Preparation of a sample for structural particle analysis

Abstract

A sample is prepared for structural particle analysis by means of transmission electron microscopy in the following manner. A quantity of liquid, which contains at least one particle to be analyzed, is sandwiched (102, 103) between a top two-dimensional material layer and a bottom two-dimensional material layer. The bottom two-dimensional material layer is supported by a transmission electron microscopy grid. The quantity of liquid is reduced (104) to an extent that at least one particle contained therein is encapsulated in a cell formed by the top two-dimensional material layer and the bottom two-dimensional material layer. Specifically, the quantity of liquid is reduced (104) to an extent that the cell has a thickness corresponding with that of the at least one particle to be analyzed with a remaining quantity of liquid less than 50 nm thick throughout the cell.

Description

[0001] Preparation of a sample for structural particle analysis.

[0002] FIELD OF THE INVENTION

[0003] An aspect of the invention relates to a method of preparing a sample for structural particle analysis by means of transmission electron microscopy. The sample may comprise nanoscale biological particles, such as, for example, proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and vesicles. In that case, the structural particle analysis may be a so-called single particle analysis. Alternatively, the sample may comprise a biological cell, such as, for example, a bacterium, a spore, a mammalian cell, plant cell, or an ensemble of such cells. Other aspects of the invention relate to a method of carrying out structural particle analysis by means of transmission electron microscopy and a sample preparation for structural particle analysis by means of transmission electron microscopy.

[0004] BACKGROUND ART

[0005] Single particle analysis is a relatively recent technique that allows producing a three-dimensional map of a nanoscale biological particle at atomic resolution. Such a three-dimensional map reveals a spatial structure of the nanoscale biological particle. Accordingly, single particle analysis has been used to create three-dimensional reconstructions of proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), vesicles, as well as other nanoscale biological particles.

[0006] Single particle analysis relies on having a set of images of a nanoscale biological particle showing the nanoscale biological particle from a wide variety of angles with only minor differences between these angles. These images are generally taken by means of transmission electron microscopy. An algorithm executed by a processor may then create a three-dimensional map of the nanoscale biological particle on the basis of the set of images. At least a thousand images, usually many more, may be required to create the three-dimensional map at atomic resolution.

[0007] A relatively great number of images usable for single particle analysis may be obtained from a sample that comprises many nanoscale biological particles, which are identical. These nanoscale biological particles should preferably have a wide variety of orientations with only minor differences between these orientations. The sample may be placed in a transmission electron microscope so as to take an image of the sample. This image then constitutes an assembly of images of the nanoscale biological particle, showing the nanoscale biological particle from a wide variety of angles with only minor differences between these angles. The latter is important for achieving atomic resolution.

[0008] A sample for single particle analysis by means of transmission electron microscopy is generally prepared as follows. A transmission electron microscopy grid is used as a substrate on which a so-called holey film is placed. A holey film typically comprises a thin carbon film with holes. A quantity of liquid containing identical nanoscale biological particles is deposited on the holey film, which is present on the transmission electron microscopy grid. The quantity of liquid that is deposited may be, for example, several microliters or several hundreds of nanoliters. The liquid containing the nanoscale biological particles may be a water-based solution, generally a physiological buffer.

[0009] The quantity of liquid on the holey film is then reduced in order to obtain a thin liquid film. To that end, liquid is gradually drained from the holey film, generally by means of an absorbent blotting paper. When sufficient liquid has been removed, a thin film of liquid containing the nanoscale biological particles remains standing within an open area of the holes in the holey film. The thin liquid film should preferably have a thickness in the order of magnitude of tens to at most hundreds of nanometers.

[0010] The transmission electron microscopy grid carrying the thin film of liquid is then immersed in a liquid at a cryogenic temperature, such as, for example liquid ethane. This is commonly referred to as plunge freezing, which causes fast freezing of the liquid containing the nanoscale biological particles. In case the liquid is water, plunge freezing makes that water molecules do not have the opportunity to form ice crystals. This allows transformation of the water into vitreous ice. The identical nanoscale biological particles are fixated in a thin film of vitreous ice.

