Abstract:
A cartridge-based cryogenic imaging system includes a sample handling system. This system uses a kinematic base and cold interface system that provides vertical loading to horizontally mounted high-precision rotation stages that are able to facilitate automated high-resolution three-dimensional (3D) imaging with computed tomography (CT). Flexible metal braids are used to provide cooling and also allow a large range of rotation. A robotic sample transfer and loading system provides further automation by allowing a number of samples to be loaded and automatically sequentially placed on the sample stage and imaged. These characteristics provide the capability of high-throughput and highly automated cryogenic x-ray microscopy and computed tomography.

Description:
RELATED APPLICATIONS 
     This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 61/096,502, filed on Sep. 12, 2008, which is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     Microscopy has played an important role in science and technology. One area where light and electron microscopy techniques have been indispensable is biological sciences. Light microscopy has allowed observation at 200 nanometer (nm) scale resolution, while electron microscopy has demonstrated atomic scale resolution with thin-sectioned specimens. Recent developments in x-ray microscopy have allowed thick hydrated samples with tens of nanometer resolution. 
     For most effective observations, cells and biological tissues must be imaged in a hydrated state in order to have the highest fidelity representation of the living state. But when imaging hydrated organic specimens using ionizing radiation, radiation damage often limits the quality and resolution of the images that can be obtained. The solution is to work with hydrated specimens that have been rapidly frozen so as to minimize the formation of crystalline ice in the specimens. 
     Cryogenic specimen handling methods were first developed in electron microscopy in 1974 by K. Taylor and R. Glaeser, see Electron diffraction of frozen, hydrated protein crystals. Science, 106:1036-1037, 1974, and by the late 1980s there was a considerable knowledge base in place regarding rapid freezing and cryo electron microscopy. Cryomicroscopy is also expected to be important in trace element mapping in fluorescence microprobes, since specimen drying is likely to affect the distribution of the diffusible ions that can play such important physiological roles. Cryogenic methods have also found wide spread use in protein crystallography, where the usual practice involves a cryogenic gas stream directed onto a specimen to cool it within an atmospheric pressure, room temperature environment. 
     SUMMARY OF THE INVENTION 
     Aspects of the present invention concern cryogenic sample handling systems for high-resolution microscopy applications, such as x-ray, optical, and/or electron microscopy. By using a cartridge sample mount and robotic sample handling system, highly automated sample transfer and loading can be achieved. These are essential components of a high-throughput automated cryogenic microscopy that maintains the temperature of the specimen at between 80 and 170 degrees Kelvin, for example, or lower. 
     This system uses a kinematic mount and cold interface system that provide vertical loading to horizontally mounted high-precision rotation stages that are able to facilitate automated high-resolution three-dimensional (3D) imaging with computed tomography (CT). Flexible metal braids are used to provide cooling and also allow a large range of rotation. A robotic sample transfer and loading system provides further automation by allowing a number of samples to be loaded and automatically sequentially placed on the sample stage and imaged. These characteristics provide the capability of high-throughput and highly automated cryogenic x-ray microscopy and computed tomography. 
     In general, according to one aspect, the invention features a cryogenic imaging system, comprising a kinematic base that receives cartridges on a cryogenic base, with each cartridge carrying a specimen. The system further includes a positioning stage and a warm-cold interface between the positioning stage and the cryogenic base. A flexible thermal linkage is included between the cryogenic base and a refrigeration source to provide conductive cooling. A robotic loading and transfer system accepts one or more cartridges and loads the cartridges onto the cryogenic base, and a microscopy system images specimens in the cartridges. 
     In one example, this microscopy system comprises an x-ray source for generating an x-ray beam that irradiates the cartridges on the cryogenic base and a detector for detecting the x-ray beam from the cartridges. 
     In embodiments, the positioning stage positions the cryogenic base along three axes and also rotates the cryogenic base. The warm-cold interface comprises a ball and groove configuration for low thermal conductivity. The flexible thermal linkage includes one or more metal wires. 
     In general, according to another aspect, the invention features, a cryogenic x-ray imaging method, comprising generating an x-ray beam that irradiates specimens, detecting the x-ray beam from the specimens, holding the specimens on a cryogenic base in the x-ray beam, positioning the specimens in the beam by moving the cryogenic base, and cooling the cryogenic base via a flexible thermal linkage between the cryogenic base and a refrigeration source. 
