Abstract:
An improved cryogenic specimen holder for imaging and analysis facilitates imaging at very high tilt angles with a large field of view. A retractable specimen holder tip protects the specimen during transport. An optimized Dewar design is positioned at a fixed, tilted angle with respect to the axis of the holder, providing a means of continuously cooling the specimen irrespective of the high tilt angle and amount of liquid nitrogen present in the vessel. The Dewar neck design reduces entrapment of nitrogen gas bubbles and its shape prevents the spilling of liquid nitrogen at high tilt angles. The specimen holder has a retractable tip that completely encapsulates the specimen within a shielded environment internal to the specimen holder body. The cooling and specimen transfer mechanisms reduce thermal drift and the detrimental effects of vibrations generated by both the evaporation of liquid nitrogen present in the Dewar and other environmental effects.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a continuation-in-part of Stabacinskiene, et al., U.S. patent application Ser. No. 12/845,486, filed Jul. 28, 2010. It claims the benefit of United States Provisional Patent Application No. 61/515,359, filed Aug. 5, 2011. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    This invention relates to specimen holders for maintaining a specimen at a low temperature during visualization, imaging or analysis. More specifically, it relates to holders utilized for transmission electron microscopy (TEM) and particularly to the Dewar used for the containment of liquid nitrogen and the subsequent cooling of the TEM specimen placed on a retractable cartridge and positioned within a cryogenically cooled shield. 
         [0004]    2. Description of the Prior Art 
         [0005]    There are a variety of imaging technologies which have been developed to observe and analyze specimens at the molecular and/or atomic level. These include optical, electron, x-ray and photon microscopy together with associated imaging and analysis. Cryo electron microscopy, or Cryo EM, is a powerful technique for studying frozen hydrated biological specimens in transmission electron microscopy. To generate results with minimum artifacts, specimens are rapidly frozen and then imaged in a fully hydrated state. This reduces the detrimental effects of fixatives or stains commonly used to prepare microscopy specimens at more ambient temperatures. Cryo EM is extremely beneficial for studying proteins, viruses, macromolecular assemblies, vesicles/liposomes, organelles, and cells in more native conditions. In order to obtain a TEM image the specimen needs to be sufficiently thin to allow for the transmission of electrons therethrough. As with all conventional TEM imaging, the TEM image is formed by electron interactions with the specimen. The quality and usability of TEM images increases with improved resolution. Biological specimen quality is highly dependent on the method of preparation. Typical preparation includes rapidly vitrifying a thin film of suspension by freezing it in an extremely cold material, such as liquid ethane. The specimen is transferred to the TEM in the frozen state, at a consistently low temperature, and is then examined in its fully hydrated state. Alternatively, a bulk specimen can be cryoprotected, high pressure frozen, cryosectioned and transferred to the imaging or analytical device. The mounting/support and transfer of specimen to a TEM, as an illustrative example, for subsequent imaging has typically been performed with the help of a cryotransfer specimen holder. These prior art TEM specimen holders, consistent with TEM specimen holders, generally, comprise longitudinal rods of a given length to support and mount the specimen near one end of the rod. The rod end of the TEM specimen holder is inserted into the microscope and placed between the components of the electron optics. As is well known to those skilled in the art, these components, by physical necessity, allow for only a very small and dimensionally constrained specimen or support contained thereon. With respect to biological specimens, as indicated above, the specimen must also be maintained at a low temperature, preferably below −155° C. during the transfer, imaging and analysis process and while located within the constrained space of the microscope. This is because it is desirable to maintain the ice component of the specimen in an amorphous state below −155° C. Above −155° C., the ice will adopt a crystalline form, which is detrimental to imaging and analysis. During transfer and while the specimen holder is not inserted into the imaging or analysis device, it is also essential to physically protect the specimen, as it is highly vulnerable to physical and environmental damage and/or contamination from water vapor and other sources. 
         [0006]    The physical port of a microscope, for example, which accepts and restrains the specimen holder is known as the goniometer. It is a micromanipulator for moving the specimen holder, and thus the specimen itself, in the X, Y, Z dimensions, and the α and β tilt directions. This helps to position the specimen at the focal point of electron beam, thus allowing the desired region of the specimen at the precise angle/orientation necessary in order to observe the relevant characteristics of the specimen. As stated, the goniometer is used to tilt the specimen holder inside the column of the microscope relative to the electron imaging beam. Angular displacement of the specimen while mounted within the microscope is an extremely important feature for cryotomography in order to obtain three-dimensional, or 3D, information for life science applications. This same methodology is applicable to the physical sciences. To generate 3D information, the sample is imaged at various tilt angles and/or orientations. The two-dimensional projections are then recombined to produce a composite 3D image. 
         [0007]    While mounted on the specimen holder, the specimen is maintained at the required low temperature through the use of a cooling medium which reduces the temperature of portions of the specimen holder and the specimen itself. This cooling medium, typically liquid nitrogen, is stored in an insulated container mounted to one end of the specimen holder, typically identified as a Dewar. The Dewar is a component of the specimen holder and it comprises a highly reflective inner vessel enclosed within an evacuated housing. The vacuum within the evacuated housing, coupled with the materials utilized for the construction of the device, thermally isolates the inner vessel from the housing. A rod or other thermal conductor assembly provides the thermal contact between the specimen and the receptacle for the cooling medium present in the Dewar. The conductor is typically constructed of a material having high thermal conductivity such as silver or copper. The cooling medium is utilized to remove the heat from the specimen support and specimen to maintain the same at the necessary low temperature. 
