontal stage  926 . Since the sample gripper  800  has already been released, the sample assembly  100  remains on the sample positioner mounting post  934  due to magnetic attraction. Since the cryostream unit  936  has already been activated, the sample  110  is released from the sample gripper  800  cryogenic interior directly into the cold stream of the cryostream unit  936 . In this manner, the sample  110  is never exposed to ambient room temperature.  
         [0147]    Initial Sample Reference Position Setup  
         [0148]    In this system, a zooming microscopic camera  948  views provides information to correctly position the mounted sample  110  (shown earlier in FIG. 1). In order to establish this spatial position, a three-dimensional position in space must be aligned with the incoming synchrotron x-ray beam  901  while the beam is active. With the x-ray beam  901  active, a pin, or other small axisymmetric alignment shape is moved into the beam until the beam is partially occluded. An x-ray imaging camera, operationally similar to the high resolution macroscopic zooming video camera  948 , except that x-rays are detected, and zooming is not likely necessary, is used to image the axisymmetric alignment shape. Either the synchrotron beam current must be greatly reduced, or more preferably the x-ray beam intensity is greatly attenuated by an attenuator to prevent burnout of the camera for this operation.  
         [0149]    With the x-ray beam  901  producing an image on the x-ray imaging camera, the axisymmetric alignment shape (typically a small bead, or pin with a point) is moved by coordinated movements of the X′ and Y′ compound stage  930 , and Z′ stage  932  axes. Eventually, the movements are manually (or possibly computer controlled) coordinated until the alignment shape enters the field of view of the x-ray imaging camera. By coordinated movements of the X′ Y′ compound stage  930 , and Z′ stage  932 , and rotation of the goniometer  928 , a three-dimensional reference location for the center of the x-ray beam relative to the X′ Y′ compound stage  930 , and Z′ stage  932  is developed. Once the alignment shape is aligned to the x-ray beam, the beam may be turned off, or blocked completely, as it is no longer necessary for the initial alignment, as the x-ray beam center relative to the X′ Y′ compound stage  930 , and Z′ stage  932  is already known.  
         [0150]    The backlighter stage  958  now raises the backlighter  956  so that the alignment shape is back-lit when viewed by the high resolution macroscopic zooming video camera  948 . Since the x-ray beam may now be turned off, it is safe for personnel to enter the potentially irradiated hutch area to manually (or by remote control of appropriate tilt or pointing actuators) align the camera  948  to view the alignment shape. Once the camera  948  is correctly positioned to view the alignment shape in roughly the center of field of view, no further camera  948  alignment should be necessary. The camera  948  is then used to view the alignment shape. The alignment shape is viewed, and the pixel location corresponding to the portion of the alignment shape previously positioned in the center of the x-ray beam is recorded. This is the pixel location of the beam center at a particular zoom magnification. The zoom magnification is then increased to a higher magnification so as to more completely fill the field of view of the camera  948 , and the pixel location again corresponding to the portion of the alignment shape previously positioned in the center of the x-ray beam is recorded.  
         [0151]    Note that the zooming camera  948 , can typically only determine position in two dimensions as imaged pixel locations. Typical imaging devices can only focus within a particular optical depth of field, which, depending on the depth of field of the optical image, can provide additional information regarding a distance from the optical objective by either being in or out of focus. In this instance, the zooming camera  948  is preferably parfocally focused on the alignment shape; so that it remains in focus at all zoom magnifications.  
         [0152]    Note that, at this time, there are two reference positions being used: the software pixel location of the sample as viewed by the zooming camera  948 , which is a two dimensional pixel reference position related to the field of view of the zooming camera  948 ; and the spatial center of the alignment shape relative to the positioner, a three dimensional reference. These software pixel locations, and the spatial position of the alignment shape relative to the positioner which has previously be collocated through the center of the x-ray beam, are subsequently used to rapidly align samples for x-ray crystallography.  
         [0153]    Sample Position Setup  
         [0154]    Once the sample assembly  100  has been positioned on the positioner as depicted in FIG. 9G, the sample  110  must be aligned to be concentric with the x-ray beam  901  when it is activated. The previously obtained software pixel locations (two dimensional information) and the spatial center of the alignment shape relative to the positioner (three dimensional information) are used to correctly center the sample crystal  110  for x-ray crystallography. These coordinates are cooperatively used to position the sample crystal  110  relative to the positioner so that the sample crystal  110  may be rotated about the point where the x-ray beam  901  passes when it is activated.  
