Abstract:
A cryotransfer holder for side entry transmission electron microscopes is provided. The cryotransfer holder design permits a specimen to be rotated about its axis as well as tilted while being maintained at a low temperature. The holder allows two sets of tilt data, preferably perpendicular to one another, to be collected from a single frozen specimen.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of Invention 
     The present invention relates to side entry specimen cryotransfer holders for transmission electron microscopy and particularly to cryoholders capable of rotating the specimen in the plane of the specimen and tilting that specimen around the electron beam axis. 
     2. Description of Prior Art 
     Electron microscope specimen holders may either take the form of a cartridge or a rod. Cartridge type holders are inserted vertically into the microscope through the upper polepiece of the objective lens, whereas rod type holders are inserted horizontally through the side of the microscope between the upper and lower polepieces. In recent years, rod type holders have become more widely used because mechanisms for tilting the specimen are simpler and more reliable. The ability to tilt a specimen is important for stereo imaging, three dimensional reconstruction, and tuning for optimum diffraction conditions. 
     Rod type specimen holders have also become the preferred type for observation of specimens at low temperatures. This is because the extraction of heat from the specimen is easily accomplished by forming the specimen tip of a thermally conductive material such as copper and extending the tip in the form of a copper rod to a dewar of liquid nitrogen or helium mounted at the other end of the rod. Cryotransfer holders are a more recent development of the rod type cooling holders in which specimens can be transferred at low temperature into the microscope from an external cryostation without frosting of the specimen. 
     Conventional resin embedding methods for biological samples have been shown to produce readily identifiable artifacts that can be interpreted in reference to structures observed in living cells. Water must be removed from specimens during resin embedment, causing diffusion artifacts and collapse of delicate structures. For these and other reasons, many laboratories use ultra-rapid freezing technologies to prepare biological specimens for examination in the transmission electron microscope. While this approach has many advantages, several technical problems must be surmounted; the specimen must be rapidly frozen to promote the formation of amorphous ice, and the sample must be maintained at temperatures below −140° C. to prevent devitrification. 
     Cryotransfer holders have been designed to maintain specimens in a frozen state and to prevent frost deposition on specimens during the transfer of the holder from a workstation into the microscope. Typically, when the specimen has been clamped into the holder, a cryo-shutter is moved to completely cover the specimen while being transferred. An example of this type of cryotransfer holder is described in Swann et al, U.S. Pat. No. 4,703,181. Once inserted into the microscope, the shutter is retracted to expose the frozen specimen. Depending on the type of specimen examined, the holder can require tilting to present the most advantageous aspect for recording. The cryoshutter can be replaced over the specimen if the specimen is destined for reexamination at a later date. 
     As the resolving power of electron microscopes has improved, efforts to resolve high resolution structures of biological organelles and macromolecules have been attempted. Three-dimensional electron microscopic imaging, or electron tomography, is one of the methods employed to gather detailed volume and surface data. This technique involves the reconstruction of individual objects from projection data collected over a large range of specimen tilts. An example of a cryotransfer holder which is useful in electron tomography is described in Swann, U.S. Pat. No. 5,753,924. A low resolution data set would include images of a specimen through a single tilt range of ±70° at 1° intervals. Ideally an object should be tilted ±90°, but current microscope design does not permit this range of movement. 
     Thus, practical limitations of tomography are set by the damage a specimen suffers with repeated irradiation and the range of specimen tilt available to generate projection data. To address the problem of specimen damage from repeated irradiation, samples are examined at low temperature in a cryoholder. The tilt range of a specimen could be extended by tilting the specimen in a second axis perpendicular to the first as is done with a double tilt holder. An example of a commercially-available double tilt holder is the Model 915 Double Tilt Cryotransfer System from Gatan, Inc., Pleasanton, Calif. For most double tilt holders, the tilt range of the second axis is limited to up to ±45° by the construction of the specimen cradle or the method by which tilting through the second axis is achieved. A data set including ±70° data in the first axis and ±45° in the second axis of tilt is not ideal. The specimen cannot be removed from the microscope and manually reoriented after one set of tilt data is taken because there is a high probability that the specimen will melt and become damaged or contaminated during this manipulation. 
