Preparation of samples for particle structure analysis

Abstract

For particle structure analysis by transmission electron microscopy, the sample is prepared in the following manner: A predetermined amount of liquid containing at least one particle to be analyzed is sandwiched between an upper two-dimensional material layer and a lower two-dimensional material layer (102, 103). The lower two-dimensional material layer is supported by a transmission electron microscope grid. The amount of liquid is reduced to such an extent that at least one particle contained in the amount of liquid is enclosed in a cell formed by the upper two-dimensional material layer and the lower two-dimensional material layer (104). Specifically, the amount of liquid is reduced to such an extent that the cell has a thickness corresponding to the thickness of at least one particle to be analyzed, and the remaining amount of liquid has a thickness of less than 50 nm in the cell (104).

Description

[Technical Field]

[0001] Aspects of the present invention relate to a method for preparing a sample for particle structure analysis by transmission electron microscopy. The sample may comprise nanoscale biological particles, such as proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), vesicles, and the like. In this case, the particle structure analysis may be so-called single particle analysis. Alternatively, the sample may comprise biological cells, such as bacteria, spores, mammalian cells, plant cells, or populations of such cells, and the like. Other aspects of the present invention relate to a method for performing particle structure analysis by transmission electron microscopy, and to a sample preparation for particle structure analysis by transmission electron microscopy. [Background Art]

[0002] Single particle analysis is a relatively recent technique that can generate three-dimensional maps of nanoscale biological particles at atomic resolution. Such three-dimensional maps reveal the spatial structure of nanoscale biological particles. Accordingly, single particle analysis has been used to generate three-dimensional reconstructions of proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), vesicles, and other nanoscale biological particles.

[0003] Single particle analysis is based on having a set of images of nanoscale biological particles that show the nanoscale biological particles from various angles with only small differences between the angles. The images are generally acquired by transmission electron microscopy. Then, an algorithm executed by a processor can generate a three-dimensional map of the nanoscale biological particles based on the set of images. At least one thousand, and usually more, images may be required to generate a three-dimensional map at atomic resolution.

[0004] A relatively large number of images usable for single-particle analysis can be obtained from a sample containing many identical nanoscale biological particles. These nanoscale biological particles should preferably have diverse orientations with only slight differences between them. The sample can be placed in a transmission electron microscope to acquire images of the sample. These images constitute a collection of images of nanoscale biological particles, showing them from diverse angles with only slight differences between them. The latter is important for obtaining atomic resolution.

[0005] Samples for single-particle analysis by transmission electron microscopy are generally prepared as follows: A transmission electron microscope grid is used as a substrate, and a so-called perforated film is placed on it. The perforated film typically contains a porous carbon thin film. A predetermined amount of liquid containing the same nanoscale biological particles is deposited on the perforated film present on the transmission electron microscope grid. The amount of liquid deposited may be, for example, several microliters or several hundred nanoliters. The liquid containing the nanoscale biological particles may be an aqueous solution, generally a physiological buffer solution.

[0006] Next, the amount of liquid on the perforated membrane is reduced to obtain a liquid thin film. To do this, the liquid is gradually removed from the perforated membrane, generally using absorbent blotting paper. Once sufficient liquid has been removed, a thin film of liquid containing nanoscale biological particles remains within the pore opening regions of the perforated membrane. This liquid thin film preferably needs to have a thickness of several tens of nanometers to a maximum of several hundred nanometers.

[0007] Then, a transmission electron microscope grid, loaded with a thin film of liquid, is immersed in a cryogenic liquid, such as liquid ethane. This is commonly known as plunge freezing and results in the rapid freezing of the liquid containing nanoscale biological particles. If the liquid is water, plunge freezing prevents water molecules from forming ice crystals. This allows the water to transform into glassy ice. The same nanoscale biological particles are then immobilized within the thin film of glassy ice.