         [0008]    Modern day transmission electron microscopes are capable of achieving atomic scale resolution. However, image quality and resolution are highly dependent upon reduction of specimen displacement through vibrations and drift induced from the holder during imaging or analysis. In practice, these environmental and other conditions need to be minimized to achieve optimum resolution. At atmospheric pressure, liquid nitrogen boils at −196° C. In many prior art specimen holder designs, a solid conductor rod within the holder is in contact with the Dewar and the specimen support in the form of a receptacle tip. As the liquid nitrogen or other cooling medium boils off under ambient atmospheric conditions, vibrations are formed by the turbulence in the medium. The conductor rod transmits these vibrations directly to the specimen tip, causing the specimen to vibrate during imaging and analysis. The rigid contact between the Dewar and the cooling assembly of the prior art devices further introduces physical stresses on the device during thermal expansion and contraction. 
         [0009]    Most Dewar devices are open to ambient atmosphere to permit the boil off of the liquid medium and to minimize the retention of expanding warm gas medium, which has deleterious effects relating to pressure within the vessel. The devices are rigidly constructed such that any displacement of the specimen holder results in a corresponding displacement of the specimen. In tomography, higher tilt angles of the specimen during imaging yield more accurate and detailed 3D reconstructions. The ability to increase the tilt of the specimen holder is limited, however, by the possibility of spillover of the liquid medium from the Dewar, as well as a thermal gradient induced into the walls of the container, which create unsatisfactory results, including vibrations, drift and, potentially, spilled liquid nitrogen in the laboratory. Avoidance of this condition substantially limits the ability to tilt the specimen. 
         [0010]    Another shortcoming of prior art cryogenic specimen holder designs is the ability to constrain thermal distortions of the device itself. Thermal variations lead to the expansion or contraction of materials. The thermal gradient present between the Dewar and the specimen cartridge, as a function of distance and time, as well as changing environmental conditions along the length of the holder, causes unpredictable and dynamic dimensional changes, resulting in specimen drift from the nominal position within the imaging device. It is desirable, therefore, to maintain the assembly at a constant, low steady state temperature with a minimum thermal gradient. Moreover, any thermal contact between the conductor assembly, which extends within, but dimensionally separate from the outer holder barrel, may introduce additional heat or, at a minimum, temperature variations within the system. In addition, since the exterior holder barrel is in contact with the microscope goniometer during imaging, any such contact between the conductor assembly and the outer holder barrel leads to an undesirable thermal path from the microscope, a large warm heat sink, and the specimen, causing additional drift. 
         [0011]    During imaging and analysis of the specimen, constant evaporation of the cooling medium also results in a drop in the volume of liquid present in the Dewar. This necessitates the physical interface between the cooling assembly and the Dewar to be located at a point most likely to be in direct contact with the cooling medium, which is the bottom of the Dewar. At any given time, a temperature gradient exists along the wall of the Dewar, being coolest at the points of contact directly adjacent the cooling medium and increasing in temperature with increasing distance from the surface of the cooling medium. Loss of direct contact with the cooling medium immediately adjacent the interface between the cooling assembly and the Dewar causes the temperature to rise in the assembly and further exacerbates drift of the specimen during imaging. 
         [0012]    Cryotransfer holders like those described in Swann et al., U.S. Pat. No. 5,753,924, have been developed to maintain samples at the desired temperature and to prevent frost from forming on the specimen during transfer. As illustrated in FIG. 1 of Swann, the holder 10 includes a holder body 12 and a specimen tip 14 with a source of cooling for the tip. The specimen tip 14 includes a support grid 16 of thermally conductive material and a tab portion 24 that is adapted to be secured to the specimen tip (see FIG. 5). A cryoshield is formed by an opening in the specimen holder tip 14. To load the specimen grid 16 into the tip 14 of holder body 12, tab 24 is inserted through a slot 40 in specimen tip holder 14 (see FIG. 4) which forms a cryoshield for the specimen. The specimen grid 16 is moved from an extended position to a retracted position by a drawbar 46, which is in thermal contact with support grid 16 and extends along the longitudinal axis of holder body 12. 
         [0013]    The cryotransfer holder of Swann suffers from several disadvantages. In this design, the drawbar is in rigid thermal contact with the support grid and the holder specimen tip, thus acting as a potential source of heat load. This requires much greater energy extraction to cool the entire assembly to the desired temperature. Extraneous heat may cause thermal expansion and contraction of the cooling rod and drawbar. The rigid contact directly transfers all such movement to the specimen resulting in a loss of image resolution because of drift and vibrations. Finally, the proprietary design of the specimen grid, which requires insertion of a tab to secure the grid into the cryoholder, makes the system incompatible with any independent, standard transmission electron microscope specimen disks, which are 3 mm in diameter, or any other shapes now in use or in development. 
         [0014]    The Dewar assembly of the type described by Gallagher et al., U.S. Pat. No. 5,302,831 has been developed in an attempt to maintain constant contact between the liquid nitrogen supply and the cold finger assembly. FIG. 2 of Gallagher illustrates a trapezoidal or truncated triangular shaped Dewar 50 with a cold finger assembly 14. A copper braided strap 122 is secured on one end through a lug 118 to the bottom wall 94 of liquid nitrogen vessel. The other end of the copper braided strap 122 is secured through a lug to a portion of the cold finger assembly 114. The triangular Dewar 50 is tapered at the bottom so that liquid nitrogen always fills the front portion of vessel 90 adjacent to the intersection of the front wall 98 and the bottom wall 94. Dewar 50 is angled so that the liquid nitrogen is forced into a portion of Dewar 50 that is in constant contact with the cold finger assembly 114 and strap 122 at a fixed angle in the range of 0° to 60°. The vessel 90 top wall 102 has an opening 103 for the addition of liquid nitrogen to Dewar 50. 