     The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings: 
         FIG. 1  is a schematic diagram of a cryogenic x-ray imaging system according to the present invention; 
         FIG. 2  is a schematic diagram of a kinematic base  150  and cryogenic shield according to the present invention; and 
         FIG. 3  is a scale drawing of a warm-cold interface of the kinematic base according to the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The radiation biology literature uses the International System (SI) unit of the Gray (equal to one Joule of absorbed energy per kilogram of mass) as its unit of radiation dose. At 100 keV in transmission electron microscopes (TEM), an electron exposure of 1 e − /nm 2  corresponds to a radiation dose of about 3×10 4  Gray. From both protein crystallography and electron microscopy or crystallography data, diffraction spots corresponding to atom resolution information begin to fade at radiation doses in the 10 7 -10 8  Gray range, with diffraction spots corresponding to 2-10 nm structural information fading at 10 8 -10 9  Gray. 
     In electron microscopy, radiation doses of about 1000 e − /nm 2  or about 3×10 7  Gray lead to the onset of “bubbling” in the specimen, where water is broken down into OH −  and H +  and the hydrogen gas will form voids in the ice matrix when it is unable to diffuse through the ice; enhanced diffusion may explain the observation that in some cases liquid nitrogen temperatures are preferred to liquid helium temperatures. 
     In cryogenic x-ray microscopy, excellent structural preservation has been observed at radiation doses as high as 10 10  Gray, without “bubbling”. The absence of “bubbling” is presumably due to some combination of the lower dose rate relative to cryogenic electron microscopy (giving more time for diffusive release of H +  through the ice matrix) and the lower ratio of absorption in water versus organic materials at the 520 eV “water window” photon energies used in these experiments. These energies are just below the energy of the oxygen absorption edge. Sensitive coherence-based “speckle” measurements have shown that there is no measurable shrinkage of frozen hydrated cryo specimens, at least at doses up to 10 10  Gray. 
     These studies emphasize the essential nature of cryogenic approaches for x-ray microscopy of hydrated organic specimens such as cells and tissues. For tomography, the specimen must remain constant as projections from different viewing angles are acquired so that all the individual views provide true representations of the object that is to be reconstructed. For spectrum imaging/spectromicroscopy image sequences, the specimen must not shrink or otherwise change its morphology so that all images can be registered to each other to yield a spectrum per pixel for subsequent analysis. For trace element mapping, it is important to not lose side groups that might be bound to the very elements one is hoping to measure and quantify. Cryogenics is essential to realize these important x-ray microscopy techniques. 
       FIG. 1  shows a cyrogenic x-ray imaging system  100  that has been constructed according to the principles of the present invention. 
     In more detail, the system has an x-ray source  110  that generates an x-ray beam  112 . In the one embodiment, the source  110  is a beamline of a synchrotron x-ray generation facility. In other embodiments, smaller sources are used, such as laboratory sources. For example, laboratory sources that generate x-rays by bombarding a solid target anode with energetic electrons are one possible source that could be used, including microfocus and rotating anode type sources. 
     In still other embodiments, the imaging modality is other than x-rays. In one such embodiment, the source generates an electron beam or an optical beam. 
     The condenser  114  collects and focuses the x-ray beam  112  from the source  110 . For the full field imaging setup, a suitable illumination of the sample  10  is required. This is most conveniently achieved by the use of a zone plate condenser, capillary, or Wolter optic. 
     When the imaging modality is an electron beam or an optical beam, other condenser systems are used such as focusing magnets or refractive/reflective optics, respectively. 
     A chamber or housing  116  is used to create a controlled environment for the specimens. The x-ray beam  112  enters the housing  116  through a housing input window  118 . In some examples, the inside of the housing  116  is cooled to cryogenic temperatures such as less than 274 Kelvin (K) and usually about 77K, the temperature of liquid nitrogen, or colder. It is therefore insulated from the surrounding atmosphere. In other examples, the housing  116  is capable of holding a vacuum. In such cases, a vacuum pump system  144 , such as a system including a turbomolecular pump, is in communication with the housing  116  via a pipe  152  in order to pull a vacuum within the housing  116 . 