[0008] This sample preparation method satisfies various requirements for single-particle analysis by transmission electron microscopy. Firstly, plunge freezing fixes a thin film of liquid containing nanoscale biological particles. This prevents the thin film from evaporating in the vacuum present within the transmission electron microscope. Secondly, a thin film of glassy ice, preferably much thinner than 100 nanometers, enables satisfactory quality images to be obtained by transmission electron microscopy. In principle, the thinner the glassy ice film, the less background noise there is in the image, and the higher the resolution of the image. Thirdly, glassy ice constitutes a uniform amorphous matrix that does not significantly disrupt the structure of the nanoscale biological particles being imaged. Furthermore, glassy ice is permeable to electrons. Thus, in the sample preparation, it is important that the liquid thin film is sufficiently thin and transformed into glassy ice rather than crystalline ice.

[0009] Several difficulties and practical problems exist in relation to the sample preparation method described herein. Firstly, the thickness of the glassy ice layer is difficult to control. Various factors, such as ambient humidity and other ambient conditions, the timing and speed of blotting, and the timing and speed of plunge freezing, affect the thickness. In practice, reproducibility between grids tends to be low. Continuous sample preparation performed in a similar manner can result in considerable variability in the thickness of the glassy ice layer.

[0010] Generally, there is an optimal thickness for glassy ice layers. This optimal thickness is related to the dimensions of the nanoscale biological particles of interest. In general, the optimal thickness corresponds to the dimensions of the nanoscale biological particles. Approximating the optimal value sufficiently is a time-consuming process, and it is further specific to each different type of particle. This is because different types of nanoscale biological particles can differ in size and have different surface properties. Furthermore, different types of nanoscale biological particles can interact differently with the liquid-air interface where the surface of the liquid containing the particles comes into contact with the surrounding air.

[0011] Further complicating matters is the delicate process of forming thin liquid films by blotting, as described herein. Within the liquid film, nanoscale biological particles are trapped between two liquid-air interfaces. When the liquid is removed, the film thins, potentially allowing at least some of the nanoscale biological particles to resist this trapping and be released from the liquid. The glassy ice film obtained by plunge freezing may contain too few nanoscale biological particles for effective imaging. Conversely, sections of sufficient thickness for imaging may contain no nanoscale biological particles at all.

[0012] Another issue concerns nanoscale biological particles with diverse orientations within a sample, so that the image of the sample shows nanoscale biological particles from various angles. Nanoscale biological particles, such as proteins, can interact with one or both of the two liquid-air interfaces. This interaction can cause certain types of nanoscale biological particles to have a preferred orientation. This complicates, if not prevents, obtaining an image of a set of nanoscale biological particles showing them from images at various angles. As mentioned above herein, obtaining such an image set is necessary for single-particle analysis.

[0013] Another difficulty and practical problem is that samples obtained after plunge freezing must be stored at cryogenic temperatures until sufficient images of the sample are obtained by transmission electron microscopy. Samples particularly sensitive to temperature fluctuations must be handled, transported, and imaged at cryogenic temperatures. Long-term storage of samples may not be economically, technically, or both feasible. This imposes significant logistical constraints on sample preparation and imaging. Furthermore, it makes single-particle analysis relatively complex and therefore costly. [Overview of the project] [Problems that the invention aims to solve]

[0014] There is a need for sample preparation techniques for particle structure analysis using transmission electron microscopy that result in improvements in at least one of the following: ease of implementation, achievable resolution in transmission electron microscopy, reproducibility, and effectiveness. [Means for solving the problem]

[0015] An aspect of the present invention as defined in claim 1 relates to a method for preparing a sample for particle structure analysis by transmission electron microscopy. The method is: - A predetermined amount of liquid containing at least one particle to be analyzed is sandwiched between an upper two-dimensional material layer and a lower two-dimensional material layer, the lower two-dimensional material layer being supported by a transmission electron microscope grid. - The method involves reducing the amount of liquid so that at least one particle contained in the liquid is sealed within a cell formed by an upper two-dimensional material layer and a lower two-dimensional material layer, and the remaining amount of liquid is less than 50 nm thick within the cell.

[0016] A further aspect of the present invention, as defined in claim 12, relates to a method for performing particle structure analysis by transmission electron microscopy, wherein the sample is prepared according to the method described herein.

[0017] A further aspect of the present invention, as defined in claim 15, relates to a sample preparation for particle structure analysis by transmission electron microscopy. The sample preparation comprises at least one particle, enclosed in a cell formed by an upper two-dimensional material layer and a lower two-dimensional material layer, such that the amount of remaining liquid in the cell is less than 50 nm thick.