         [0015]    The Dewar described in Gallagher is used to cool a radiation detector cold finger, which has different physical constraints than a TEM specimen holder, including, most importantly, that a specimen is not cooled thereby, merely a detector. This Dewar is not built to satisfy any of the specimen vibration and drift requirements necessary for proper TEM imaging. Also, cooling a radiation detector does not require or even permit the Dewar to be tilted about the longitudinal axis of the cold finger. Gallagher&#39;s trapezoidally shaped Dewar will not contain the liquid nitrogen at higher tilt angles, causing the liquid nitrogen to boil and spill over leading to substantial sample drift. The three corners present near the Dewar neck will also trap nitrogen gas, causing vibrations, which in turn would also limit image resolution. 
         [0016]    There remains a need, therefore, for a cryogenic holder adapted to be rotated about the longitudinal axis of the holder, which will help tilt the specimen within the microscope while maintaining optimum thermal transmission between the liquid cooling medium and the specimen at all times. The specimen support cartridge has to be vibrationally isolated from the cooling medium and the exterior sections of the holder have to be thermally isolated from the cooling medium to provide additional reduction of sample drift and vibration and to reduce thermal stresses within the device. In addition, there is a need for a holder that includes a cartridge specimen tip that receives a standard sized specimen and optionally and controllably shields the specimen during transport to prevent contamination. 
       SUMMARY OF THE INVENTION 
       [0017]    A cryogenic specimen holder is disclosed for a variety of imaging and analytical applications. The holder improves the quality of imaging and analytical information by overcoming major challenges related to holder drift and vibration. In accordance with one embodiment of the invention, a specimen holder includes a Dewar for receiving and storing a cooling medium, such as liquid nitrogen. The Dewar is constructed as a conventional vacuum flask with an interposed partial vacuum between an inner liquid support vessel and an outer protective housing. A set of thermal conductor ribbons, braids or the like, preferably flexible, including combinations thereof, are incorporated into the thermal transfer system which extracts heat from the specimen. In one embodiment, one such member is attached to the lowest point of the Dewar. The other end of this assembly is connected to a conductor rod which is in thermal contact with the sample cartridge. A similar flexible assembly may form the thermal junction between the conductor rod and the cartridge housing. Each of the thermal ribbons, for example, in the preferred embodiment, forms a vibration damper between the various components of the cooling assembly. Additional points of thermal contact may also be utilized to enhance cooling of the specimen. In the preferred embodiment, a secondary, insulating cooling conduit is formed from at least one non-structural tube, which extends throughout the length of the holder shaft. Longitudinal manipulation of the specimen is accomplished with a separately cooled actuation rod extending within the primary cooling conduit which provides additional cooling for the specimen stage. An actuation assembly mechanism allows longitudinal displacement of the actuation rod while maintaining a thermal interface with the Dewar. The actuation assembly mechanism is preferably a rack and pinion gear mechanism, but may also alternatively include a resilient member such as a bellows, in conjunction with a cam actuator. The actuation rod may be actively cooled by a flexible thermal ribbon or from the surrounding support. The inner vessel of the Dewar is preferably cylindrical and positioned at a fixed, tilted angle offset from the longitudinal axis of both the holder rotation and the exterior protective housing of the Dewar itself. This forms, in the preferred embodiment, an arcuate V-shaped bottom surface of the inner vessel which is dimensionally unchanged in orientation during tilt of the holder assembly. The flexible conductive ribbons are affixed such that they are in contact with the inner vessel at the lowest point of the arcuate bottom surface of the cylindrical shape. As a result, the cooling medium, despite reductions in volume during use, is always in contact with mounting point of the conductor ribbons to the Dewar regardless of the holder angle. Any shape or conformation, however, is acceptable which achieves this containment of the liquid cooling medium adjacent to the thermal conductor. The Dewar therefore ensures stable operating conditions at tilt angles ranging from 0° to +/−90°, and in most applications, up to +/−80°, limited only by the design of the opening in the Dewar. Additional angular displacement could therefore be possible with unique Dewar exterior shape designs which may be applicable to particular applications. 
         [0018]    In an additional embodiment, the neck of the inner vessel is profiled such that it does not trap any nitrogen gas through the tilt range of the holder. The vessel design also prevents the liquid nitrogen from spilling over at high holder tilt angles. This provides stable, vibration free operating conditions. 
         [0019]    The holder further may include a sample cartridge that can be retracted into a cryogenically cooled shield located within the holder tip. Moreover, the sample cartridge may be modular and removable from its support carrier within the holder. When in a retracted position, the cartridge acts as a thermal shield around the cryogenic sample and prevents sample contamination and damage. The present sample holder can therefore be successfully utilized to transfer the cryogenically prepared samples, which may be of any shape or conformation, including but not limited to, the current standard 3 mm disk, from any mounting or storage facility to an electron microscope. Additionally, the modular sample cartridge may be loaded or otherwise manipulated prior to insertion into the holder with the specimen in place. After insertion into the protected environment of the electron microscope, the cartridge can then be extended to the operating position for observations within the microscope. The preferred sample tip is adapted to receive and restrain standard sized and shaped samples and/or specimens, including grids, which are well known in the art. 
