Patent Publication Number: US-2020303162-A1

Title: System and method for preparing cryo-em grids

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Patent Application No. 62/821,857, filed on Mar. 21, 2019 and entitled NOVEL DEVICE AND METHOD FOR PREPARING CRYOEM GRIDS WITH HIGH RELIABILITY, REPEATABILITY AND REPRODUCIBILITY, which is incorporated herein by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     This invention relates generally to electron microscopy. More particularly, the invention relates to a method and apparatus for depositing and vitrifying liquid samples for use in electron microscopy. 
     BACKGROUND OF THE INVENTION 
     Part of the research and development cycle of pharmaceuticals is determining how an experimental drug interacts with the body. This is a critical step in learning not only if the pharmaceutical is effective for its intended purpose but also what other unintended side effects it may cause. One way for identifying the effects of a new pharmaceutical is to conduct laboratory experiments that expose the pharmaceutical to biological samples (e.g., proteins) and then observing the results of that interaction. Among other things that may be observed during these experiments, the shape of the sample may change and these changes in shape can provide an indication of the effects that the pharmaceutical would have on a body. In other words, these structural characterization studies show the site and strength of drug molecule binding. To ensure that the results of these tests are accurate, it is important to begin the experiment with a biological sample that is as close to its natural state and shape as possible with high structural and functional integrity. If the shape of a biological sample is altered or damaged before a test is conducted, the results of the test may not accurately reflect how the pharmaceutical being tested would affect the body in real life. Similar arguments are also valid in other fields such as materials science that involve looking at component interactions in the liquid state. 
     Cryogenic electron microscopy (cryo-EM) is an electron microscopy technique involving the imaging of biological materials in a transmission electron microscope under cryogenic conditions. In cryo-EM, high-voltage electrons are generated in a vacuum by an electron source (i.e., an electron gun). Those electrons are focused into a fine beam and are then directed towards and through a sample located on a movable stage. After passing through the sample, the electrons either scatter or hit an image recording system that includes an electron detector to generate an image. However, before any imaging or analysis can occur in the cryo-EM process, the samples must be prepared. During the sample preparation stage, sample proteins in an aqueous environment are captured in a thin layer of vitreous ice by being cooled very quickly (generally, in less than a millisecond) to cryogenic temperatures. When samples are prepared properly, the vitreous ice layer can trap biological matter in its natural form and provides a thin (generally, less than 3 micrometers thick), clear sample that is well suited for cryo-EM imaging and analysis. Cryo-EM may also be used in other scientific fields, including materials science, nanomedicine, and renewable energy. 
     Thus, sample preparation is a very important step in cryo-EM analyses. However, sample preparation is often complex, difficult, and costly. One common issue is the inability to reliably and precisely control the thickness of the vitreous ice formed when preparing a sample on a cryo-EM grid. Since electrons must transmit through an EM sample for an image to be formed, it is necessary that the sample be thin enough to transmit sufficient electrons to form an image with minimum energy loss and a high enough signal-to-noise ratio. On the other hand, if the sample is too thin, the sample may not be fully encapsulated by the vitreous ice layer and may extend through and become exposed at the water-air interface, which can cause their shape or composition to be adversely impacted. Proteins can aggregate and become grouped too closely together if the ice layer is too thin, or they may become disassociated (i.e., torn apart) or spread too far apart from or stack on top of one another if the ice layer is too thick. Other issues, such as the formation of ice artifacts and crystallization within the ice that cloud the ice, can make obtaining an image difficult or impossible. Therefore, carefully forming the vitreous ice layer with a particular thickness and clarity is critical to obtaining good samples that are suitable for use in cryo-EM imaging and analysis. 
     Conventional vitrification processes rely on trial and error to achieve an acceptable sample. Typically, several cryo-EM samples are prepared on EM grids under a variety of conditions, with the hope that one of those conditions will produce a vitrified sample having the desired ice thickness and clarity. With reference to  FIGS. 1-3 , there is illustrated an example of a conventional EM grid  100  that may be used to suspend biological samples. Grid  100  includes a flat disk  102 , which is provided with an array of grid openings  104  that are formed by intersecting metallic rails  106 . These rails  106  are made from a material having high thermal conductivity such as copper, nickel, aluminum, etc. A single grid opening  104 ′ is highlighted in  FIG. 1  and is enlarged in  FIG. 2 . A film  108 , sometimes called a holey carbon film, is placed on top of and is adhered to the disk  102  and covers the grid openings  104 . The film  108  is provided with an array of very small holes  110  that extend through the film and across the entire surface of the disc  102 . 