[0018] In each of these embodiments, the particles in the sample are enclosed in a cell formed by an upper two-dimensional material layer and a lower two-dimensional material layer. Therefore, plunge freezing is not required, although it can be used. Since plunge freezing is not required, the sample can be stored at non-cryogenic temperatures. Relatively stable samples can be handled, transported, and imaged at non-cryogenic temperatures. Samples can be stored for relatively long periods at an acceptable cost. All of these result in improved ease of implementation.

[0019] Furthermore, the amount of liquid can be reduced relatively safely to the extent that the sample has a thickness that approximates the dimensions of the target particles. By sandwiching the particles between the upper two-dimensional material layer and the lower two-dimensional material, the risk of particles being released from the liquid is reduced, if not eliminated. This allows for a sufficient approximation of the optimal thickness without significant risk and therefore in a relatively easy manner. Moreover, the thickness is relatively well controlled. For a given type of particle, the variation in thickness between samples can be relatively small. The amount of remaining liquid in the sample is relatively small. This allows for imaging of one or more particles contained within the sample with relatively high resolution and relatively low background noise. All of this results in improvements in ease of implementation, achievable resolution, reproducibility, and effectiveness in transmission electron microscopy.

[0020] In the embodiment of claim 2, a plurality of particles are enclosed in a cell formed by an upper two-dimensional material layer and a lower two-dimensional material layer.

[0021] In the embodiment of claim 3, the plurality of particles encapsulated in the cell are nanoscale biological particles, the term nanoscale biological particles includes proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and vesicles.

[0022] In the embodiment of claim 4, a single particle is enclosed in a cell formed by an upper two-dimensional material layer and a lower two-dimensional material layer.

[0023] In the embodiment according to claim 5, the single particle enclosed in the cell is a biological cell, and the term biological cell includes bacteria, spores, mammalian cells, plant cells, and populations of such cells.

[0024] In the embodiment according to claim 6, a porous membrane is included between the lower two-dimensional material layer and a transmission electron microscope grid.

[0025] In the embodiment according to claim 7, the upper two-dimensional material layer and the lower two-dimensional material layer comprise graphene.

[0026] In the embodiment according to claim 8, when sandwiching the volume of liquid, a further two-dimensional material layer is included between the volume of liquid and at least one of the two-dimensional material layers that is the upper two-dimensional material layer and the lower two-dimensional material layer.

[0027] In the embodiment according to claim 9, the further two-dimensional material layer comprises graphene oxide.

[0028] In the embodiment according to claim 10, sandwiching the volume of liquid between the upper two-dimensional material layer and the lower two-dimensional material layer comprises: - placing the upper two-dimensional material layer onto the liquid comprising at least one particle to be analyzed, and - using a loop to deposit a droplet of the liquid with the upper two-dimensional material layer floating thereon onto the lower two-dimensional material layer supported by a transmission electron microscope grid.

[0029] In the embodiment according to claim 11, reducing the volume of liquid comprises blotting with a liquid-absorbing material.

[0030] In the embodiment according to claim 13, the prepared sample is stored under non-cryogenic conditions.

[0031] In the embodiment according to claim 14, particle structure analysis is performed under non-cryogenic conditions.

[0032] For illustrative purposes, some embodiments of the present invention will be described in detail with reference to the accompanying drawings. Further features will be presented in this description, some of which are defined in the dependent claims, and their advantages will become apparent. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 is a flowchart illustrating the method for preparing a sample for particle structure analysis using transmission electron microscopy. [Figure 2] Figure 2 is a schematic cross-sectional view of a sample preparation of the first example for particle structure analysis by transmission electron microscopy. [Figure 3] Figure 3 is an image obtained by transmission electron microscopy, showing a sample preparation corresponding to the sample preparation of the first example. [Figure 4] Figure 4 is a schematic cross-sectional view of a sample preparation for a second example of particle structure analysis by transmission electron microscopy. [Figure 5] Figure 5 is an image obtained by transmission electron microscopy, showing a sample preparation corresponding to the sample preparation of the second example. [Modes for carrying out the invention]

[0034] Figure 1 schematically illustrates a method for preparing a sample for particle structure analysis by transmission electron microscopy. Figure 1 provides a flowchart of the method, including several steps. The method uses two-dimensional material layers. The two-dimensional material may include, for example, graphene, graphene oxide, hexagonal boron nitride, molybdenum sulfide, ultrathin amorphous carbon, or any other suitable two-dimensional material layer. For the sake of clarity in describing the method, we will assume, as an example, that each two-dimensional material layer contains graphene. Thus, it should be understood that the term graphene layer means any suitable two-dimensional material layer, and any suitable superposition of two-dimensional material layers.