         [0020]    In an additional embodiment, the retractable cartridge is spring loaded with respect to its mounting on the actuation rod as well as the entire shaft within the holder tip. This allows for the cartridge to settle in a stress free equilibrium position during the initial thermal cycling and improves thermal contact and transfer between the associated components. In another embodiment, a spring pushes the cartridge on a pair of rollers made from insulating material that provide complete thermal isolation from the outer barrel of the holder. To provide further vibration damping the thermal contact between the cartridge and the cooling conductor rod is provided with a flexible thermally conductive ribbon assembly. The flexible ribbons also compensate for the dimensional change of the cooling rod assembly due to various thermal stress and strains. 
         [0021]    The holder, together with its particular features and advantages, will become more apparent from the following detailed description and with reference to the appended drawings. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0022]      FIG. 1  is a side view of the specimen holder of the present invention. 
           [0023]      FIG. 1   a  is an isometric view of a second embodiment of the specimen holder of the present invention. 
           [0024]      FIG. 2  is cross sectional side view of a first embodiment of the specimen holder of the present invention. 
           [0025]      FIG. 2   a  is a cross sectional side view of a portion of a second embodiment of the specimen holder of the present invention. 
           [0026]      FIG. 2   b  is a cross sectional side view of a portion of a third embodiment of the specimen holder of the present invention. 
           [0027]      FIG. 2   c  is a cross sectional side view of a portion of the third embodiment of the specimen holder of the present invention. 
           [0028]      FIG. 2   d  is a cross sectional isometric view of a portion of the third embodiment of the specimen holder of the present invention. 
           [0029]      FIG. 3  is an isometric view of the specimen holder of the present invention. 
           [0030]      FIG. 3   a  is a side sectional view of the actuator assembly of the first embodiment of the present invention. 
           [0031]      FIG. 3   b  is a longitudinal sectional view of a portion of the second embodiment of the specimen holder of the present invention. 
           [0032]      FIG. 3   c  is a longitudinal sectional view of a portion of the third embodiment of the specimen holder of the present invention. 
           [0033]      FIG. 4   a  is a cross sectional side view of the first embodiment of the specimen holder tip with the sample cartridge in the extended position. 
           [0034]      FIG. 4   b  is a cross sectional side view of the first embodiment of the specimen holder tip with the sample cartridge in the retracted position. 
           [0035]      FIG. 4   c  is an end view of the first embodiment of the specimen holder tip. 
           [0036]      FIG. 4   d  is an isometric, sectional view of the second embodiment of the specimen holder tip with the sample cartridge in the retracted position. 
           [0037]      FIG. 4   e  is an isometric, sectional view of the second embodiment of the specimen holder tip with the sample cartridge in the extended position. 
           [0038]      FIGS. 5   a - f  are various isometric views of the Dewar inner vessel containing liquid cooling medium at different tilt angles. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0039]    Referring now to  FIGS. 1 and 1   a , generally, the holder  200  is comprised of three main sections. A housing  202  supports the Dewar assembly and is provided with an extension for the addition of liquid cooling medium and the escape of gas, formed by neck  207  and opening  209 . A holder middle barrel  210  forms the middle section which is adapted for insertion into and restraint by the goniometer, together with the front barrel  212 . Further, the middle barrel  210  and front barrel  212  are designed by size and shape to place the specimen in the appropriate location within the microscope. It is to be specifically noted that the size and particular arrangement of the barrel components may vary based upon the physical constraints of microscopes manufactured by different suppliers. A front barrel  212  extends outwardly from the middle barrel  210  in a direction opposite that of the housing  202  and contains the specimen cartridge. As shown in  FIG. 1   a , a front section  202 A is provided in association with housing  202  having a particular contoured transition from the housing  202  and middle barrel  210 . This particular shape is intended to increase structural integrity while maintaining a low weight. 
         [0040]    Referring now to  FIGS. 1 ,  1   a  and  3 , the housing  202  further comprises a valve  504  which is utilized to evacuate the insulating space within the housing  202 , as will be described further with reference to  FIGS. 2 and 2   a . Electrical connectors  502 ,  509  are provided to permit the electrical interface between internal heating assembly  502   a  and internal temperature sensor  246 , respectively, described more fully with reference to  FIG. 2  and an external monitoring/control device (not shown). An actuation assembly  249  is provided for the longitudinal displacement of the specimen holder cartridge assembly with respect to the front barrel  212 , as will be described more fully with respect to  FIGS. 2   d ,  3   a ,  3   b ,  3   c  and  4 . The actuation assembly  249  may be constructed of any known design, including a slidably displaceable armature or through rotational knob  248  in a gear or cam system, as would be interchangeable to those skilled in the art. 