     As discussed above, if a conventional single tilt cryotransfer holder is used to collect a tomographic series, current data sets typically cover a range up to ±70° of tilt through a single axis. When used for applications requiring high tilt such as tomography, this limited tilt range prohibits obtaining the necessary images for three-dimensional reconstruction of a specimen&#39;s top and bottom surfaces. This comprises roughly one-third of the data set for the structure being examined, while ideally for a single tilt axis stage, a complete data set from ±90° is needed to minimize directional distortions. However, this is impossible with currently-available cryotransfer holders. 
     In a known cryotransfer holder, designed and manufactured by Gatan Inc., a frozen specimen on a standard circular  3  mm diameter grid may be transported from a cryostation to the transmission microscope for examination. The cryo-holder is designed to maintain a specimen at a temperature of less than −160° C. at all times. The specimen tip is configured to produce a primary axis tilt of ±70° in a microscope polepiece gap with a “Z” axis distance of 3 mm or larger from the centerline of the holder to the nearest contacting surface. The tip of the specimen rod contains a moveable cryo-shutter which prevents frost formation on the specimen during transfer from a workstation to the microscope. The cryoshutter is manipulated at will by means of a manual control positioned outside of the holder. The specimen grid is held in place by a specimen clamping device, for example, a Clipring (trademark of Gatan, Inc.). 
     Accordingly, the need still exists in this art for a cryotransfer holder which is capable of acquiring tilt data at high angles at more than one specimen orientation without disturbing the frozen specimen. 
     SUMMARY OF THE INVENTION 
     The present invention meets that need by providing a cryotransfer holder which takes advantage of the design features of the standard cryotransfer holders, including a moveable cryoshutter, maintaining a specimen temperature of less than −160° C., and secure specimen clamping, but which also is capable of acquiring tilt data at high angles at more than one specimen orientation without disturbing the frozen specimen (i.e., without removing the specimen from the microscope). The present invention adds the ability to rotate the specimen in the plane of the specimen at cryotemperatures during observation in the microscope which extends the useful maximum tilt range of the holder through another tilt axis, ideally perpendicular to the original axis. 
     In accordance with one aspect of the present invention, a side-entry specimen cryoholder for an electron microscope is provided and includes a specimen holder including a specimen cradle having a specimen grid adapted to carry a specimen to be analyzed; a translation mechanism for rotating the specimen cradle within the specimen holder in the plane of the specimen; a mechanism for tilting the specimen cradle; and a cryoshutter which is adapted to protect the specimen during cryotransfer and moveable from a first position covering the specimen to a retracted position. Preferably, the translation mechanism includes an actuation mechanism which is operable externally of the electron microscope. Also preferably, the specimen cryoholder includes a source of cooling. 
     In a preferred embodiment of the invention, the translation mechanism comprises a motion exchange mechanism linked to the specimen cradle and a translation shaft linked to the motion exchange mechanism. The translation shaft preferably comprises a rod having first and second ends and extending along the long axis of the specimen holder, with the first end of the rod being connected to the motion exchange mechanism and the second end adapted to extend outside the transmission electron microscope. In one embodiment, the motion exchange mechanism comprises a toothed rack communicating with corresponding toothed gears on the periphery of the specimen cradle. The cryoholder further includes a knob threadably connected to the specimen holder, with the second end of the translation shaft being coupled linearly to the knob such that rotation of the knob causes linear movement of the translation shaft. 
     A sliding vacuum seal communicating with the translation shaft is also provided such that the vacuum inside the transmission electron microscope is maintained during movement of the motion exchange mechanism. The cryoshutter also includes a pin connected therewith such that linear movement of the translation mechanism engages the pin and causes the cryoshutter to move from the first position covering the specimen to a retracted position. The source of cooling preferably comprises a dewar of liquid nitrogen connected to the specimen holder by a thermally insulated conductor. 