[0035] In the first step 101, the graphene layer is placed on a transmission electron microscope grid. A perforated film may be placed on the transmission electron microscope grid beforehand. In this case, the graphene layer is placed on the perforated film, which is then placed on the transmission electron microscope grid. The perforated film primarily functions to provide structural support to the graphene layer. In contrast, in the prior art sample preparation, the perforated film primarily functions to form a thin liquid layer as described herein in the background art section.

[0036] For ease of reading, the graphene layer placed on the transmission electron microscope grid will be referred to as the lower graphene layer below. One side of the lower graphene layer is in contact with either the transmission electron microscope grid or a perforated film. Below, this side will be referred to as the outside, and the opposite side as the inside.

[0037] The interior of the lower graphene layer may be covered with another two-dimensional material layer. That is, a superposition of two different two-dimensional material layers, including the lower graphene layer and the other two-dimensional material layer, may be placed on a transmission electron microscope grid. The other two-dimensional material layer may be, for example, graphene oxide. Covering the interior of the lower graphene layer with another two-dimensional material layer can be advantageous, and this will be explained below. For the sake of readability, the lower graphene layer below refers to the lower graphene layer itself and, where applicable, the superposition including the lower graphene layer covered with the other two-dimensional material layer.

[0038] In the second step 102, a predetermined amount of liquid containing at least one particle to be analyzed is added to the top of the lower graphene layer. The amount of liquid may be, for example, several hundred nanoliters or microliters. The amount of liquid may have a particle concentration of, for example, 1 to 10 nanomoles / milliliter.

[0039] Adding a predetermined amount of liquid to the top of the lower graphene layer can be done in various ways, some of which are described herein. For example, droplets of liquid containing particles or several particles can be deposited by pipetting. Another example is spraying a liquid mist onto the lower graphene layer. Yet another example is immersing a transmission electron microscope grid, along with the perforated film and lower graphene layer present on it, in the liquid. Alternatively, the lower graphene layer can be brought into surface contact with the liquid. In yet another example, one or more target particles can first be deposited, fixed, or grown on the lower graphene layer. Then, one or more particles can be incorporated into the liquid by bringing the lower graphene layer, supported, for example by a transmission electron microscope grid, into contact with the liquid.

[0040] In the third step 103, an additional graphene layer is placed on top of the liquid added to the lower graphene layer. For ease of reading, this additional graphene layer will be referred to as the upper graphene layer below. One side of the upper graphene layer is in contact with the liquid. Hereafter, this side will be referred to as the inside and the opposite side as the outside. Like the inside of the lower graphene layer, the inside of the upper graphene layer may be covered with another two-dimensional material layer, such as graphene oxide. Here again, covering the inside of the upper graphene layer with another two-dimensional material layer can be advantageous, and this will be explained below. For ease of reading, the upper graphene layer below refers to the upper graphene layer itself and, where applicable, the superposition including the upper graphene layer covered with another two-dimensional material layer.

[0041] The upper graphene layer can be added in various ways, some of which are described herein. For example, the upper graphene layer can be placed on top of a volume of liquid using an additional transmission electron microscope grid as a support. In this way, a sandwich structure containing two transmission electron microscope grids is temporarily formed. As another example, the lower graphene layer can be manipulated using a transmission electron microscope grid that supports the volume of liquid present above it, so that the liquid comes into contact with the upper graphene layer which may be suspended on the fluid. As yet another example, a loop can be used to receive droplets of liquid on which the upper graphene layer is suspended. The loop holds the droplets together with the upper graphene layer suspended above it. The droplets are then moved and added to the volume of liquid already present on the lower graphene layer. This loop-assisted transfer technique is described in detail in International Publication No. 2021 / 123458.