     These grids  100  are commonly used in a conventional sample preparation method known as the blotting and plunge freezing method, which can be done manually or semi-automatically with devices currently on the market. In preparing a sample for cryo-EM imaging and analysis, a droplet of a sample material is often deposited onto the film by hand using a pipette. A cross section of two of the holes  110  of grid opening  104 ′ is shown in  FIG. 3 . As seen there, the sample solution  112  fills the holes  110  but a large amount of the solution collects on top of the film  108 . At the blotting step, filter paper is brought into contact with the sample solution  112  and a portion of the sample solution is absorbed into the filter paper. The left hole  110  shown in  FIG. 3  shows the sample solution  112  before the filter paper is used. The right hole  110  shown in  FIG. 3  shows the sample solution  112  after a portion of the sample solution  112  has been removed from the grid  102  at the blotting step. The grid  100  is then vitrified by plunge freezing into a cryogen, such as liquid ethane, liquid propane, or a mixture of the two cooled by liquid nitrogen. 
       FIGS. 4-6  are enlarged side views of the right hole  110  shown in  FIG. 3  under three different scenarios. In each case, sample solution  112  is held in the hole  110  by surface tension (i.e., capillary action) and a meniscus having a peak  114  formed at the center of the layer of sample solution on both the top and bottom of the layer of sample solution. The vertical distance between these peaks  114  defines a height H of the ice layer. In  FIG. 4 , too much sample solution  112  was left remaining at the blotting stage, which resulted in a vitreous ice layer having a thickness H that is too great. This is evidenced by the stacking of proteins  116  on the left-hand side and the dissociated protein shown on the right-hand side. In  FIG. 5 , an insufficient amount of sample solution  112  was left at the blotting stage, which resulted in an ice layer having a thickness H that is too small. This is evidenced by the exposure of the proteins  116 , which extend through the water-air interface  118 . In  FIG. 6 , an ideal amount of sample solution  112  was left at the blotting stage, which resulted in an ice layer having an ideal thickness H. This is evidenced by proteins  116  that are fully encapsulated by the ice layer and are well-dispersed throughout. Ideally, proteins  116  are homogeneous and well-dispersed in a single layer across throughout the vitreous ice layer and adopt random orientations. These random orientations allow the proteins to be viewed from multiple angles in a single view, enabling the three-dimensional structural reconstruction at a later step. 
     As shown above, the conventional blotting and plunge freezing method is unreliable, labor intensive, and slow. Each stage of sample preparation, namely pipetting, blotting, and plunge freezing, is carried out sequentially or by hand. The actual amount of time separating each of these steps may be only seconds, but it is long enough for the samples to be adversely impacted as molecules tumble around in solution. For example, when sample solutions are initially deposited onto a grid, they may have well-dispersed and randomly-oriented individual proteins. However, while the blotting occurs and before the plunge freezing step, those proteins may coalesce, disperse, readjust their configurations in solution, and adopt a preferential alignment (i.e., proteins align themselves in a particular manner and are not randomly oriented). Each of these behaviors negatively impacts the sample and makes EM imaging and the determination of protein structures more difficult. 
     Another major drawback to this conventional process is the cost associated with waste sample material. Generally, a 2-4 μl sample volume is required to prepare a single sample grid  100 . However, 99.9% of the sample volume is lost during grid preparation. Much of this loss occurs at the blotting stage of sample preparation, but evaporation is also a cause of loss of the sample solution. Sample solutions are often difficult and expensive to obtain due to extensive work in synthesis, extraction, and purification, etc. For that reason, attempts have been made to reduce these losses. For example, samples are sometimes prepared in an environment having a high humidity level, such as in an enclosed chamber, such that sample loss due to evaporation is minimized or eliminated. However, as explained above, EM imaging occurs in a vacuum. For that reason, it has been impossible to prepare a sample using the conventional blotting and plunge freezing method and then image that sample in the same environment. 
     Therefore, what is needed, is an improved method and apparatus for preparing biological samples for cryo-EM imaging and analysis. 