[0011] This sample preparation method meets various requirements for single particle analysis by means of transmission electron microscopy. Firstly, plunge freezing fixates the thin film of liquid, which comprises the nanoscale biological particles. This prevents the thin film from evaporating in a vacuum that exist in a transmission electron microscope. Secondly, the thin film of vitreous ice, preferably much thinner than a hundred nanometers, allows obtaining images of satisfactory quality by means of transmission electron microscopy. In principle, the thinner the film of vitreous ice is, the less background noise will be present in the images and the higher the resolution these images will have. Thirdly, the vitreous ice constitutes a uniform and amorphous matrix that does not significantly disrupt the structure of the nanoscale biological particles being imaged. What is more, the vitreous ice is transparent to electrons. Thus, it is critical in sample preparation that the thin liquid film is sufficiently thin and transforms into vitreous ice rather than crystalline ice.

[0012] There are several drawbacks and practical problems associated with the sample preparation method described hereinbefore. Firstly, the thickness of the vitreous ice layer is difficult to control. Various factors affect thickness, such as, for example, ambient humidity, as well as other ambient conditions, timing and speed of blotting, and timing and speed of plunge freezing. In practice, grid-to-grid reproducibility tends to be low. Successive sample preparations, which are carried out similarly, may result in quite large variations in vitreous ice layer thicknesses.

[0013] In general, there is an optimum for the thickness of the vitreous ice layer. This optimum thickness is related to the dimensions of the nanoscale biological particle of interest. Generally, the optimum thickness corresponds with the dimensions of the nanoscale biological particle. Sufficiently approximating the optimum can be a timeconsuming process, which, moreover, is unique for every different type of particle. This is because different types of nanoscale biological particles may differ in size and may have different surface properties. Moreover, different types of nanoscale biological particles may interact differently with a liquid-air interface, where a surface of the liquid in which the particles are contained is in contact with ambient air.

[0014] Further complicating matters is that creating a thin film of liquid by blotting, as described hereinbefore, is a delicate process. In a film of liquid, nanoscale biological particles are confined between two liquid-air interfaces. While liquid is removed, the film may become so thin that at least some of the nanoscale biological particles resist this confinement and are expelled from the liquid. The thin film of vitreous ice obtained by plunge-freezing may contain too few nanoscale biological particles for effective imaging. It may also happen that a section with a suitable thickness for imaging does not contain any nanoscale biological particle.

[0015] Another issue concerns nanoscale biological particles having a wide variety of orientations in a sample so that an image of the sample shows the nanoscale biological particles from a wide variety of angles. Nanoscale biological particles, such as, for example, proteins, may interact with one of the two liquid-air interfaces, or both. This interaction may make that certain types of nanoscale biological particles have a preferred orientation. This complicates, if not prevents, obtaining a set of images of a nanoscale biological particle showing the nanoscale biological particle from a wide variety of angles images. As mentioned hereinbefore, obtaining such as set of images is required for single particle analysis.

[0016] Yet another drawback and practical problem is that a sample that has been obtained after plunge freezing should be kept at cryogenic temperature until sufficient images of the sample have been taken by means of transmission electron microscopy. The sample, which is particularly sensitive to temperature fluctuations, should be handled, transported, and imaged at cryogenic temperature. Long-time storage of the sample may not be economically or technically feasible, or both. This imposes significant logistical constraints on sample preparation and imaging. Also, this makes that single particle analysis is relatively complicated and thus costly.

[0017] SUMMARY OF THE INVENTION

[0018] There is a need for a sample preparation technique for structural particle analysis by means of transmission electron microscopy that offers an improvement in at least one of the following aspects: ease of implementation, attainable resolution in transmission electron microscopy, reproducibility, and effectiveness.

[0019] An aspect of the invention, which is defined in claim 1, relates to a method of preparing a sample for structural particle analysis by means of transmission electron microscopy. The method comprises: sandwiching a quantity of liquid, which contains at least one particle to be analyzed, between a top two-dimensional material layer and a bottom two- dimensional material layer, the bottom two-dimensional material layer being supported by a transmission electron microscopy grid; reducing the quantity of liquid to an extent that at least one particle contained therein is encapsulated in a cell formed by the top two-dimensional material layer and the bottom two-dimensional material layer with a remaining quantity of liquid less than 50 nm thick throughout the cell.

[0020] A further aspect of the invention, which is defined in claim 12, relates to a method of carrying out structural particle analysis by means of transmission electron microscopy, whereby a sample is prepared in accordance with the method cited hereinbefore. Yet a further aspect of the invention, which is defined in claim 15, relates to a sample preparation for structural particle analysis by means of transmission electron microscopy. The sample preparation comprises at least one particle that is encapsulated in a cell formed by a top two-dimensional material layer and a bottom two-dimensional material layer with a remaining quantity of liquid less than 50 nm thick throughout the cell.