[0042] The loop-assisted transfer technique can be combined with the second step 102 and the third step 103. The liquid in which the upper graphene layer is suspended may contain one or more particles to be analyzed. Using the loop, a droplet of liquid containing one or more particles to be analyzed can be received along with the upper graphene layer suspended on the droplet. The droplet is then moved and deposited onto the lower graphene layer by the loop. Thus, a single operation is sufficient to achieve that a predetermined amount of liquid containing at least one particle is present on the lower graphene layer and the upper graphene layer is present on top of the predetermined amount of liquid.

[0043] When the third step 103 is completed, whether performed separately or in combination with the second step 102, a graphene-liquid-graphene sandwich structure is obtained. The amount of liquid containing at least one particle to be analyzed is sandwiched between the upper graphene layer and the lower graphene layer. The lower graphene layer is supported by a transmission electron microscope grid, thereby allowing a perforated membrane to form the intermediate portion.

[0044] In the fourth step 104, the amount of liquid sandwiched between the upper and lower graphene layers is reduced. The amount of liquid can be reduced, for example, by blotting with liquid-absorbing paper or another liquid-absorbing material. The amount of liquid is reduced to the extent that, in a particular region, the upper and lower graphene layers are attracted to each other by van der Waals forces. As a result, one or more cells can be formed that contain the liquid along with at least one particle. That is, one or more particles, along with the remaining amount of liquid, can be trapped within such cells. Specifically, this involves reducing the amount of liquid to the extent that the remaining amount of liquid has a thickness of less than 50 nm in the cell. The cells generally have a thickness corresponding to the thickness of the one or more particles trapped within the cell. Two cases can be distinguished in this regard.

[0045] When multiple particles are trapped within a cell, the density of these particles is relatively low, while the corresponding remaining liquid can fill the spaces between them. The upper graphene layer between two adjacent particles is relatively flat and may exhibit relatively little or even insignificant sagging. The upper graphene layer can be thought of as the roof of a tent that can be stretched relatively tightly, and the multiple particles can be thought of as the tent poles supporting the roof. In this example, the amount of remaining liquid may be roughly the thickness of the cell.

[0046] When a single particle is confined within a cell, the upper graphene layer can tightly surround the particle. The amount of remaining liquid can be concentrated, on the one hand, near the lower graphene layer in the region surrounded by the upper graphene layer in contact with the lower graphene layer, and on the other hand, around the upper graphene layer in contact with, or at least in close proximity to, the particle. In this case, the amount of remaining liquid can be orders of magnitude thinner than the cell.

[0047] Figure 2 shows a sample preparation 200 of the first example for particle structure analysis by transmission electron microscopy. Figure 2 provides a schematic cross-sectional view of this sample preparation 200 of the first example. The sample preparation 200 of the first example can be obtained by the method described herein with reference to Figure 1. The sample preparation 200 of the first example includes a cell 201 formed by an upper graphene layer 202 and a lower graphene layer 203. The cell 201 contains a plurality of particles 204 and a quantity of remaining liquid 205 between these plurality of particles 204. The cell 201 is supported by a perforated membrane 206, which is further supported by a transmission electron microscope grid 207.

[0048] The sample preparation 200 of the first example corresponds to the example described herein, in which multiple particles are confined within a cell, but the density of these particles is relatively low. The upper graphene layer 202 is relatively flat, as is the lower graphene layer 203. Thus, the cell 201 has a relatively uniform thickness corresponding to the thickness of the multiple particles 204. That is, the multiple particles 204 are in a relatively flat environment. This situation is generally advantageous for creating a three-dimensional reconstruction of the particles of interest.

[0049] Figure 3 is an image 300 obtained by transmission electron microscopy containing a sample preparation corresponding to sample preparation 200 of the first example. Image 300 was obtained at room temperature. In this sample preparation, the particles are the same protein. Image 300 shows cells containing multiple proteins arranged in a single layer; that is, no stacked protein layers are visible. This indicates that the cells have a thickness that closely approximates the dimensions of the proteins.

[0050] The sample prepared according to the method described herein with reference to Figure 1 is advantageous in many respects. The cell formed by the upper and lower graphene layers can spontaneously exhibit a thickness corresponding to the dimensions of the target particles, in which many particles can be encapsulated by the cell. Thus, the cell contains a relatively small amount of liquid and is relatively thin, sufficient to accommodate many of the target particles. This allows imaging to be acquired by transmission electron microscopy with relatively low background noise. In general, when the cell thickness corresponds to the dimensions of the target particles, the maximum resolution is achieved, or at least can be very close to it.