         [0041]    Referring now to  FIGS. 2 ,  2   a ,  2   b  and  2   c , the Dewar assembly is comprised of a series of component elements within housing  202 , which, as shown in the second and third embodiments, may comprise a domed rear wall for additional structural support. In all embodiments, an inner vessel  204  is disposed within the housing  202  generally within an evacuated space  211 . Inner vessel  204  is provided with a highly reflective outer surface to minimize the impact of any radiated heat within the housing  202 . Inner vessel  204  is adapted to receive and store a liquid cooling medium, such as liquid nitrogen. The inner surface of the inner vessel  204  is highly polished, smoothed or otherwise coated to avoid surface irregularities which provide nucleation points for the formation of gas bubbles. An adsorbent or absorbent material may be mounted within inner vessel  204  to improve retention of the cooling medium at a particular location. A vacuum is created within the evacuated space  211  in order to provide thermal insulation between the cooling medium and the housing  202 , as is well known to those skilled in the art. This vacuum increases stable performance by minimizing heat effects from convection of heat between the ambient atmosphere beyond the housing  202  and the cooling medium. Inner vessel  204  is supported entirely by the connection to the housing  202  located at opening  209  through serpentine reentrant tubing  208 , as described more fully below. The extreme top of the inner vessel  204  and reentrant tubing  208  is sealed and secured with by joint  209   a  at the opening  209 , which may be of any known type including glue, solder, welding and the like. This reentrant tubing  208  minimizes any physical interface and creates a long path length connection utilizing a poorly conducting material between inner vessel  204  and housing  202  which would permit thermal transfer of heat. Inner vessel  204  is surrounded with a vacuum adsorption medium  501  such as Zeolite. A heater assembly  502   a  is further provided within the housing  202  and affixed to inner vessel  204 . Vacuum adsorption medium  501  adsorbs any moisture present within evacuated space  211 . Heater assembly  502   a  is utilized to rapidly heat the vacuum adsorption medium  501 , which then releases the adsorbed vapor and helps enhance the vacuum quality within the evacuated space  211 . Valve  504  selectably connects evacuated space  211  to an external vacuum system (not shown) through port  504   a  for evacuation purposes while being heated. Valve  504  is constructed of a mounting flange  504   b  and a removable valve body  504   c  to facilitate easy replacement and/or servicing of the valve components. The evacuation process is performed periodically, when the holder is not in use, to help optimize the vacuum conditions. Housing  202  includes additional mechanisms for securing the actuation rod  240 , discussed in more detail below. 
         [0042]    Inner vessel  204  is mounted within the housing  202  at a fixed angle with respect to the horizontal axis of the holder, for example about 20°. This angle may be in the range of 10° and 50°. The cylindrical shape of inner vessel  204 , combined with its off axis mounting, allows any remaining volume of liquid nitrogen to maintain a near constant center of mass along the arcuate surface at the lowest, or other collection point of the inner vessel  204 , even at high tilt angles. This applies a symmetric load distribution on the goniometer and improves the holder performance during tilting. 
         [0043]    The housing  202  has an opening  209  for filling liquid nitrogen in the inner vessel  204 . The reentrant tubing  208  connects the inner vessel  204  to the housing  202 . The reentrant tubing  208  provides a long thermally insulating path, minimizing the heat gain from the housing  202  which is at ambient temperatures to the inner vessel  204  which is maintained at cryogenic temperatures. More particularly, reentrant tubing  208  is formed from a plurality of concentric cylindrical components joined alternately at the tops and bottoms of the cylindrical components forming a sinuous support. The neck  508 , as more fully illustrated with additional reference to  FIG. 5 , is designed to provide a smooth transition for the nitrogen gas to escape from the inner vessel  204  through the opening  209 . This design ensures that inner vessel  204  does not trap any liquid nitrogen bubbles when the holder  200  is tilted to an angle of up to approximately 80°. The elimination of trapped liquid nitrogen bubbles in turn prevents vibrations, which are detrimental to image resolution. The inner vessel  204  is designed to hold approximately 200 ml of liquid nitrogen. The inner vessel design helps maintain up to 80% of the liquid nitrogen volume without spilling at the extreme tilt angles of +/−80°. 
         [0044]    Referring again to  FIGS. 2 ,  2   a ,  2   b  and  2   c , one end of a thermally conductive ribbon assembly  214  is connected to the lower arcuate section  206  of the lower circumferential surface of inner vessel  204  while the other end is connected to conductor rod  505 , in the first embodiment, and conductor conduit  505   a  in the second embodiment and actuation housing  1002  in the third embodiment. Conductor conduit  505   a  is preferably constructed of silver or other high thermally conductive material. In the second embodiment, conductor rod  505  is separately cooled by ribbon  214   a,  which is affixed to ribbon assembly  214 . Conductor rod  505  may also serve as an actuator rod for the translation of the specimen, as described below. As a result, liquid nitrogen present in inner vessel  204  is always in proximal contact with the ribbon assembly  214 , through the wall of inner vessel  204 , regardless of the holder angle. The device therefore ensures stable operating conditions at high tilt angles. The flexible nature of ribbon assembly  214  further minimizes any vibrations that are generated either by the cooling medium or by any environmental conditions, such as acoustical noise. Ribbon assembly  214  also compensates for the change in length of the cooling rod assembly resulting from thermal changes and reduces the strain on the overall device. 
         [0045]    Ribbon assembly  214  extends away from arcuate section  206  of inner vessel  204  where it is affixed to one of conductor rod  505  or conductor rod  505   a,  as described above, by a weld or similar joint which ensures thermal conductivity. Conductor rod  505 , which may be of unitary or modular construction, extends through the body of middle barrel  210  and front barrel  212 . In the first embodiment, radiation shield  218  concentrically surrounds and seals conductor rod  505  along a substantial portion of its length, but is not in physical or thermal contact therewith, forming an evacuated space therebetween. Radiation shield  218  is constructed from a thin walled stainless steel tube or other poor thermal conductor that provides additional thermal insulation to the cold conductor rod  505 . The radiation shield  218  and conductor rod  505  are mounted within, but not in physical or thermal contact with middle barrel  210  and front barrel  212 . As shown in the second embodiment of  FIG. 2   a , a plurality of radiation shields  218   a  in a spaced apart relation to conductor rod  505  are illustrated within middle barrel  210 . These preferably comprise three concentric, coaxial shields. Similar to reentrant tubing  208 , radiation shields  218   a  are alternatively connected at each end, providing a lengthy, circuitous path which thermally separates but structurally supports conductor conduit  505   a  within middle barrel  210 . Referring again to  FIG. 4   a , conductor rod  505  is then connected to the specimen cartridge housing  506  at the holder tip  230  using a second ribbon assembly  214   a.  This ribbon assembly  214   a  acts similarly to ribbon assembly  214  and provides additional vibration damping between the inner vessel  204  and the specimen. Referring to the embodiment shown in  FIG. 2   a , conductor rod  505  is mounted axially within conductor conduit  505   a,  which is independently cooled by ribbon assembly  214   a.  The third embodiment, as shown in  FIG. 2   c , no radiation shields are present. 