     In operation, the specimen cryoholder is inserted into an electron microscope and the translation mechanism is actuated to retract the cryoshutter. A first set of data is taken with the specimen in a first orientation and the specimen cradle tilted through approximately ±70°. Then, the specimen is rotated, preferably 90° in the plane of the specimen using the motion exchange mechanism, and a second set of tilt data is taken through approximately ±70°. This procedure adds a second set of tilt data to compensate for information which would otherwise be missing from prior art specimen holders unless the holder were removed from the microscope and the specimen manually rotated. The present invention facilitates the production of a high resolution reconstruction of the object being studied without further handling of the frozen specimen. 
     Accordingly, it is a feature of the invention to provide a side-entry cryotransfer holder for electron microscopes that allows a specimen to be rotated in a plane perpendicular to the electron beam axis in a transmission electron microscope without removing the specimen from the microscope. It&#39;s a further feature of the invention to provide a rotation mechanism which is activated externally of the microscope. It is yet a further feature of the invention to provide a moveable cryoshutter which protects the specimen during transfer from the specimen loading workstation into a microscope, which also protect regions of the grid when the holder is positioned in the microscope before viewing of the sample, and which is removed prior to a viewing session. These, and other features and advantages of the present invention, will become apparent from the following detailed description, the accompanying drawings, and the appended claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     Reference is made by way of example to the drawings in which like elements are represented by like reference numerals and in which: 
     FIG. 1 is a schematic top view, in section, of the side-entry cryotransfer holder, including the moveable cryoshutter, a translation mechanism which rotates the specimen cradle, and an externally operated rotation knob to move the translation mechanism; 
     FIG. 2 schematically illustrates the side-entry cryotransfer holder with the cryoshutter completely covering the specimen; and 
     FIG. 3 schematically illustrates the side-entry cryotransfer holder with the cryoshutter actuated to expose the specimen for viewing, tilting, and rotation. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The present invention will be described in detail with respect to its preferred embodiment which is as a side-entry specimen holder for a transmission electron microscope. It will be apparent that the specimen holder may be adapted to operate in other types of electron microscopes as well. Further, the specimen holder of the present invention, in its preferred form, includes cooling (e.g., cryogenic) capabilities. The specimen holder is designed to be inserted into the column of an electron microscope so that the specimen carried thereon may be aligned with an electron beam which traverses the column. 
     For purposes of this invention, and as illustrated in FIG. 1, the X- and Z-axes of movement of the specimen holder are defined as follows. The X-axis refers to the longitudinal axis of specimen holder  10 , with movement toward the distal end or tip of specimen holder  10  being defined as the minus (−) direction and movement in the opposite direction being defined as the positive (+) direction. Tilting of the specimen bolder may occur in a first axis (sometimes has referred to as X-axis tilt) in either a clockwise (+) or counterclockwise (−) direction as shown. 
     Specimen holder  10  may be rotated about the vertical (Z-) axis for a full 360° in either the positive (+) clockwise direction of minus (−) counterclockwise direction. In FIG. 1, the Z-axis is a line perpendicular to the plane of the holder. 
     As shown in FIG. 1, a distal end or tip  11  of specimen holder  10  includes a specimen cradle  12 . As is conventional, frozen specimen  13 , which has been previously prepared under cryogenic conditions, is mounted within a recess in the specimen holder tip  11  and secured in cradle  12 , for example by a clamping ring or grid  14 . The specimen tip is cooled in a conventional manner such as by means of a copper conductor connected to a source of cooling such as a liquid nitrogen dewar (not shown). The specimen cradle  12  is constructed of a rigid material of high thermal conductivity. 
     Also as shown in FIG. 1, the specimen holder  10  includes a translation mechanism shown generally at  16  for rotating specimen cradle  12  (and specimen  13 ) about the Z-axis in the plane of the specimen. Translation mechanism  16  is operable externally of the microscope as will be explained in detail below. The translation mechanism includes a motion exchange mechanism, shown generally at  18 . In the embodiment illustrated, motion exchange mechanism  18  includes a rack  28  which slides along a recess in holder  10 . Rack  28  includes a series of teeth  30  which engage a corresponding series of teeth  32  on cradle  12 . Translation shaft  20 , which includes a first end  22  and a second end  24 , links rack  28  with the external actuation mechanism. The first end  22  of shaft  20  is connected to rack  28 , while second end  24  is linearly connected to rotation knob  36 . 