     NOTES ON CONSTRUCTION 
     The use of the terms “a”, “an”, “the” and similar terms in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having”, “including” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. The terms “substantially”, “generally” and other words of degree are relative modifiers intended to indicate permissible variation from the characteristic so modified. The use of such terms in describing a physical or functional characteristic of the invention is not intended to limit such characteristic to the absolute value which the term modifies, but rather to provide an approximation of the value of such physical or functional characteristic. 
     Terms concerning attachments, coupling and the like, such as “connected” and “interconnected”, refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both moveable and rigid attachments or relationships, unless specified herein or clearly indicated by context. The term “operatively connected” is such an attachment, coupling or connection that allows the pertinent structures to operate as intended by virtue of that relationship. 
     The use of any and all examples or exemplary language (e.g., “such as” and “preferably”) herein is intended merely to better illuminate the invention and the preferred embodiment thereof, and not to place a limitation on the scope of the invention. Nothing in the specification should be construed as indicating any element as essential to the practice of the invention unless so stated with specificity. 
     BRIEF SUMMARY OF THE INVENTION 
     The above and other needs are met by an electron microscope (EM) preparation and imaging system that includes an EM device and a sample preparation apparatus for forming a vitreous ice layer containing liquid (e.g., biological) samples through vitrification that may both be optionally placed into a sealable environment. The sample preparation apparatus includes a cryogenically-cooled stage that is configured to removably receive a sample deposit surface such that the deposit surface is cryogenically cooled through direct contact with the stage. The sample preparation apparatus further includes a sample dispenser that is at least one of laterally, longitudinally, vertically, or rotationally movable with respect to the stage. The sample dispenser is configured to deposit a liquid sample onto the sample deposit surface at a selected rate of deposition. Once the liquid sample is deposited onto the sample deposit surface by the sample dispenser, it is vitrified automatically in place. The sealable environment is configured to be placed under at least one of a positive pressure or a negative pressure. At least a portion of the EM device and sample preparation apparatus is located inside the sealable environment such that a sample may be vitrified by the sample preparation device and imaged by the EM device inside of and without being removed from the sealable environment and without changing the pressure of sealable environment. 
     In order to facilitate an understanding of the invention, the preferred embodiments of the invention, as well as the best mode known by the inventor for carrying out the invention, are illustrated in the drawings, and a detailed description thereof follows. It is not intended, however, that the invention be limited to the particular embodiments described or to use in connection with the apparatus illustrated herein. Therefore, the scope of the invention contemplated by the inventor includes all equivalents of the subject matter described herein, as well as various modifications and alternative embodiments such as would ordinarily occur to one skilled in the art to which the invention relates. The inventor expects skilled artisans to employ such variations as seem to them appropriate, including the practice of the invention otherwise than as specifically described herein. In addition, any combination of the elements and components of the invention described herein in any possible variation is encompassed by the invention, unless otherwise indicated herein or clearly excluded by context. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The presently preferred embodiments of the invention are illustrated in the accompanying drawings, in which like reference numerals represent like parts throughout, and in which: 
         FIG. 1  is a plan view depicting a sample grid that has been conventionally used in connection with electron microscopy; 
         FIG. 2  is a plan view depicting a single grid opening of the grid of  FIG. 1 ; 
         FIG. 3  is a partial sectional view of the grid of  FIG. 2 , shown along line  3 - 3 ; 
         FIGS. 4-6  are detail views of the boxed portion of  FIG. 3  that depict a vitreous ice layer that is too thick, too thin, and is ideal, respectively; 
         FIG. 7  is a perspective view depicting an EM sample preparation apparatus according to an embodiment of the present invention; 
         FIGS. 8 and 9  are side and front elevation views depicting the EM sample preparation apparatus of  FIG. 7 , respectively; 
         FIG. 10  is a detail view of a sample dispenser having a reservoir and nozzle according to an embodiment of the present invention; 
         FIG. 11  is a second detail view of the sample dispenser shown in  FIG. 10  having a reservoir and nozzle according to an alternative embodiment of the present invention; 
         FIG. 12  is a plan view illustrating a multi-sample grid according to an embodiment of the present invention; 
         FIG. 13  is a perspective view depicting an EM sample preparation apparatus according to an alternative embodiment of the present invention; 
         FIG. 14  is a top plan view depicting a nanometer-scale deposit of liquid sample deposited onto a sample deposit surface of the apparatus of  FIG. 13 ; and 
         FIG. 15  is an elevation view depicting an EM preparation and imaging system according to an embodiment of the present invention, namely a fully integrated cryo-EM sample preparation and electron microscopy system. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This description of the preferred embodiments of the invention is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description of this invention. The drawings are not necessarily to scale, and certain features of the invention may be shown exaggerated in scale or in somewhat schematic form in the interest of clarity and conciseness. 