         [0046]    Referring again to the second embodiment of  FIG. 2   a , conductor rod  505  and conductor conduit are received and supported by bellows assembly  511 . Bellows assembly  511  is a flexible, insulating assembly for structurally supporting, at the tip end  508   a,  the Dewar end of conductor conduit  505   a,  and at the support end  508   b,  the dewar end of conductor rod  505  within barrels  210 ,  212 . Additionally, bellows assembly  511  is intended to separate evacuated space  211  from the remainder of the holder. Evacuated space  211  includes a vacuum controlled by the user through valve  504 . Barrels  210 ,  212  are alternatively vented to ambient pressure while the holder is not in use, with the interior space of barrels  210 ,  212  being evacuated by the microscope pumping mechanism while inserted in the goniometer and in use. 
         [0047]    Referring now to  FIGS. 4   a - 4   e,  front barrel  212  is provided with an independent front insulating tube  258  which is spatially isolated from front barrel  212 , preferably constructed of a non-magnetic nickel chromium alloy such as Inconel, manufactured by Special Metals Corporation. In the embodiment illustrated in  FIGS. 4   a - 4   c,  holder tip  230  includes a specimen cartridge  232 , which is slidably mounted within and in good thermal contact with cartridge housing  506 . Spring  236   b  is interposed between specimen cartridge  232  and cartridge housing  506  within spring well  236   c  to create a frictional interface which resists slippage therebetween, other than as specifically controlled by actuating pin  242 , as will be more fully described below. Specimen cartridge  232  and cartridge housing  506  are jointly interposed between isolating mounting block  255  and isolation rods  238 . Isolating mounting block  255  is itself spring loaded by spring  236   a  within front insulating tube  258  in conjunction with isolation rods  238 . Isolation rods  238  and isolating mounting block  255  jointly serve to thermally isolate cartridge housing  506  from front insulating tube  258  and the remainder of the device. Front insulating tube  258  further thermally isolates specimen cartridge  232 . Disposed at the distal end of specimen cartridge  232  is a recess forming specimen cup  233 , which includes a supporting surface  234  for receiving and restraining specimen  235  to be viewed or imaged in a microscope system. Supporting surface  234  is adapted to receive a standard circular grid, 3 mm in diameter, or other specimen appropriate for electron microscopy or other imaging applications. 
         [0048]    In the second embodiment illustrated in  FIGS. 4   d - e , front insulating tube  258  receives and supports an alternative cartridge holder  506 ′, which may be comprised of several components or formed integrally preferably constructed of silver or other material having a high thermal conductivity. Each may be press fit or welded, as necessary. As illustrated in  FIGS. 4   d - e , front insulating tube  258  receives and supports conductive sheath  260 . Conductive sheath  260  is closed at the tip end by end cap  259 , and extends axially inwardly by supporting conductive support  261 . 
         [0049]    Cartridge holder  506 ′, through conductive support  261 , further receives and supports conducting support  505   b,  which is also constructed of silver or other thermally conductive material, and the slidably mounted tip  230  in an axial position within front barrel  212 . Conducting support  505   b  supports conducting conduit  505   a  in an axial position within barrels  210 ,  212 , as well as temperature sensor  246 . End cap  259  is provided with vent  259   a,  which provides the pathway for cartridge  232  to be extended from within the holder. End cap  259 , coupled with the close fitting end portion of cartridge  232 , provides a substantial barrier for ambient air flow within front barrel  212 . Vent  259   a  is sized and shaped to provide a close but low friction interface with cartridge  232 . All components are sized and shaped to slidable, press fit engagement to minimize, if not eliminate independent movement or vibration. A wave spring  236   c  exerts and inward force on tip  230  to further reduce vibration and ensure proper location of the axial movement of conductor rod  505 . 
         [0050]    When in the forward, or operative, position as illustrated in  FIGS. 4   a  and  4   e , the left lateral end of cartridge  232 , and thus specimen cup  233  and specimen  235  extend outside front barrel  212 . In the embodiment illustrated in  FIGS. 4   a - c , cartridge  232  and cartridge housing  506 , which are located inside front insulating tube  258  and front barrel  212 , are restrained by the force of spring  236   a  against isolation rods  238 . Springs  236   a  and  236   b  are preferably formed from a non-magnetic material such as beryllium-copper. Springs  236   a  and  236   b  are aligned such that they each exert a compressive force onto isolating mounting block  255  and specimen cartridge  232 , respectively, and thus, in addition to minimizing unwanted shifts of the components, serve to dampen any stray vibrations introduced to specimen cartridge  232 . In addition, springs  236   a  and  236   b  allow for specimen cartridge  232  to settle in a stress free equilibrium position during the initial thermal cycling. In the embodiment illustrated in  FIGS. 4   d  and  4   e , spring  236   c  exerts a similar force on tip  230 . 
