Integrated crystal mounting and alignment system for high-throughput biological crystallography

Abstract

A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.

1 . A sample assembly comprising: 
 a. a sample base,    b. a sample tube, wherein one of two ends of the sample tube is affixed to the sample base;    c. a sample loop affixed to the other end of the sample tube, wherein    d. a sample is affixed by the sample loop.    
   
   
       2 . The sample assembly of  claim 1  wherein said sample base is ferromagnetic.  
   
   
       3 . The sample assembly of  claim 2  wherein said sample base is essentially axisymmetric.  
   
   
       4 . An apparatus for transporting cryogenically frozen biological crystal samples with the sample assembly of  claim 1 , comprising: 
 a. a cryogenic transport container, and    b. a cassette carrier with a plurality of shelves retained by a sheet, the cassette carrier removably securing a plurality of sample cassettes within the cassette carrier unit, with each of the sample cassettes independently secured,    wherein the cassette carrier is packaged in the cryogenic transport container for shipping at cryogenic temperatures to transport a plurality of cryogenically frozen biological crystal samples with the sample assembly of  claim 1     
   
   
       5 . The apparatus for transporting cryogenically frozen biological crystal samples of  claim 4  wherein the cassette carrier comprises: one or more of the sample assemblies of  claim 1 .  
   
   
       6 . The apparatus for transporting cryogenically frozen biological crystal samples of  claim 4  wherein the sample cassette further comprises: 
 a. a cassette base with at least one through opening for loosely receiving a sample assembly;    b. a magnet inserted into a recess in the cassette base, the magnet containing an opening aligned with, and opened to the corresponding cassette base through opening;    c. at least one keyed shaft mounted in the cassette base; wherein said cassette base can loosely accept one of the sample assemblies in each cassette base through opening and is removably retained by said magnet; and    d. a cassette cover with 
 1. a keyed opening to accept the cassette base keyed shaft to substantially prevent rotation, and  
   2. a recess corresponding to each cassette base through opening, wherein each of said recesses positively retains the corresponding sample assembly and protects the sample attached to the sample assembly.    
   
   
       7 . The cassette base of  claim 6  wherein said cassette base is ferromagnetic.  
   
   
       8 . The cassette base of  claim 7  wherein said magnet is further comprised of a flexible machinable magnetic material.  
   
   
       9 . A sample gripper comprising: 
 a. a grasping means for retractably capturing one of the sample assemblies of  claim 1 ,    b. a cooling means for drawing liquid nitrogen up and around the captured sample assembly, and    c. a flowing means for sheathing the captured sample assembly from direct contact with ambient moist air.    
   
   
       10 . A sample gripper capable of gripping and moving one of the sample assemblies of  claim 1 , comprising: 
 a. a retractable split collet with a collet actuation surface, a collet interior, and a collet recess;    b. an inner tube, mounted to a gripper flange, which compresses the collet actuation surface upon retraction of the split collet, causing the collet recess to contract upon the sample assembly of  claim 1 , thus forcibly retaining the sample assembly;    c. a shroud tube of larger diameter than the inner tube, forming an outer shroud area between the inner tube and the shroud tube; and    d. a plurality of small openings in the gripper flange connecting to a port, communicating a flow from the port to the inner shroud area; wherein a warm dry gas can be applied to the port to keep frost from forming on the inner tube and the split collet, and a gage vacuum can be applied to the port to force liquid nitrogen into the collet interior when the outer shroud area is immersed in liquid nitrogen.    
   
   
       11 . A sample repository comprising: 
 a. a storage Dewar, with an external referencing feature substantially immovably attached to said Dewar;    b. an internal referencing feature substantially immovably attached to the storage Dewar;    c. wherein said internal referencing feature is substantially aligned with said external referencing feature such that a translation and rotation of said external referencing feature produces in the internal referencing feature a known movement substantially the same as said translation and rotation.    
   
   
       12 . A sample assembly comprising: 
 a. a sample base,    b. retaining means, whereby a sample is affixed to the sample base.    
   
   
       13 . The sample assembly of  claim 1  wherein said retraining means comprises: 
 a. a loop of material retained by a tube affixed in the sample base;    b. wherein the loop of material retains the sample.    
   
   
       14 . The sample assembly of  claim 12  wherein said sample base is essentially axisymmetric.  
   
   
       15 . An apparatus for transporting cryogenically frozen biological crystal samples on the sample assemblies of  claim 12 , comprising: 
 a. a cryogenic transport container, and    b. a transport means removably inserted into the cryogenic transport container, whereby a plurality of the sample assemblies of  claim 12  may be transported in a cryogenic state.    
   
   
       16 . The apparatus for transporting cryogenically frozen biological crystal samples of  claim 15  wherein the transport means further comprises: 
 a. a cassette base with at least one through opening for loosely receiving one or more of the sample assemblies of  claim 12;     b. a magnet inserted into a recess in the cassette base, the magnet containing an opening aligned with, and opened to the corresponding cassette base through opening;    c. at least one keyed shaft mounted in the cassette base; wherein said cassette base can loosely accept one of the sample assemblies in each cassette base through opening and is removably retained by said magnet; and    d. a cassette cover with 
 1. a keyed opening to accept the cassette base keyed shaft to substantially prevent rotation, and  
 2. a recess corresponding to each cassette base through opening, wherein each of said recesses positively retains the corresponding sample assembly and protects the sample attached to the sample assembly.