     With reference now to  FIG. 7 , there is provided an EM sample preparation apparatus  200  that includes a cryogenically-cooled stage  202  and a sample dispenser  204  for dispensing a liquid sample onto the stage according to an embodiment of the present invention. The sample dispenser  204  is movable and rotatable with respect to the stage  202 . A cryogen loop, including an inlet  206  and an outlet  208 , circulates a cryogen, such as liquid ethane, helium or nitrogen, through the stage  202  in order to cool it to cryogenic temperatures (i.e. from absolute zero up to sub-135 Kelvin). In this case, the stage  202  is formed as a stationary metallic block having a low heat capacity and high thermal conductivity (e.g., copper, aluminum, etc.), which supports a fast time scale of vitrification. 
     In preparing a cryo-EM sample with apparatus  200 , a liquid sample solution  210  may be deposited onto a sample deposit surface  212 , which is preferably removably positioned on top of the stage  202 . Preferably and advantageously, the sample deposit surface  212  utilized with apparatus  200  can be a conventional sample grid, such as the grid  100  that is shown in  FIG. 1 . This could include, for example, current commercially-available grids made from materials such as holey carbon, holey gold, or other materials with a high thermal conductivity, such as C-flat™ and Quantifolil® brand grids. As such, the term “grid” may also be used interchangeably to refer to sample deposit surface  212 . 
     Advantageously, using this apparatus  200 , the liquid sample  210  deposited onto the stage  202  (or a grid  212  in this case) is vitrified automatically and almost immediately in place. (i.e., the liquid sample may be deposited and then vitrified in the same position on the stage without being moved). For example, in certain embodiments, vitirication of the sample solution  210  occurs within one microsecond after contacting stage  202  or the grid  212 . By minimizing the time between deposition and vitrification, many of the adverse and unwanted effects, such as proteins becoming dissociated, unfolded, or moving to the water-air interface can be avoided. The type of cryogen selected to cool the stage  202  may vary depending on the type of liquid sample that is being vitrified. For example, liquid nitrogen boils at around 77 K, and may be used to vitrify any liquids that vitrify above that temperature. On the other hand, for liquids that vitrify below 77 K, a different cryogen, such as liquid helium, which boils at around 4 K, may be used as the cryogen. 
     Preferably, sample dispenser  204  is movable side-to-side along a lateral axis AL, movable front-to-back along a longitudinal axis AT, and movable vertically along a vertical axis AV with respect to the stage  202  and grid  212 . For example, in this particular non-limiting embodiment, relative movement of up to ±6 inches is permitted along the AL and AT axes and up to 6 inches is permitted along the AV axis. Either the stage  202 , the sample dispenser  204 , or both may be moved with respect to the other in the manner discussed above. The presence of the cryogen loop in the stage  202  makes moving the stage more difficult and, therefore, it is preferable that only the sample dispenser  204  is moved. These degrees of freedom enable the sample dispenser  204  to deposit liquid sample  210  at any location across the stage  202  and grid  212 . Additionally, sample dispenser  204  is rotatable about at least the lateral axis AL by an angle α (as shown in  FIG. 8 ) and may also be rotatable about the longitudinal axis AT or vertical axis AV. Preferably, sample dispenser  204  may be rotated freely about any of the above-described axes up to 360®. The sample dispenser  204  is configured to deposit the liquid sample solution  210  onto the grid  212  at a selected rate of deposition. In certain cases, the rate of deposition is in the range of 150-350 μL/min. Liquid sample solution  210  may include samples of many types, including biological macromolecules including but not limited to soluble and membrane proteins, protein complexes, nucleic acids, and lipid bilayers; nanomaterials; protein nanocrystals; cellular and subcellular components and soft tissues; etc. The liquid sample solution  210  may also include other types of solutions, including high viscosity solutions, such as liposomes, ionic liquids, and solubilized membrane proteins. Thus, the actual rate of deposition may change based on a number of factors, including the viscosity and viscosity index of the liquid sample solution  210 . Viscosity is a material property which describes the resistance of a fluid to shearing flows. The factors that affect viscosity include intermolecular forces, temperature, and the shape of the molecules. Since apparatus  200  works with both liquids and gases at low temperature, sample viscosity is expected to be an important property and parameter to consider. On the other hand, viscosity index (VI) is an arbitrary, unitless measure of the change of viscosity with temperature. In general, the greater the VI, the smaller the change in fluid viscosity for a given change in temperature. In this case, the ideal samples or sample solutions should be ones with the highest VI for they will remain stable and not vary much in viscosity during the sudden drop in temperature in the deposition and vitrification step. For example, the viscosity of the liquid sample solution  210  may range from 0 Pa·s to 2.758×107 Pa·s. Lastly, the rate of movement of the sample dispenser  204  with respect to the stage  202  may be adjusted. The above-described parameters, namely relative movement and rotation of the stage  202  to the sample dispenser  204 , the speed of that movement, and the rate of deposition, in combination, enable apparatus  200  to form an ice layer having a precisely-controlled thickness that can be customized by modifying these parameters. 