[0021] In each of these aspects, a particle in a sample is encapsulated in a cell formed by a top two-dimensional material layer and a bottom two-dimensional material layer. Accordingly, there is no need of plunge freezing, although plunge freezing may be used. Since no plunge freezing is required, the sample may be kept at non-cryogenic temperature. The sample, which is relatively stable, may be handled, transported, and imaged at non-cryogenic temperature. The sample may be stored for a relatively long time at acceptable cost. All this offers an improvement in ease of implementation.

[0022] Moreover, the quantity of liquid can be relatively safely reduced to an extent that the sample has a thickness that approximates the dimensions of the particle of interest. The risk of the particle being expelled from the liquid is reduced, if not eliminated, by the particle being sandwiched between the top two-dimensional material layer and the bottom two-dimensional material. This allows sufficiently approximating an optimum thickness without much risk and thus in a relatively easy manner. Moreover, the thickness is relatively well-controlled. For a given type of particle, variations in thickness between samples may be relatively small. The remaining quantity of liquid in the sample is relatively small. This allows imaging one or more particles, which are contained in the sample, with a relatively high resolution and with relatively low background noise. All this offers an improvement in ease of implementation, attainable resolution in transmission electron microscopy, reproducibility, and effectiveness.

[0023] In an embodiment according to claim 2, multiple particles are encapsulated in the cell formed by the top two-dimensional material layer and the bottom two- dimensional material layer.

[0024] In an embodiment according to claim 3, the multiple particles that are encapsulated in the cell are nano-scale biological particles, the term nano-scale biological particles including: proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and vesicles.

[0025] In an embodiment according to claim 4, a single particle is encapsulated in the cell formed by the top two-dimensional material layer and the bottom two-dimensional material layer. In an embodiment according to claim 5, the single particle that is encapsulated in the cell is a biological cell, the term biological cell including: a bacterium, a spore, a mammalian cell, a plant cell, and an ensemble of such cells.

[0026] In an embodiment according to claim 6, a holey film is comprised between the bottom two-dimensional material layer and the transmission electron microscopy grid.

[0027] In an embodiment according to claim 7, the top two-dimensional material layer and the bottom two-dimensional material layer comprise graphene.

[0028] In an embodiment according to claim 8, in sandwiching the quantity of liquid, a further two-dimensional material layer is comprised between the quantity of liquid and at least one of the following two-dimensional material layers: the top two-dimensional material layer and the bottom two-dimensional material layer.

[0029] In an embodiment according to claim 9, the further two-dimensional material layer comprises graphene oxide.

[0030] In an embodiment according to claim 10. sandwiching the quantity of liquid between a top two-dimensional material layer and a bottom two-dimensional material layer comprises: placing the top two-dimensional material layer on a liquid containing the at least one particle to be analyzed; and using a loop to deposit a droplet of the liquid with the top two- dimensional material layer floating thereon, on the bottom two-dimensional material layer supported by the transmission electron microscopy grid.

[0031] In an embodiment according to claim 11, reducing the quantity of liquid includes blotting with a liquid-absorbent material.

[0032] In an embodiment according to claim 13, the sample that has been prepared is kept under non-cryogenic conditions.

[0033] In an embodiment according to claim 14, the structural particle analysis is carried out under non-cryogenic conditions.

[0034] For the purpose of illustration, some embodiments of the invention are described in detail with reference to accompanying drawings. In this description, additional features will be presented, some of which are defined in the dependent claims, and advantages will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. l is a flow chart diagram of a method of preparing a sample for structural particle analysis by means of transmission electron microscopy.

[0036] FIG. 2 is a schematic cross-sectional diagram of a first exemplary sample preparation for structural particle analysis by means of transmission electron microscopy.

[0037] FIG. 3 is an image taken by a transmission electron microscope that comprises a sample preparation corresponding with the first exemplary sample preparation.

[0038] FIG. 4 is a schematic cross-sectional diagram of a second exemplary sample preparation for structural particle analysis by means of transmission electron microscopy.

[0039] FIG. 5 is an image taken by a transmission electron microscope that comprises a sample preparation corresponding with the second exemplary sample preparation.