     The x-ray or other beam  112  is projected onto the specimen that is contained within a cartridge  10 . The cartridge  10  is held on a horizontally extending base  150 . This base  150  is a kinematic unit that positions the sample cartridge  10  along both the x, y, and z. axes. The kinematic base  150  further has the capability of rotating the specimen/cartridge  10  around the y axis to enable the acquisition of tomographic projections at different angles to the axis  115  of the x-ray beam  112 . 
     The kinematic base  150  is held on a mounting plate  122 . Then, on top of the kinematic base  150  and mounting plate  122 , a cryogenic shield  140  surrounds the cartridge  10 . This cryogenic shield  140  includes a shield input beam port  142  through which the beam  112  passes to the sample cartridge  10 . A shield output beam port  144  of the cryogenic shield  140  allows the beam to exit after passing through the specimen/cartridge  10 . 
     A refrigeration source  124  is preferably located within the housing  116 . It is connected via a heat transfer element  125  to the cryogenic shield  140 . In one example, this refrigeration source  124  is a refrigeration unit. In other examples, the refrigeration source  124  is a dewar or other container containing liquid nitrogen. The heat transfer element  125  is constructed from a high thermal transfer material such as braided copper cable. 
     The beam  112  from the sample cartridge  10  exits the cryogenic housing  116  through a housing output port  126 . An x-ray objective  128  collects x-rays  112  from the specimen and images the x-ray beam  112  onto a detector system  130 . In a current embodiment, the objective  128  is a Fresnel zone plate. 
     In examples where the beam  112  is an optical beam, the image is formed with refractive or reflective optics. 
     The detector system  130  is preferably a high-resolution, high-efficiency scintillator-coupled CCD (charge coupled device) camera system for detecting x-rays from the specimen. In one example, a camera system  130  as described in U.S. Pat. No. 7,057,187, which is incorporated herein by this reference in its entirety, is used. 
     A robotic loading and unloading system is provided in the preferred embodiment. Microscopy specimens are delicate and have a poor chance of surviving repeated handling. For this reason it is good practice to mount them once in a cartridge, and then handle that cartridge in subsequent operations. Cartridges  10  are loaded into the system  100  on a shuttle  176 . A robot system  170  then individually loads and unloads the cartridges  10  onto kinematic base  150 . 
     Cartridge covers are preferably used to prevent contamination buildup on the specimen during the various handling steps. Further the cartridges  10  preferably all share a common design in the top that is grabbed by the robot system&#39;s gripper  174  and for the end that is inserted into the kinematic base  150 . Preferably, a unified base can support a variety of specimen mounting schemes. For example, one type of cartridge might use clamping rings for standard 3 mm TEM grids, another might use a micro-fabricated silicon stalk to minimize x-ray fluorescence background while maintaining good dimensional stability and thermal conductivity, while yet another might use a thin-walled capillary for the mounting of tomography specimens. 
     A horizontal linear travel stage  175  is used to move the shuttle base  176  from a position well out of the way of the kinematic base  150 , to a series of locations that put each of the cartridges  10  or cartridges slots in the shuttle  176  directly above the center of the kinematic base  150  and the loading port  146  formed in the cryo shield  140 . A robot arm  172  the picks the cartridges with the gripper  174  and transfers the cartridges  10  between the kinematic base  150  and the cartridge slots of the shuttle  176 , accessing the kinematic base  150  via the loading port  146 . 
     The robot system  170  preferably has a vertical linear travel stage  172  upon which the gripper  174  is mounted. A fiberglass insert provides thermal isolation for the gripper end  174  which is in turn conductively cooled using a copper braid to a dewar, in one example. This requires access to the specimen from above, and either enough “headroom” in the chamber  116  to hold the vertical linear travel stage upon which the robot grabber is mounted, or a port with a linear feedthrough. 
       FIG. 2  shows the details of the kinematic base  150  and shield  140 . 