         [0155]    In this system, a zooming microscopic camera  948  initially views the sample  100  (shown earlier in FIG. 1) at minimum magnification, and produces video images of the mounted sample crystal  110 . The images are read by a video frame-grabber to provide a digital image of the sample. By either manual or computer algorithmic operation, the frame pixel coordinates of the center of the sample may be determined. The sample positioner (comprised of X′ Y′ compound stage  930 , Z′ stage  932 , and the goniometer  928 ) is then actuated to translate the sample  110  to the software reference pixel location arrived at during initial zooming camera  948  alignment in a plane roughly (preferably within 45°, more preferably within 30°, yet more preferably within 15°, and most preferably within 5 20  ) parallel to the image plane of the camera  948 . The sample positioner then rotates the sample  110  through angular movement of the goniometer  928 , and the process is repeated. At each rotation, the translational increments of the X′ and Y′ compound stage  930 , and Z′ stage  932  axes are recorded. Subsequently, these coordinates are used to arrive at the true three-dimensional spatial center of the sample  110  crystal relative to the positioner. The entire process is then optionally repeated at higher zoom magnification levels as necessary. This sample  110  spatial center may be determined relative to the positioner in as few as two rotations due to the short depth of field of the camera  948  at maximum zoom.  
         [0156]    The distance from the alignment shape center to the sample  110  center forms an offset vector. The offset vector is used to coordinate the movement of the sample  110  by relative movements of the X′ Y′ compound stage  930 , and Z′ stage  932  at each rotation. In this manner, the sample  110  may be rotated in space through a point collocated with the center of the x-ray beam  901  when it is operated. The x-ray beam  901  is not allowed to strike the sample  110  during alignment, so as to minimize any synchrotron-produced x-ray  901  heating or x-ray induced chemical degradation.  
         [0157]    Sample X-ray Crystallography  
         [0158]    In the normal operation of the system, the sample positioner is moved so that the sample  110  is positioned to a location where synchrotron generated x-rays  901  will be emitted after sample  110  alignment. After the sample  110  is aligned as described above, the synchrotron x-ray beam  901  is unblocked, allowing x-rays to irradiate the sample  110 , which can then be rotated to any arbitrary angular position while remaining centered within the x-rays beam  901 . After x-ray crystallography is complete, the synchrotron x-ray  901  source is again blocked or shuttered so as to interrupt delivery of the x-ray beam, effectively turning off the x-ray beam. This blocking and unblocking of the x-ray source is important since the x-rays can induce damage to the crystalline sample, thereby degrading the data collected.  
         [0159]    Sample Dismounting  
         [0160]    Following the FIGS.  9 G- 9 A in reverse is essentially the sequence of motions used by the unmounting protocol, where the sample assembly  100  initially mounted on the sample positioner mounting post  934  is finally replaced in the storage Dewar  908 .  
         [0161]    For the remaining sample assemblies  100 , the sequence of steps previously described is performed in reverse, from  9 G (the present data collection state), to  9 F,  9 E,  9 D,  9 C,  9 B, and  9 A where the sample assembly  100  is replace in the Dewar  908 . The sample gripper  800  is retracted sufficiently to clear the sample assemblies  100 , but still partially immersed in the liquid nitrogen  910 . The Y 1 , linear stage  904  and rotary stage  906  are actuated to position the next sample assembly  100  beneath the sample gripper  800 . At this point, the process repeats for sampling of the remaining sample assemblies  100 .  
         [0162]    After a period of use, the sample gripper  800  may become frost covered. A warm up or defrost protocol is used to remove any accumulated frost from the sample gripper  800 . Although the spatial configuration for defrosting is not directly shown in any Figure, it is readily visualized. During the defrost cycle, a heater  950  is extended by an In/Out stage  952 , and warm dry nitrogen gas is emitted from the heater  950  onto the sample gripper  800 , which has previously been moved into position for defrosting.  
         [0163]    Conclusion  
         [0164]    All publications, patents, and patent applications mentioned in this application are herein incorporated by reference to the same extent as if each individual publication or patent application were each specifically and individually indicated to be incorporated by reference.  
         [0165]    The description given here, and best modes of operation of the invention, are not intended to limit the scope of the invention. Many modifications, alternative constructions, and equivalents may be employed without departing from the scope and spirit of the invention. In particular, the sequence of motions used in mounting and demounting sample assemblies  100  may be re-sequenced in a myriad of permutations without deviating from the general goal to be achieved so long as components and subsystems do not destructively interfere with each other.

Technology Category: 7