[0051] Furthermore, the cells formed by the upper and lower graphene layers can spontaneously exhibit a thickness corresponding to the dimensions of the target particles, thus providing high reproducibility. The cells within the sample preparation generally have a uniform thickness that closely matches the thickness of other sample preparations for the same target particles. This uniform thickness is largely independent of ambient conditions or the specific details of the sample preparation procedure. In particular, variations in the timing and extent of blotting generally do not result in significant variations in cell thickness.

[0052] Another advantage is the relative stability of the cells within the sample preparation. Once the cells are formed, the upper and lower graphene layers ensure that the cells are airtight and liquid-tight. Furthermore, these cells remain stable over a wide temperature range, including room temperature, cryo temperatures, and even temperatures higher than room temperature. Therefore, the sample can be prepared well in advance of imaging, even in a location different from, or even remote from, where imaging will take place. In contrast to conventional sample preparations based on plunge freezing, there is no need for the potentially cumbersome storage, transport, and imaging of the sample under cryogenic conditions. Nevertheless, samples prepared according to the method described herein, with reference to Figure 1, can be plunge-frozen and then thawed again, if desired.

[0053] Since the sample preparation is stable at room temperature and also stable in a vacuum, imaging by transmission electron microscopy can be performed at room temperature. This also simplifies the structural analysis of the particles. Furthermore, particles within the sample preparation can move, perform functions, aggregate, rotate, and react. This allows for real-time dynamic imaging of the particles, particularly imaging of processes within the sample involving the particles. Three-dimensional reconstruction of dynamic properties can be performed.

[0054] Another advantage relates to the desirable need for particles within a sample to have diverse orientations, so that images of the sample show the particles from various angles. In the sample preparation described herein, the upper and lower graphene layers may prevent particles from interacting with these layers so that the particles have a preferred orientation. As described herein, the insides of the upper and lower graphene layers may be covered with another two-dimensional material. This other two-dimensional material may more strongly prevent particles of the desired type from interacting with the upper and lower graphene layers, and therefore more strongly prevent these particles from having a preferred orientation. For example, the insides of the upper and lower graphene layers may be covered with graphene oxide, which is more hydrophilic than graphene. Thus, a sample can be prepared according to the method described herein with reference to Figure 1 so that the particles have a variety of different orientations. In this way, imaging of the sample will provide a set of images showing the particles from various angles, which is advantageous for structural analysis.

[0055] Figure 4 shows a sample preparation 400 of a second example for particle structure analysis by transmission electron microscopy. Figure 4 provides a schematic cross-sectional view of this sample preparation 400 of the second example. The sample preparation 400 of the second example is particularly suitable for structural analysis of biological cells, such as bacteria, spores, mammalian cells, plant cells, or populations of such cells. The sample preparation 400 of the second example can be obtained by the method described herein with reference to Figure 1. The sample preparation 400 of the second example includes a graphene cell 401 formed by an upper graphene layer 402 and a lower graphene layer 403. The upper graphene layer 402 closely surrounds the biological cell 404. The graphene cell 401 is supported by a perforated membrane 406, which is further supported by a transmission electron microscope grid 405.

[0056] The graphene cell 401 can encapsulate biological cells 404 along with a relatively small amount of remaining liquid 407. The amount of remaining liquid 407 is concentrated on the one hand near the lower graphene layer 403 in the region surrounded by the upper graphene layer 402 in contact with the lower graphene layer 403, and on the other hand around the upper graphene layer 402 in contact with, or at least in close proximity to, the biological cells 404. The amount of remaining liquid 407 can be orders of magnitude thinner than the graphene cell 401. The amount of remaining liquid 407 may be, for example, less than 50 nm thick.

[0057] The amount of remaining liquid 407 is relatively small due to the following: the upper graphene layer 402 and the lower graphene layer 403 are attracted to each other. As a result, when preparing the sample as described above with reference to Figure 1, a relatively large amount of liquid may be discharged around the biological cells 404. The liquid can be discharged, for example, by blotting as described above.