[0040] DESCRIPTION OF SOME EMBODIMENTS

[0041] FIG. 1 schematically illustrates a method of preparing a sample for structural particle analysis by means of transmission electron microscopy. FIG. 1 provides a flow chart diagram of this method, which comprises several steps. In the method, two- dimensional material layers are used. A two-dimensional material may be, for example, graphene, graphene oxide, hexagonal boron nitride, molybdenum sulfide, ultrathin amorphous carbon, or any other suitable two-dimensional material layer. In describing the method, it will be assumed that each two-dimensional material layer comprises graphene by way of example and for ease of reading. The term graphene layer should thus be understood as meaning any suitable two-dimensional material layer, as well as any suitable superposition of two-dimensional material layers.

[0042] In a first step 101, a graphene layer is placed on a transmission electron microscopy grid. A holey film may have previously been placed on the transmission electron microscopy grid. In that case, the graphene layer rests on the holey film, which, in turn, rests on the transmission electron microscopy grid. The holey film primarily serves to provide structural support to the graphene layer. In contrast, in the prior-art sample preparation, the holey film primarily serves in forming a thin liquid layer as described hereinbefore in the background art section.

[0043] The graphene layer that is placed on the transmission electron microscopy grid will be referred to hereinafter as the bottom graphene layer for the sake of ease of reading. One side of the bottom graphene layer contacts the transmission electron microscopy grid, or the holey film, whichever applies. This side will be referred to hereinafter as the outer side, while the opposite side will be referred to as the inner side.

[0044] The inner side of the bottom graphene layer may be lined with another two- dimensional material layer. That is, a superposition of two different two-dimensional material layers may be placed on the transmission electron microscopy grid, which comprises the bottom graphene layer and another two-dimensional material layer. The other two-dimensional material layer may be, for example, graphene oxide. Lining the inner side of the bottom graphene layer with another two-dimensional material layer may be advantageous, which will be explained hereinafter. For ease of reading, the bottom graphene layer hereinafter refers to the bottom graphene layer as such, as well as to a superposition comprising the bottom graphene layer lined with another two-dimensional material layer, wherever applicable.

[0045] In a second step 102, a quantity of liquid, which contains at least one particle to be analyzed, is added op top of the bottom graphene layer. The quantity of liquid may be in the order of, for example, hundreds of nanoliters or microliters. The quantity of liquid may have a particle concentration that is, for example, in the order of 1 to 10 nanomol e / milliliter.

[0046] Adding the quantity of liquid on top of the bottom graphene layer can be done in a variety of ways, some of which are described here. For example, a droplet of a liquid containing a particle, or several particles, may be deposited by pipetting. As another example, a mist of the liquid may be sprayed on the bottom graphene layer. Yet another example is dipping the transmission electron microscopy grid with the holey film and the bottom graphene layer present thereon into the liquid. Alternatively, the bottom graphene layer may be brought superficially into contact with the liquid. In yet another example, one or more particles of interest may first be deposited, fixated, or grown on the bottom graphene layer. Subsequently, the one or more particles may be incorporated into the liquid by, for example, bringing the bottom graphene layer supported by the transmission electron microscopy grid into contact with the liquid.

[0047] In a third step 103, a further graphene layer is placed on the quantity of liquid that has been added on top of the bottom graphene layer. This further graphene layer will be referred to hereafter as the top graphene layer for ease of reading. One side of the top graphene layer contacts the liquid. This side will be referred to hereinafter as the inner side, while the opposite side will be referred to as the outer side. Like the inner side of the bottom graphene layer, the inner side of the top graphene layer may be lined with another two-dimensional material layer, such as, for example, graphene oxide. Here too, lining the inner side of the top graphene layer with another two-dimensional material layer may be advantageous, which will be explained hereinafter. For ease of reading, the top graphene layer hereinafter refers to the top graphene layer as such, as well as to a superposition comprising the top graphene layer lined with another two-dimensional material layer, wherever applicable.

[0048] The top graphene layer can be added in a variety of ways, some of which are described here. For example, a further transmission electron microscopy grid may be used as a support to place the top graphene layer on the quantity of liquid. By doing so, a sandwich structure comprising two transmission electron microscopy grids is temporarily formed. As another example, the transmission electron microscopy grid, which supports the bottom graphene layer with the quantity of liquid present thereon, may be manipulated so that the liquid is brought into contact with the top graphene layer, which may be floating on a fluid. As yet another example, a loop may be used to take a droplet of a liquid on which the top graphene layer is floating. The loop then holds the droplet with the top graphene layer floating thereon. The droplet is transferred and added to the quantity of liquid already present on the bottom graphene layer. This loop assisted transfer technique is described in detail in patent publication WO2021123458A1.