     A small, low-mass cryo base  152  is mounted on a high-temperature rotation and/or nanopositioning stage  154  that is supported on the mounting plate  122 . The nanopositioning stage  154  positions and moves the cryo base  152  and thus the specimen in the cartridge  10  along the x, y and z axes to position the region of interest of the specimen within the x-ray beam  115 , and also preferably rotates the specimen about the y axis. 
     A warm-cold interface  156  separates the nanopositioning stage  154  from the cryo base  152 . It is constructed from an interface material and has a geometry with low mechanical creep and low thermal conductance. 
     The cold cryo base  152  mainly “sees” the area of the cryo shield  140 , radiative heat transfer into the specimen  10  is thus greatly reduced. The dominant heat transfer path becomes that of the warm-cold interface  156 , which has both high mechanical stiffness and low thermal conductivity. In this way only modest cooling power (well below 100 milliWatts (mW)) must be supplied to the cryo base  152 . This is preferably supplied by “weak” heat conductors  158  which involve very low mechanical coupling force between the cryo base  152  and the cryo shield  140  for rotations up to ±90 of the cryo base by the movement of the nanopositioning stage  154  or translations of several millimeters. (Initial cool-down involves moving a raised surface on the cryo base into strong contact with a cold “finger” from the cryo shield). This cryo base  152  is normally kept cold in the microscope  100  at all times. 
     Key thermal design considerations for this approach include the following: Gas conductivity becomes negligible at pressures of below about 10 −4  torr, and pressures well below this are needed to minimize ice buildup on cryo specimens. Because the thermal conductivity of high-purity copper increases at lower temperatures, weak heat conductors  158  comprise a number of copper wires that can provide good thermal cooling power, such as less than 500 wires. As an example, 150 wires in parallel, each 100 micrometers (μm) in diameter and 50 millimeters (mm) long, can provide 120 mW of cooling power over a temperature difference of 10 K between the cryo shield  140  that is cooled by the heat conduction through heat transfer element  125  to the refrigeration source  124 . The wire conductors  158  are much longer than the distance (D) between the outer wall of the base  152  and the shield  140  so that the base  152  is moved and rotated freely by the nanopositioning stage  154 . In one example, the length of the wire conductors  158  are more than 5 times distance D. 
       FIG. 3  shows an embodiment of the warm-cold interface  156 . 
     In more detail, the warm-cold interface  156  comprises an upper member  312  on which the cryogenic base  152  is placed and lower member  310  that is secured to the nanopositioning stage  154 . 
     Both the upper member  312  and lower member  310  have low emissivity coatings, especially on the two surfaces that face each other. For example, using a conservative estimate for the emissivity (ε) of highly polished gold of ε=0.05 (as opposed to the ε=0.018-0.035 values given in published tables), the radiative heat transfer between two 25 mm disks when one is at 100 K and the other at 300 K is only about 11 mW. Thermal conductivity then becomes the dominant path. This is controlled by using a ball-on-flat mounting approach and with both low conductivity materials. Preferably, both the upper member  312  and lower member  310  are constructed from fused silica or an infrared glass. AMTIR-1, from Amorphous Materials Inc, for example, has 5× lower conductivity and nearly equal stiffness. 
     The current ball-on flat approach to thermal isolation uses three recesses  316 A,  316 B,  316 C are formed in the lower member  310 . In the preferred embodiment, each of these three recesses  316 A,  316 B,  316 C comprises three planar surfaces in the general form of a pyramid. In an alternative design, the three recesses  316 A,  316 B,  316 C are in the form of a cone. Low thermal conductivity balls or spheres  314 A,  314 B,  314 C are each placed in a corresponding one of the three recesses  316 A,  316 B,  316 C. The upper member  312  has a corresponding, mirrored series of recesses that receive the balls  314 A,  314 B,  314 C. This creates rigid yet low thermal conductivity interface. 
     Depending on the choice of materials and the force applied, a thermal conduction power of no more than 20-50 mW can be obtained between the cryo base  152  and the warm nanopositioning stage  154 . This ball-on-flat system implemented in the upper member,  312 , lower member  310  and balls or spheres  314 A,  314 B,  314 C also has the advantage of being naturally suited to a kinematic mounting system, where no mechanical stress is induced that would otherwise lead to mechanical drift. Other design configurations can include the commonly used ball-groove-flat kinematic mounting system. 
     While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.