[0058] Because the amount of remaining liquid 407 is relatively small, the biological cells 404 can be imaged by transmission electron microscopy with relatively high resolution and low background noise. Furthermore, this imaging can be performed at room temperature. The graphene cell 401 protects the biological cells 404 from the vacuum present in the transmission electron microscope used for imaging. The graphene cell 401 further supports the biological cells 404 in this process. In fact, many of the advantages described herein in relation to the sample preparation 200 of the first example apply equally to the sample preparation 400 of the second example.

[0059] Figure 5 is an image 500 obtained by transmission electron microscopy containing a sample preparation corresponding to sample preparation 400 of the second example. Image 500 was acquired at room temperature. The biological cell is embedded between two graphene layers, one of which is the upper graphene layer and the other is the lower graphene layer. The upper graphene layer covers the biological cell. The upper graphene layer is observed in the image as wrinkles radiating outward from the periphery of the biological cell. The lower graphene layer is observed to be flattened and therefore not observed.

[0060] To obtain a three-dimensional reconstruction of biological cells, the sample preparation can be tilted within a transmission electron microscope, and images can be acquired from various angles. These images acquired from various angles of the biological cells can then be used to construct a three-dimensional image of the cells.

[0061] The sample preparations 200 of the first example and 400 of the second example share several common features. By encapsulating the particles between the upper and lower graphene layers, the particles are protected from ambient conditions, particularly the vacuum within the transmission electron microscope, without the need to rely on cryogenic techniques. Furthermore, this encapsulation allows for relatively easy removal of excess liquid during sample preparation. Since any excess liquid can reduce image resolution and cause background noise, this also allows for imaging of the particles with relatively high resolution and low background noise.

[0062] In the sample preparations of both examples, a relatively small amount of liquid remains, which generally does not significantly degrade image quality. Specifically, in the sample preparation 200 of the first example, which is particularly suitable for structural analysis of nanoscale biological particles, the amount of remaining liquid 205 may be approximately the thickness of the cell 201. Furthermore, the cell 201 may have a thickness corresponding to the dimensions of the nanoscale biological particles 204. As a result, the nanoscale biological particles 204 are encapsulated within a thin but flat fixed structure. In the sample preparation 400 of the second example, which is particularly suitable for structural analysis of biological cells 404, the amount of remaining liquid 407 may be orders of magnitude thinner than the dimensions of the biological cells 404. In the sample preparations of both examples, the amounts of remaining liquid 205 and 407 may be less than 50 nm thick in the cells 201 and 401. More specifically, the amounts of remaining liquid 205 and 407 may be less than 40 nm, 30 nm, less than 20 nm, or even less than 10 nm thick. The thickness can be measured between the upper two-dimensional material layer and the lower two-dimensional material layer, as the extent to which the remaining liquid exists.

[0063] Precautions The embodiments described herein with reference to the drawings are shown as examples. The present invention can be carried out in numerous different ways. To illustrate this, some alternative forms are briefly shown.

[0064] The present invention can be applied to a wide variety of products or methods relating to particle structure analysis. In the presented embodiments, sample preparations are examined in relation to single-particle analysis, which is a form of particle structure analysis. In other embodiments, for example, samples can be prepared according to the present invention for particle structure analysis based on three-dimensional tomography, four-dimensional electron diffraction, and microelectron diffraction.

[0065] There are numerous different methods for reducing the amount of liquid in the method of the present invention. In the embodiments described herein, the amount of liquid is reduced by blotting using a liquid absorbent material. In other embodiments, the amount of liquid may be reduced using other techniques.

[0066] The term "grid for transmission electron microscopy" should be interpreted broadly. This term may be used in transmission electron microscopy and encompasses any type of support, including but not limited to metals, and may include any suitable type of material.

[0067] The matters described herein indicate that the embodiments described with reference to the drawings are illustrative of the invention, not limiting it. The invention can be carried out in a number of alternative ways within the appended claims. All modifications that fall within the equivalent meaning and scope of the claims are encompassed within that scope. Any reference numerals in the claims should not be construed as limiting the claims. The verb “comprise” in the claims does not preclude the existence of other elements or steps other than those described in the claims. The same applies to similar verbs such as “include” and “contain.” The description of a singular element in a claim relating to a product does not preclude the product from comprising more than one such element. Similarly, the description of a singular step in a claim relating to a method does not preclude the method from comprising more than one such step. The mere fact that each dependent claim defines its own additional features does not preclude combinations of additional features other than those reflected in the claims.