[0049] The loop assisted transfer technique allows combining the second and third step 102, 103. A liquid on which a top graphene layer is floating may contain one or more particles to be analyzed. A loop may then be used to take a droplet of the liquid that contains one or more particles to be analyzed with the top graphene layer floating on the droplet. The droplet is transferred and deposited on the bottom graphene layer by means of the loop. Accordingly, a single operation is sufficient to achieve that a quantity of liquid containing at least one particle is present on the bottom graphene layer and that the top graphene layer is present on the quantity of liquid.

[0050] Once the third step 103 has been completed, whether carried out separately or in combination with the second step 102, a graphene-liquid-graphene sandwich structure is obtained. The quantity of liquid, which contains at least one particle to be analyzed, is sandwiched between the top graphene layer and the bottom graphene layer. The bottom graphene layer is supported by the transmission electron microscopy grid, whereby the holey film may constitute an intermediate.

[0051] In a fourth step 104, the quantity of liquid, which is sandwiched between the top graphene layer and the bottom graphene layer, is reduced. The quantity of liquid may be reduced by, for example, blotting with a liquid-absorbent paper or another liquidabsorbent material. In certain areas, the quantity of liquid is reduced to an extent that the top graphene layer and the bottom graphene layer are attracted to each other through Van der Waals forces. As a result, one or more cells may be formed encapsulating the liquid together with at least one particle. That is, one or more particles may become trapped in such a cell with a remaining quantity of liquid. Specifically, the quantity of liquid may be reduced to an extent that the remaining quantity of liquid is less than 50 nm thick throughout the cell. The cell generally has a thickness that corresponds with that of the one or more particles that are trapped in the cell. In this respect, two cases may be distinguished.

[0052] In case multiple particles are trapped in the cell, while the density of these particles is relatively low, the remaining liquid corresponds may fill spaces between the multiple particles. The top graphene layer between two adjacent particles may be relatively flat, presenting relatively little or even insignificant sag. In a manner of speaking, the top graphene layer may be regarded as a tent roof, which may be relatively tightly stretched, and the multiple particles may be regarded as multiple tent poles upholding the tent roof. In that case, the remaining quantity of liquid may be almost as thick as the cell.

[0053] In case a single particle is trapped in the cell, the top graphene layer may closely surround the particle. The remaining quantity of liquid may be concentrated near the bottom graphene layer in a zone bounded by, on the one hand, the top graphene layer touching the bottom graphene layer and, on the other hand, around the top graphene layer touching the particle, or at least being in close proximity to the particle. In that case, the remaining quantity of liquid may be orders of magnitude thinner than the cell.

[0054] FIG. 2 illustrates a first exemplary sample preparation 200 for structural particle analysis by means of transmission electron microscopy. FIG. 2 provides a schematic cross-sectional diagram of this first exemplary sample preparation 200. The first exemplary sample preparation 200 may be obtained by the method described hereinbefore with reference to FIG. 1. The first exemplary sample preparation 200 comprises a cell 201 formed by a top graphene layer 202 and a bottom graphene layer 203. The cell 201 encapsulates multiple particles 204 and contains a remaining quantity of liquid 205 between these multiple particles 204. The cell 201 is supported by a holey film 206, which, in turn, is supported by a transmission electron microscopy grid 207.

[0055] The first exemplary sample preparation 200 corresponds to the case described hereinbefore in which multiple particles are trapped in a cell, while the density of these particles is relatively low. The top graphene layer 202 is relatively flat like the bottom graphene layer 203. The cell 201 has thus a relatively uniform thickness corresponding to that of the multiple particles 204. That is, the multiple particles 204 are within a relatively flat environment. This situation is generally favorable for making a three-dimensional reconstruction of a particle of interest.

[0056] FIG. 3 is an image 300 taken by a transmission electron microscope that comprises a sample preparation corresponding with the first exemplary sample preparation 200. The image 300 has been taken at room temperature. In this sample preparation, the particles are identical proteins. The image 300 shows a cell comprising multiple proteins, which are arranged in a single layer. That is, no stacks of heaped up proteins appear. This indicates that the cell has a thickness closely approximating the dimensions of the proteins.