     Preferably, the grid  212  is in thermal contact with the stage  202  and both are pre-cooled to cryogenic temperatures by the cryogen loop before liquid sample solution  210  is deposited from sample dispenser  204 . Thus, as the liquid sample solution  210  is deposited onto the grid  212 , it is immediately vitrified in place (i.e., without being removed from the stage  202 ) to form an ice layer having a precisely-controlled thickness without requiring the removal of excess sample solution from the sample deposit surface (e.g. blotting) and without requiring moving the grid from a sample solution deposition location to another location for plunge freezing. Thus, apparatus  200  and the related method of use provide superior cryo-EM sample grids more efficiently, quickly and consistently with less material and time than was possible using conventional apparatuses and methods, including particularly the blotting followed by plunge freezing method discussed above. 
     With continued reference to  FIG. 7  and with further reference to  FIG. 10 , in this particular embodiment, the sample dispenser  204  is configured to form a microfluidic jet of sample solution  210  that is comprised of a first liquid sample  214  that is flattened into a sheet. In this particular embodiment, sample dispenser  204  includes a sheet nozzle  216  that flattens the first liquid sample  214  into a sheet by directing gas jets  218  towards opposing sides of the jet of first liquid sample. In some embodiments, this sheet can be as thin as 1 nm. Preferably, gas jets  218  are formed using a non-reacting gas such as nitrogen or argon. Nozzle  216  includes a first liquid channel  220  for carrying the first liquid sample  214  and an aperture  222  for forming jet of first liquid sample  214  and separate gas channels  224  for carrying the non-reactive gas and separate apertures  226  for forming gas jets  218 . Preferably, channels  224 , nozzles  226 , or both are angled with respect to channel  220 , nozzle  222 , both such that gas jets  218  are directed symmetrically towards opposing sides of the jet of first liquid sample  214 . In other embodiments, nozzle  216  is configured to steer and mix the liquid sample solution  210  or the individual components thereof prior to forming the sheet shown in  FIG. 7 . 
     With reference to  FIG. 11 , in certain embodiments, an alternative nozzle  216 ′ is almost structurally and functionally identical to nozzle  216  and includes first liquid channel  220  and gas channels  224 . However, nozzle  216 ′ also includes one or more auxiliary or second liquid channels  228 , each with a nozzle, for carrying other liquid samples. The liquid sample carried by second liquid channels  228  may be identical to first liquid sample  214 . However, in other embodiments, a second, different liquid sample  230  is carried through second liquid channels  228 . Preferably, the jets of first and second liquid samples  214 ,  230  are formed between the jets of non-reacting gas  218 , such that the jets of non-reacting gas flatten the jets of first and second liquid samples into a single sheet of sample solution  210 ′ that is comprised of the first and second liquid samples. In still other embodiments, additional channels may be provided to combine and mix even more liquid samples together. 