         [0051]    Specimen cartridge  232  can be displaced in a direction parallel to a longitudinal axis of holder  200  and thus will move laterally to the left and the right as seen in the Figures. In a retracted position as illustrated in  FIGS. 4   b  and  4   d , the left lateral end of specimen cartridge  232  and thus specimen cup  233 , supporting surface  234  and specimen  235  reside in a position inside cartridge housing  506  in the  FIG. 4   b  embodiment and within end cap  259  in  FIG. 4   d . Although holder tip  230  is shown in  FIGS. 4   a  and  4   b  as having two springs  236   a  and  236   b,  it will be appreciated by those of skill in the art that holder tip  230  may be provided with additional springs without departing from the scope of the invention. Temperature sensor  246  is mounted directly to cartridge housing  506  in  FIGS. 4   a - c  and to conducting support  505   b  in  FIG. 4   e  to monitor the temperature of specimen  235 . Referring now only to  FIGS. 4   a - c , in order to facilitate displacement of specimen cartridge  232  relative to cartridge housing  506 , actuating pin  242  is inserted within slot  242   a  within specimen cartridge  232 . Actuating pin  242  is affixed to actuator rod  240  which extends throughout the length of the holder  200 . 
         [0052]    Referring now to  FIGS. 4   d  and  4   e , specimen cartridge  232 ′ is mounted to a mounting tongue  230   a  provided on tip  230  for receiving specimen cartridge  232 ′. Spring  236   d  is interposed therebetween and provides a snug fit between the components and further reduced vibration and independent motion of the two components. Specimen cartridge  232 ′ is provided with a clamshell design having a hinge, which is pivotable to open and close in order to load the specimen  235  therein on supporting surface  234 . Cartridge base  232   a  provides the interface with tip  230  and supporting surface  234  is disposed thereon. Cartridge clamp  232   b  is hinged and pivots to engage tongue  230   a  in a snap fit and is restrained in a closed position by rod  232   c.  As with the previously described components, the specimen cartridge components are sized to ensure close interference fits which minimize if not eliminate vibration and independent movement. 
         [0053]    With additional reference to  FIGS. 2 ,  3  and  3   a , a first embodiment of actuation assembly  249  is affixed to the end of actuator rod  240  proximate to the housing  202 . Actuator rod  240 , which may be optionally comprised of several components or modules interconnected with each other, extends within the interior space of middle barrel  210  and front barrel  212  to terminate with actuator pin  242  at the operative end of the device in order to displace specimen cartridge  232 . As will be appreciated by those skilled in the art, such displacement may be in any direction or dimension. Specimen cartridge  232  is adapted for slidable displacement in a lateral direction in and out of cartridge housing  506  when a lateral force is applied to the actuator rod  240  by turning rotational knob  248 . Knob  248  is affixed to actuator shaft  252  which is rotatably mounted within handle  249 . O-ring seal  251  is utilized to separate the interior of the holder assembly  200 , which may be under vacuum conditions, from the ambient atmosphere. It is noted that actuator shaft  252  is rotated to engage and adjust cam  253  with respect to actuator rod  240  and that the rotational motion of knob  248  causes lateral displacement of actuator rod  240  while maintaining an effective seal of O-ring  251 . Actuator rod  240  is restrained in place and thermally isolated by isolation rod  238   a  and actuator isolation block  238   b.  Cam  253  and actuating rod  240  are designed such that actuating pin  242  does not physically contact the cartridge in the final extended and retracted positions of specimen cartridge  232 . This eliminates a major source of heat flow, thus facilitating a more stable cartridge temperature. 
         [0054]    With reference to  FIGS. 2   a  and  3   b , a second embodiment of actuation assembly  249  is affixed to the end of bellows assembly  511  and conductor rod  505  proximate to the housing  202 . Conductor rod  505 , which may be optionally comprised of several components or modules interconnected with each other, extends within the interior space of middle barrel  210  and front barrel  212  to terminate at tip  230  in order to displace specimen cartridge  232 . As will be appreciated by those skilled in the art, such displacement may be in any direction or dimension. Specimen cartridge  232  is adapted for slidable displacement in a lateral direction in and out of end cap  259  when a lateral force is applied to the conductor rod  505  by turning rotational knob  248 . Knob  248  is affixed to actuator shaft  252  which is rotatably mounted within handle  249 . O-ring seal  251  is utilized to separate the interior of the holder assembly  200 , which may be under vacuum conditions, from the ambient atmosphere. It is noted that actuator shaft  252  is rotated to engage and adjust cam  253  with respect to conductor rod  505  and that the rotational motion of knob  248  causes lateral displacement of conductor rod  505  while maintaining an effective seal of O-ring  251 . Conductor rod  505  is slidingly supported and thermally isolated by isolation block  238   b.  Rotation of knob  248  and shaft  252  cause cam  253  to be laterally displaced along the axis of holder  200 . Actuator  242  is therefore also laterally displaced and engages bellows assembly  511 . Such displacement compresses bellows assembly  511  in the extended direction. 