[0057] A sample prepared according to the method described hereinbefore with reference to FIG. 1 is advantageous in many respects. A cell formed by the top graphene layer and the bottom graphene layer may spontaneously take on a thickness corresponding to dimensions of a particle of interest of which many instances may be encapsulated by the cell. The cell is thus relatively thin containing a relatively small quantity of liquid, just enough to accommodate the many instances of the particle of interest. This allows taking images by means of transmission electron microscopy in which there is relatively little background noise. In general, maximum resolution can be achieved or, at least, closely approximated, when the cell thickness corresponds to the dimensions of the particle of interest.

[0058] Moreover, since the cell formed by the top graphene layer and the bottom graphene layer may spontaneously take on a thickness corresponding to dimensions of a particle of interest, there is a high degree of reproducibility. Cells within a sample preparation will generally have a uniform thickness that will closely match that of another sample preparation concerning the same particle of interest. This uniform thickness does not significantly depend on ambient conditions or specific procedural details of sample preparation. In particular, variations in the timing and extent of blotting will generally not result in significant variations in cell thickness.

[0059] Another advantage is that cells in the sample preparation are relatively stable. Once a cell has formed, the top graphene layer and the bottom graphene layer make that the cell is air and liquid tight. What is more, the cell is stable in a wide temperature range including room temperature, cryo-temperature, and even above room temperature. Accordingly, a sample can be prepared well in advance of sample imaging and at a location that is different, and even remote from, where the imaging takes place. In contrast with conventional sample preparation based on plunge freezing, there is no need to store, transport, and image the sample under cryogenic conditions, which may be quite cumbersome. Notwithstanding, a sample prepared in accordance with the method described hereinbefore with reference to FIG. 1 may be plunge-frozen, if so desired, and even thawed again.

[0060] Since the sample preparation, in addition to being stable at room temperature, is also stable in vacuum, imaging by means of transmission electron microscopy may be carried out at room temperature. This too simplifies structural analysis of a particle. What is more, particles in the sample preparation may be able to move, perform a function, aggregate, rotate, and react. This allows real-time dynamic imaging of particles, in particular imaging of processes within the sample involving the particles. A three-dimensional reconstruction of a dynamic nature can be made.

[0061] Yet another advantage relates to the desirability of particles in a sample having a wide variety of orientations so that an image of the sample shows particles from a wide variety of angles. In the sample preparation described hereinbefore, the top graphene layer and the bottom graphene layer may prevent particles from interacting with these layers in a manner that the particles have a preferred orientation. As mentioned hereinbefore, the inner side of the top graphene layer as well as the inner side of the bottom graphene layer may be lined with another two-dimensional material. This other two- dimensional material may more strongly prevent particles of the type of interest from interacting with the top graphene layer and the bottom graphene layer and thus more strongly prevent these particles from having a preferred orientation. For example, the inner side of top graphene layer and that of the bottom graphene layer may be lined with graphene oxide, which is more hydrophilic than graphene. Accordingly, it is possible to prepare a sample in accordance with the method described hereinbefore with reference to FIG. 1 so that the particles therein have a wide variety of different orientations. Imaging of the sample will thus provide a set of images showing the particle from a wide variety of angles, which is favorable for structural analysis.

[0062] FIG. 4 illustrates a second exemplary sample preparation 400 for structural particle analysis by means of transmission electron microscopy. FIG. 4 provides a schematic cross-sectional diagram of this second exemplary sample preparation 400. The second exemplary sample preparation 400 is particularly suited for structural analysis of a biological cell, such as, for example, a bacterium, a spore, a mammalian cell, a plant cell, or an ensemble of such cells. The second exemplary sample preparation 400 may be obtained by the method described hereinbefore with reference to FIG. 1. The second exemplary sample preparation 400 comprises a graphene cell 401 formed by a top graphene layer 402 and a bottom graphene layer 403. The top graphene layer 202 closely surrounds a biological cell 404. The graphene cell 401 is supported by a holey film 405, which, in turn, is supported by a transmission electron microscopy grid 406.

[0063] The graphene cell 401 encapsulates a biological cell 404 with a relatively small remaining quantity of liquid 407. The remaining quantity of liquid 407 is concentrated near the bottom graphene layer 403 in a zone bounded by, on the one hand, the top graphene layer 402 touching the bottom graphene layer 403 and, on the other hand, around the top graphene layer 402 touching the biological cell 404, or at least being in close proximity to the biological cell 404. The remaining quantity of liquid 407 may be orders of magnitude thinner than the graphene cell 401. The remaining quantity of liquid 205 may be, for example, less than 50 nm thick.