Claims

1. A method for preparing samples (200, 400) for particle structure analysis by transmission electron microscopy, - A predetermined amount of liquid containing at least one particle (204, 404) to be analyzed is sandwiched (102, 103) between an upper two-dimensional material layer (202, 402) and a lower two-dimensional material layer (203, 403), wherein the lower two-dimensional material layer is supported by a transmission electron microscope grid (207, 405). A method for preparing a sample for particle structure analysis, comprising: encapsulating at least one particle contained in the amount of liquid in a cell (201, 401) formed by the upper two-dimensional material layer and the lower two-dimensional material layer, wherein the cell has a thickness corresponding to the thickness of the at least one particle to be analyzed, and reducing the amount of liquid (104) to such an extent that the remaining amount of liquid (205, 407) has a thickness of less than 50 nm in the cell.

2. A method for preparing a sample for particle structure analysis according to claim 1, wherein a plurality of particles (204) are enclosed in the cell (201), and the cell has a uniform thickness.

3. A method for preparing a sample for particle structure analysis according to claim 2, wherein the plurality of particles (204) enclosed in the cell (201) are nanoscale biological particles, the term nanoscale biological particles includes proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and vesicles.

4. A method for preparing a sample for particle structure analysis according to claim 1, wherein a single particle (404) is enclosed in the cell (401).

5. A method for preparing a sample for particle structure analysis according to claim 4, wherein the single particle (404) enclosed in the cell (401) is a biological cell, and the term biological cell includes bacteria, spores, mammalian cells, plant cells, and populations of such cells.

6. A method for preparing a sample for particle structure analysis according to any one of claims 1 to 5, comprising a perforated film (206, 406) between the lower two-dimensional material layer (203, 403) and the transmission electron microscope grid (207, 405).

7. A method for preparing a sample for particle structure analysis according to any one of claims 1 to 6, wherein the upper two-dimensional material layer (202, 402) and the lower two-dimensional material layer (203, 403) contain graphene.

8. A method for preparing a sample for particle structure analysis according to any one of claims 1 to 7, wherein when the amount of liquid is sandwiched between (102, 103) the amount of liquid, a further two-dimensional material layer is included between the amount of liquid and at least one of the two-dimensional material layers, which are the upper two-dimensional material layer (202, 402) and the lower two-dimensional material layer (203, 403).

9. The method for preparing a sample for particle structure analysis according to claim 8, wherein the further two-dimensional material layer comprises graphene oxide.

10. The amount of the aforementioned liquid is sandwiched between (102, 103), - Placing the upper two-dimensional material layer (202, 402) on a liquid containing the at least one particle (204, 404) to be analyzed, A method for preparing a sample for particle structure analysis according to any one of claims 1 to 9, comprising using a loop to deposit droplets of the liquid in which the upper two-dimensional material layer is suspended onto the lower two-dimensional material layer (203, 403) supported by the transmission electron microscope grid (207, 405).

11. A method for preparing a sample for particle structure analysis according to any one of claims 1 to 10, wherein reducing the amount of the liquid (104) comprises blotting with a liquid absorbent material.

12. A method for performing particle structure analysis by transmission electron microscopy, comprising preparing a sample (200, 400) for particle structure analysis according to any one of claims 1 to 11.

13. A method for performing particle structure analysis according to claim 12, wherein the prepared sample (200, 400) is stored under non-microtemperature conditions.

14. A method for performing particle structure analysis according to claim 13, wherein the particle structure analysis is performed under non-ultra-low temperature conditions.

15. A sample preparation for particle structure analysis by transmission electron microscopy, wherein the sample preparation comprises at least one particle (204, 404) to be analyzed, enclosed in a cell (201, 401) formed by an upper two-dimensional material layer (202, 402) and a lower two-dimensional material layer (203, 403), the cell having a thickness corresponding to the thickness of the at least one particle to be analyzed, and the amount of remaining liquid (205, 407) having a thickness of less than 50 nm in the cell.

Images