     As shown, knob  36  includes internal threads  38  which mate with corresponding external threads  40  on the specimen holder. Translation shaft  20  extends through an opening  42  in knob  36  and is able to rotate freely in that opening. As shown, the second end  24  of shaft  20  is secured to knob  36  by flanges  44  and  46  so that it is coupled to knob  36  linearly. When knob  36  is rotated clockwise, for example, the rotary motion is transferred to forward linear motion of shaft  20  and rack  28 . Rotating knob  36  in a counterclockwise direction, for example, results in a rearward linear movement of shaft  20  and rack  28 . The flat sides of rack  28  mate with surfaces in holder  10  to prevent rack  28  from rotating. 
     Forward linear movement of rack  28  causes rotation of cradle  12  through the meshing gear teeth  30  (on the rack) and  32  (on cradle  12 ). Cradle  12  is maintained in proper position by cradle bearing pins  34 . Thus, rotation of knob  36  externally of the microscope by an operator cause the linear motion of shaft  20  to be translated into rotary motion of cradle  12 . The vacuum in the microscope is maintained by the presence of sliding O-ring vacuum seal  26  which permits linear movement of shaft  20  while maintaining an air-tight seal between holder  10  and the exterior of the microscope. Further, while a manually-actuated mechanism has been described, it will be apparent that a motorized drive mechanism may be substituted. 
     While motion exchange mechanism  18  has been illustrated by the rack and gear embodiment shown in FIG. 1, it will be apparent to those skilled in this art that other drive mechanisms are possible which would still be within the scope of the invention. For example, manual or motorized control of the motion exchange mechanism could be accomplished using a drive belt, a friction drive, a spherical drive, a ribbon drive, piezo-motors, or mechanical gears. 
     Referring now again to FIG. 1, a moveable cryoshutter  48  completely surrounds the frozen specimen  13  and is removed from the specimen cradle area by the action of rack  28  pressing against cryoshutter pin  50 . Specimen cradle  12  is in close contact with cradle bearing pins  34  on one side and rack  28  with gear teeth  30  on the opposite side. The teeth  30  of rack  28  mesh with corresponding teeth  32  on the outer periphery of specimen cradle  12 . 
     Referring now to FIG. 2, cryoshutter  48  completely surrounds specimen  13  and specimen cradle  12 , protecting the frozen specimen from frosting (by exposure to the ambient atmosphere) during transfer into the electron microscope. As one of the last actions before inserting the holder into the electron microscope, the operator manually moves cryoshutter  48  over specimen  13  while holder  10  is maintained at cryotemperatures in a specimen holder workstation. Once inside the microscope, the operator turns rotation knob  36  attached to translation shaft  20  to move rack  28 . Linear movement of rack  28  causes it to contact cryoshutter pin  50  and moves cryoshutter  48  in the direction of the specimen tip to expose the specimen. Thus, actuation of knob  36  operates both cryoshutter  48  as well as causing rotation of specimen cradle  12 . 
     Referring now to FIG. 3, cryoshutter  48  is completely withdrawn to expose the specimen to the electron beam emanating down the microscope column. To rotate the specimen  13  and specimen cradle  12 , the operator (or a motor drive) turns rotation knob  36  attached to translation shaft  20  to cause rack  28  to move linearly. Rotational movement of the knob is translated through the meshing teeth  30  of rack  28  and teeth  32  of the specimen cradle  12 . 
     While the above description contains many specific details of structure and operation, one should not construe these as limitations on the scope of the invention but merely as exemplifications of the preferred embodiments thereof. Those skilled in the art will envisage other possible variations within the scope of this invention. For example, the cryoholder could include two drive shafts, one for specimen rotation and one to move the cryoshutter forward to expose the specimen and backwards to protect it during holder insertion and extraction from the microscope. A manual or motorized control of the cryoshutter mechanism using a drive belt, friction drive, piezo-motors or mechanical gears could also be utilized. The moveable cryoshutter could also be moved by a mechanism internal to the microscope.