[0064] The remaining quantity of liquid 407 is relatively small due to the following. The top graphene layer 402 and the bottom graphene layer 403 mutually attract each other. As a result, when making the sample preparation as described hereinbefore with reference to FIG. 1, a relatively large amount of liquid may be expelled from around the biological cell 404. The liquid may be expelled by, for example, blotting as described hereinbefore.

[0065] Since the remaining quantity of liquid 407 is relatively small, the biological cell 404 may be imaged by means of transmission electron microscopy with relatively high resolution and little background noise. Moreover, this imaging can be done at room temperature. The graphene cell 401 protects the biological cell 404 against the vacuum that exists in a transmission electron microscope used for the imaging. The graphene cell 401 further supports the biological cell 404 in this process. In fact, many of the advantages mentioned hereinbefore in connection with the first exemplary sample preparation 200 equally apply to the second exemplary sample preparation 400.

[0066] FIG. 5 is an image 500 taken by a transmission electron microscope that comprises a sample preparation corresponding with the second exemplary sample preparation 400. The image 500 has been taken at room temperature. A biological cell is encapsulated between two graphene layers, one being a top graphene layer, the other one being a bottom graphene layer. The top graphene layer is draped over the biological cell. The top graphene layer is a visible in the image as creases that radiate outwardly from a periphery of the biological cell. The bottom graphene layer is flatly stretched and, therefore, appears invisible.

[0067] To obtain a three-dimensional reconstruction of the biological cell, the sample preparation may be tilted inside a transmission electron microscope to take images from a range of different angles. These images taken from different angles of the biological cell may then be used to construct a three-dimensional view of the cell.

[0068] The first exemplary sample preparation 200 and the second exemplary sample preparation 400 have various features in common. Encapsulating a particle between a top graphene layer and a bottom graphene layer protects the particle against ambient conditions, in particular against vacuum in a transmission electron microscope, without having to resort to cryogenic techniques. Moreover, this encapsulation allows relatively easily expelling an excess quantity of liquid in preparing a sample. This, in turn, allows imaging the particle with a relatively high resolution and low background noise, because any excess quantity of liquid potentially may lower image resolution and cause background noise.

[0069] In both exemplary sample preparations, a relatively small amount of liquid remains, which does generally not significantly degrade image quality. Specifically, in the first exemplary sample preparation 200, which is particularly suited for structural analysis of a nanoscale biological particle, the remaining quantity of liquid 205 may be almost as thick as the cell 201. In turn, 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 in a thin yet flat fixating structure. In the second exemplary sample preparation 400, which is particularly suited for structural analysis of a biological cell 404, the remaining quantity of liquid 407 may be orders of magnitude thinner than the dimensions of the biological cell 404. In both exemplary sample preparations, the remaining quantity of liquid 205, 407 may be less than 50 nm thick throughout the cell 201, 401. More specifically, the remaining quantity of liquid 205, 407 may be less than 40 nm, 30 nm, 20 nm, or even less than 10 nm thick. Thickness may be measured between the top two-dimensional material layer and the bottom two-dimensional material layer as a range where the remaining liquid is present. NOTES

[0070] The embodiments described hereinbefore with reference to the drawings are presented by way of illustration. The invention may be implemented in numerous different ways. In order to illustrate this, some alternatives are briefly indicated.

[0071] The invention may be applied in numerous types of products or methods related to structural particle analysis. In the presented embodiments, sample preparations were discussed in relation with single particle analysis, which is a form of structural particle analysis. In other embodiments, samples may be prepared in accordance with the invention for structural particle analysis based on, for example, three-dimensional tomography, four-dimensional electron diffraction, and micro-electron diffraction.

[0072] There are numerous different ways of reducing a quantity of liquid in a method in accordance with the invention. In the embodiments presented hereinbefore, a quantity of liquid by means of blotting using a liquid-absorbent material. In other embodiments, the quantity of liquid may be reduced using other techniques.

[0073] The term transmission electron microscopy grid should be interpreted broadly. This term encompasses any type of support that may be used in transmission electron microscopy, which may comprise any suitable type of material, including, but not limited to, metal.