         [0055]    In a third embodiment of actuator assembly  249 , as illustrated in  FIGS. 2   b - 2   d  and  3   c , actuation assembly  249  is affixed to the end of conductor rod  505  proximate to the housing  202 . Knob  248  is affixed to actuator shaft  252  which is rotatably mounted. O-ring seal  251 ′ is utilized to separate the interior of the holder assembly  200 , which may be under vacuum conditions, from the ambient atmosphere. It is noted that actuator shaft  252  is rotated to engage and adjust pinion gear  1001  with respect to rack gear  1004  and that the rotational motion of knob  248  causes lateral displacement of rack gear  1004 , conductor rod  505  and conductor isolation rod  238   c,  which is affixed to rack gear  1004  by clamp  1005 . Conductor rod  505  is slidingly supported and thermally isolated by conductor isolation rod  238   c.  O-ring  1003  provides a sealing interface between the interior of actuation housing  1002  and the interior of holder  200 . The interior of actuation housing  1002  is evacuated by the TEM device, while the interior of the holder  200  is evacuated as described above. O-ring  1003  is maintained at near ambient temperature to minimize any freezing effects on the elastomeric material by isolation block  238   b . Ribbon assembly  214  may be of unitary construction or in multi part construction as illustrated in  FIGS. 2   c  and  3   c . As illustrated, a connector  1006 , which may be of any known design, such as a bolt, nut and washer, may be utilized to affix the components. 
         [0056]    Thermally conductive ribbon assembly  214  is connected to the cartridge housing  506 . Ribbon assembly  214  provides a thermal conduction path from inner vessel  204 , through connecting rod  505  and conducting conduit  505   a  to cartridge housing  506 , in a first embodiment and tip  230  in the second, and subsequently to specimen cartridge  232 . As a result, in the  FIG. 4   a - c  embodiment the remaining holder tip  230  components, for example, actuating rod  240 , actuator pin  242 , spring-loaded isolating rods  238  and  238   a  and front barrel  212 , remain at ambient temperature. Unlike the prior art references discussed previously, the ribbon assembly  214  cools only the cartridge housing  506  and in turn specimen cartridge  232 . This results in an efficient cooling system. In addition, the thermal isolation of many of the holder tip  230  components greatly reduces the negative effect of thermal gradient and helps eliminate specimen drift. 
         [0057]    Referring now to  FIGS. 5   a - f , the inner vessel  204  is illustrated having varying amounts of liquid cooling medium. With respect to  FIGS. 5   a  and  5   b , inner vessel  204  is illustrated in the upright, neutral position having 0° tilt. Liquid cooling medium is filled to a level corresponding to 80% of the nominal volume of inner vessel  204 , the surface of such liquid cooling medium is represented by reference A. Referring now to  FIG. 5   c , inner vessel  204  is illustrated at a tilt angle corresponding to the approximate limit of functionality and utility associated with the holder. The figure illustrates that even at the extreme tilt angle, the surface of the liquid cooling medium A remains within inner vessel  204  without spillage. Referring now to  FIGS. 5   d  and  5   e , inner vessel  204  is illustrated in the upright, neutral position, having 0° tilt. Liquid cooling medium is filled to a level corresponding to 10% of the nominal volume of inner vessel  204 , the surface of such liquid cooling medium is represented by reference B. Lastly,  FIG. 5   f  illustrates inner vessel  204  at the extreme tilt angle with the surface of cooling medium B at the 10% level.  FIGS. 5   d - f  represent the likely state of inner vessel  204  after substantial use of the device and the corresponding depletion of the liquid cooling medium through boiling. The figures illustrate the substantial degree of contact between the bulk of the remaining liquid cooling medium and the lower arcuate section  206  of inner vessel  204  proximate to the thermally conductive ribbons (not shown). It is noted that at all tilt positions and at all levels of cooling medium that substantial contact is made by the cooling medium with the collection point at the lower arcuate section  206  of inner vessel  204 , facilitating maximal heat transfer between the cooling medium and the thermally conductive ribbons mounted adjacent thereto. 
         [0058]    In operation, liquid nitrogen is placed into inner vessel  204  through the opening  209 . The unique design of inner vessel  204  and neck  508  help to obtain high holder tilt angles while retaining the liquid nitrogen and without trapping nitrogen gas within inner vessel  204 . 
         [0059]    The operator prepares a specimen  235  on a standard 3 mm grid in a cryogenically protected atmosphere. Other types of specimens may also benefit from this technique and can be readily position within specimen cup  233 . Specimen cup  233  may also be adjusted with respect to its geometry to accommodate other shaped specimens so long as the physical constraints of the relevant imaging device are observed. Preparation and mounting of such specimens, and appropriate cryogenic chambers to perform these tasks, are well known in the art and will not be discussed at this time. The specimen  235  is then placed on supporting surface  234  of specimen cup  233  when the specimen cartridge  232  is in the forward position as illustrated in  FIGS. 4   a  and  4   d . The operator then turns the knob  248  ( FIG. 2 ) which applies lateral force to either actuating rod  240  or conducting rod  505 , as described above for each of the two embodiments. The force causes cartridge  232  to move in a direction laterally to and generally parallel to front barrel  212  and to the right into a retracted position as shown particularly in  FIGS. 4   b  and  4   e . Specimen cartridge  232  is thus contained within cartridge housing  506  in the first embodiment and within conducting sheath  260  in the second. The cartridge housing  506  and conducting sheath  260  provide a protective and thermally cooled environment for the specimen and the holder  200  may be transported to the relevant imaging or analysis device, such as an electron microscope. 
         [0060]    Once inside the microscope, the operator uses the knob  248  to apply lateral force to specimen cartridge  232 , moving it into the appropriate position with respect to the electron optics. At the final extended position, in the first embodiment, actuating cam  253  is utilized to disconnect the actuating pin  242  from the cartridge. The specimen is then imaged by the electron microscope or by other imaging or analysis devices. 
         [0061]    The terms and expressions which have been employed herein are used as terms of description and not as limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention claimed. Although particular embodiments of the present invention have been illustrated in the foregoing detailed description, it is to be further understood that the present invention is not to be limited to just the embodiments disclosed, but that they are capable of numerous rearrangements, modifications and substitutions.