     Referring again to  FIG. 10 , in preferred embodiments, sample dispenser  204  includes one or more biocompatible reservoirs  232  that each holds a quantity of liquid sample (e.g., liquid sample  214 ,  230 ). These reservoirs  232  are in fluid communication with a liquid channel  220 ,  228  that has been designated for that particular liquid sample. In the illustrated embodiment, reservoir  232  is formed as an integral part of sample dispenser  204  that is in direct contact with nozzle  216 . However, in other embodiments, reservoir  232  is remote from nozzle  216  of sample dispenser  204 , which are connected together by microfluidic connectors, tubing or capillaries. The microfluidic connectors can have “n” number of ports or liquid channels, where “n” is any number greater than zero. Whichever number of channels used in a study depends on the particular experimental needs of the users; namely, however many chemical components and/or species are being investigated in a sample mixture and thus however many sample reservoirs  232  are needed to be connected in parallel to the microfluidic dispenser  204  through a microfluidic connector. In each case, a pressurizing force acts on the liquid sample so that the liquid sample flows from the reservoir  232  through the liquid channel  220 ,  228  and out of the appropriate nozzle  222 . This pressure may be created, for example, manually, by a pump, a pressurized gas supply (e.g., non-reactive gas  218 ), high performance liquid chromatography (HPLC), etc. In this case, separate supplies  234  of pressurized gas are connected to the sample dispenser  204 . One of the supplies  234  of gas provides pressure within sample dispenser  204  to push liquid sample from reservoir  232  and out of nozzle  216 . The second supply  234  of gas provides gas jets  218 . In this particular embodiment, nozzle  216  is formed using glass that can safely withstand pressures of up to 4000 psi. Similarly, in this particular embodiment, fluid lines, including those in and connected between the reservoir  232  and sample dispenser  204  can safely withstand internal pressure of up to 4000 psi. However, in other embodiments, components of apparatus  200  may be formed using other materials (e.g., metals, etc.), which can safely withstand even higher internal pressures. In general, the components of apparatus  200  are preferably formed using biocompatible materials, such as 316 stainless steel, polyether ether ketone (PEEK), silica, etc. This ensures and protects the chemical, biological, and physical integrity of the apparatus  200  as well as the stability of the samples used. 
     Using apparatus  200 , cryo-EM grids having an ice layer having a consistent and controllable thickness may be quickly and repeatedly created. The thickness of the ice layer may be adjusted by adjusting one or more of the following parameter: (i) the relative positioning and angle of the stage  202  and sample dispenser  204 ; (ii) the speed of relative movement between the stage and sample dispenser; and (iii) the flow rate of sample solution from the sample dispenser. As shown in  FIG. 7 , apparatus  200  can be used to simultaneously create several grids  212 . Additionally, as shown in  FIG. 12 , apparatus  200  can form separate deposits  236  of the same sample solution  210  or of different sample solutions on a single grid  212 . The arrows show the direction of relative movement of the sample dispenser (not shown) with respect to the grid  212 . Producing a multi-sample grid  210  would, for example, eliminate the need to prepare a separate grid for each sample when multiple samples need to be tested, measured, examined, screened, etc. Since grid  212  is cryogenically pre-cooled, deposits  236  are vitrified in place and almost immediately upon contact. No additional time or processing steps, such as blotting and plunge freezing, are required after sample deposition. As such, formation of the vitrified ice layers using apparatus  200  are much faster, more predictable, and less labor intensive than conventional methods. After the ice layers have been formed, the grid  212  may be removed from the apparatus  200  and transferred, according to standard cryogenic sample handling procedures, to an EM or other device for imaging and analysis. 
     With reference now to  FIG. 13 , there is provided an EM sample preparation apparatus  300  that includes a rotating cryogenically-cooled stage  302  and a sample dispenser  304  according to an alternative embodiment of the present invention. Sample dispenser  304  is movable side-to-side along a lateral axis AL, movable front-to-back along a longitudinal axis AT, and movable vertically along a vertical axis AV with respect to the stage  302 . For example, in this particular non-limiting embodiment, relative movement of up to ±6 inches is permitted along the AL and AT axes and up to 6 inches is permitted along the AV axis. Either the stage  302 , the sample dispenser  304 , or both may be moved with respect to the other in the manner discussed above. 