[0074] The remarks made hereinbefore demonstrate that the embodiments described with reference to the drawings illustrate the invention, rather than limit the invention. The invention can be implemented in numerous alternative ways that are within the scope of the appended claims. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. Any reference sign in a claim should not be construed as limiting the claim. The verb “comprise” in a claim does not exclude the presence of other elements or other steps than those listed in the claim. The same applies to similar verbs such as “include” and “contain”. The mention of an element in singular in a claim pertaining to a product, does not exclude that the product may comprise a plurality of such elements. Likewise, the mention of a step in singular in a claim pertaining to a method does not exclude that the method may comprise a plurality of such steps. The mere fact that respective dependent claims define respective additional features, does not exclude combinations of additional features other than those reflected in the claims.

Claims

CLAIMS:

1. A method of preparing a sample (200, 400) for structural particle analysis by means of transmission electron microscopy, the method comprising: sandwiching (102, 103) a quantity of liquid, which contains at least one particle to be analyzed (204, 404), between a top two-dimensional material layer (202, 402) and a bottom two-dimensional material layer (202, 402), the bottom two-dimensional material layer being supported by a transmission electron microscopy grid (207, 405); reducing (104) the quantity of liquid to an extent that at least one particle contained therein is encapsulated in a cell (201, 401) formed by the top two-dimensional material layer and the bottom two-dimensional material layer, whereby the cell has a thickness corresponding with that of the at least one particle to be analyzed with a remaining quantity of liquid (205, 407) less than 50 nm thick throughout the cell.

2. A method of preparing a sample for structural particle analysis according to claim 1, wherein multiple particles (204) are encapsulated in the cell (201), the cell having a uniform thickness.

3. A method of preparing a sample for structural particle analysis according to claim 2, wherein the multiple particles (204) that are encapsulated in the cell (201) are nano-scale biological particles, the term nano-scale biological particles including: proteins, viruses, virus-like particles, ribosomes, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and vesicles.

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

5. A method of preparing a sample for structural particle analysis according to claim 4, wherein the single particle (404) that is encapsulated in the cell (401) is a biological cell, the term biological cell including: a bacterium, a spore, a mammalian cell, a plant cell, and an ensemble of such cells.

6. A method of preparing a sample for structural particle analysis according to any of claims 1-5, wherein a holey film (206, 406) is comprised between the bottom two- dimensional material layer (203, 403) and the transmission electron microscopy grid (207,405).7 A method of preparing a sample for structural particle analysis according to any of claims 1-6, wherein the top two-dimensional material layer (202, 402) and the bottom two-dimensional material layer (203, 403) comprise graphene.

8. A method of preparing a sample for structural particle analysis according to any of claims 1-7, wherein, in sandwiching (102, 103) the quantity of liquid, a further two- dimensional material layer is comprised between the quantity of liquid and at least one of the following two-dimensional material layers: the top two-dimensional material layer (202, 402) and the bottom two-dimensional material layer (203, 403).

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

10. A method of preparing a sample for structural particle analysis according to any of claims 1-9, wherein sandwiching (102, 103) the quantity of liquid comprises: placing the top two-dimensional material layer (202, 402) on a liquid containing the at least one particle (204, 404) to be analyzed; and using a loop to deposit a droplet of the liquid, with the top two-dimensional material layer floating thereon, on the bottom two-dimensional material layer (203, 403) supported by the transmission electron microscopy grid (207,405).

11. A method of preparing a sample for structural particle analysis according to any of claims 1-10, wherein reducing (104) the quantity of liquid includes blotting with a liquid-absorbent material.

12. A method of carrying out structural particle analysis by means of transmission electron microscopy, the method comprising preparing a sample (200, 400) for structural particle analysis according to any of claims 1-11.

13. A method of carrying out structural particle analysis according to claim 12, wherein the sample (200, 400) that has been prepared is kept under non-cryogenic conditions.14 A method of carrying out structural particle analysis according to claim 13, wherein the structural particle analysis is carried out under non-cryogenic conditions.

15. A sample preparation for structural particle analysis by means of transmission electron microscopy, the sample preparation comprising at least one particle (204, 404) to be analyzed that is encapsulated in a cell (201, 401) formed by a top two- dimensional material layer (202, 402) and a bottom two-dimensional material layer (203, 403), whereby the cell has a thickness corresponding with that of the at least one particle to be analyzed with a remaining quantity of liquid (205, 407) less than 50 nm thick throughout the cell .

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