     The cryogenically-cooled stage  302  includes a stationary bottom portion  306  and a rotatable top portion  308  that is rotatable with respect to the bottom portion about AV axis. Bottom portion  306  may include a cryogenically-cooled motor (not shown) for rotating top portion  308 . The top portion  308  preferably includes two or more placement sites  310  that accept and securely hold a sample deposit surface (also referred to herein as a “grid”). For example, each placement site  310  may be slightly indented below a top surface of the top portion  308  in order to provide recessed area that is sized for the grid. In  FIG. 13 , one sample deposit surface  312 A is centrally located at the center of rotation along axis AV. Another sample deposit surface  312 B is located at one of several placement sites  310  along the periphery of the top portion  308  of the stage  302 . The sample dispenser  304  includes a capillary tube, microfluidic droplet dispenser, etc. that is configured to deposit nanometer-scale deposits  314  of said liquid sample onto the stage  302 . Advantageously, the placement sites  310  may be positioned proximate the sample dispenser  304  by rotating the rotatable top portion  308  with respect to the stationary bottom portion  306  such that liquid sample may be dropped from the capillary tube  304  onto each of the placement sites  310 , including on the grid  312  positioned there, in an identical manner without moving the sample dispenser. Alternatively, a bottom end  324  of the capillary tube may be lightly touched to the rotating grid to dispense sample solution onto the grid. 
     A cryogen loop, including an inlet  316  and an outlet  318 , circulates a cryogen through the rotatable stage  302  in order to cool the stage down to cryogenic temperatures. Thus, once drop  314  of sample solution contacts the stage  302  or a grid  312  placed on the stage, the sample vitrifies almost immediately to form an ice layer. The relative position of the stage  302  and sample dispenser  304  as well as the rotational speed of the stage may be used to adjust the thickness of the ice layer that is formed. In this particular case, top portion  308  can be rotated up to 50,000 revolutions per minute with respect to bottom portion  306 . With reference to  FIG. 14 , there is provided an overhead view of a grid  312  that is located at the center of rotation of the top portion  306  (i.e., centered on axis Av) and showing a drop  314  of liquid sample having an initial periphery  320 . The drop  314  preferably spreads evenly and uniformly across grid  312  in a direction that is orthogonal to the axis Av to a final periphery  322 . On the other hand, drops falling onto grids  312  located on the periphery of the top portion  306 , such as drop  314 ′, will spread across the surface of the grid in a non-uniform, linear manner away from the center of rotation of the top portion due to the centrifugal force caused by the rotation of the top portion. Preferably, drops  314 ,  314 ′ vitrify on the grids  312  immediately after being spread in the manner discussed above. 
     Lastly, with reference to  FIG. 15 , there is provided an electron microscope preparation and imaging system  400  according to an embodiment of the present invention. System  400  includes an EM device  402 , such as a transmission electron microscope, and a sample preparation apparatus  404 , such as apparatus  200  (shown in  FIG. 7 ) or apparatus  300  (shown in  FIG. 13 ), which may both be optionally located in a sealable environment  406 . System  400  also includes a workstation  408  for controlling the EM device  402  and sample preparation apparatus  404  that is located outside of the sealable environment  406 . 
     Sealable environment  406  can be placed under at least one of a positive pressure or a negative pressure, such that a sample grid  410  may be prepared (including both the deposition and vitrification steps), imaged, and analyzed by the sample preparation device  404  and EM device  402  entirely inside of the sealable environment. A pump apparatus  412  may be provided to create the positive and negative pressure with the sealable environment  406 . In addition to a vacuum, vitrification can occur in a variety of positive pressure atmospheres, including water-free air (e.g., humidity level less than 10%), backfilled with hydrophilic gas (e.g., sulfur dioxide), backfilled with hydrophobic gas (e.g., nitrogen), backfilled with noble gases (e.g., argon), or other water-less atmospheres. During the preparation, imaging and analysis processes, sample grid  410  can remain within the sealable environment and the internal atmosphere of the seal environment can remain unchanged. Additionally, no direct human interaction or handling is required in preparing, transferring, or imaging of the grid  410 . For example, the sealable environment may be maintained at a positive, negative, or neutral pressure relative to the environment outside the sealable environment. 
     After the vitreous ice layer has been formed using the above-described devices, the thickness of that ice layer can be measured according to several methods. One method that may be used to measure the thickness of the ice layer is ellipsometry, which is an analytical technique that utilizes thin-film interference to measure properties of thin films, including their thickness, at cryogenic temperatures. In a typical ellipsometry experiment, polarized light is reflected off a film surface to create a spectrum of colored bands. From this, the thickness of the ice layer can be determined based on an analysis of those color bands. 
     Although this description contains many specifics, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments thereof, as well as the best mode contemplated by the inventor of carrying out the invention. The invention, as described and claimed herein, is susceptible to various modifications and adaptations as would be appreciated by those having ordinary skill in the art to which the invention relates.