Fabrication of thin film liquid cells

Abstract

A thin film liquid cell suitable for transmission electron microscopy at room temperature is prepared as follows: A thin film is provided that floats on a liquid. The droplet over which the thin film floats is transferred to a support by a loop. The loop holds the droplet, which holds the thin film during this transfer. Sufficient liquid is removed from the droplet on the support to form a thin film liquid cell.

Description

[Technical Field]

[0001] The present disclosure relates to methods and systems for producing one or more substantially two-dimensional membrane liquid cells, particularly graphene liquid cells. [Background technology]

[0002] A graphene liquid cell (GLC) is a femtoliter-sized pocket in which water is confined between two films of graphene. This ultrathin graphene-water structure provides a nanoscale environment for water-based processes, enabling real-time transmission electron microscopy (TEM) at room temperature. While the development of liquid cells has enabled atomic-resolution imaging of metal nanoparticle growth, visualizing organic processes remains a major challenge. Organic molecules composed of elements with low atomic numbers are weaker electron scatterers than, for example, metal atoms, resulting in weaker contrast in electron images. Furthermore, the interaction of the electron beam with biomolecules, organic molecules, and all liquids can radiolyze the chemical bonds in the sample. Therefore, the electron beam volume used for imaging organic materials is limited, typically resulting in a poor signal-to-noise ratio.

[0003] Conventionally, silicon-based liquid cells confine water between two SiN nano-membranes, and the passage of electrons to both the SiN and the water causes significant electron scattering.

[0004] On the other hand, graphene is a single atomic membrane that minimizes background electron scattering. Furthermore, graphene is a thermal and electrical conductor that facilitates rapid energy loss, thereby preventing the beam from causing damage. The advantage of graphene in electron imaging of soft materials has been demonstrated: graphene-coated TEM samples offer an order of magnitude greater spatial resolution than graphene-free samples fixed by rapid freezing.

[0005] GLC therefore offers the possibility of dynamic imaging of biological processes, such as protein function and lipid membrane fusion, at high resolution. The first GLC studies of bioorganic systems at room temperature include SKBR3 breast cancer cells, H3N2 influenza viruses, microtubules, and polystyrene chains dissolved in water at the individual molecular level.

[0006] Beyond this initial success, the demonstration of reproducible data acquisition is a necessary next step toward application to specific case studies in the life sciences. Currently, the low reproducibility of GLC fabrication methods has hindered this development. A key challenge for the fabrication procedure is the transfer of graphene without the use of a polymer support film, since the transfer polymer inevitably leaves traces of contamination visible during TEM imaging. A recent review lists the current methods for assembling GLCs on TEM grids, but the effectiveness of each method for GLC fabrication remains unclear (M. Texto and N. De Jonge, Nano Lett. 2018 (18): 3313).

[0007] Several methods for assembling GLCs are known. For example, one method involves sandwiching water between two TEM grids, each holding graphene on a porous support layer. Because the graphene is supported throughout the sample assembly procedure, this method offers the greatest opportunity to preserve the integrity of the graphene, even at the expense of losing its flexibility. Another drawback is the dual support layers sandwiched together, obscuring much of the imaging area with the support material.

[0008] Another method involves first placing a top graphene film floating on water (or an aqueous sample solution / dispersion) from the water surface onto a graphene-coated TEM grid. This assembly can be achieved in two ways. First, the top graphene film can be scooped from the liquid surface onto the grid from below, i.e., the TEM grid can be scooped out of the water. This method is therefore called the scoop method. This scoop method induces mechanical stress in a freely floating graphene film and has so far only been demonstrated with multilayer graphene. Second, a TEM grid can be placed on top of the floating graphene film. This method is therefore called the "touchdown" method. In the touchdown method, the sample liquid can be added as droplets to the grid or sprayed as microdroplets onto either graphene film. The latter method has been shown to provide large areas of pristine, intact graphene.

[0009] It should be noted that multilayer graphene is most frequently used in this method due to its superior stability. Both multilayer graphene or defect-free single crystals of graphene have been shown to increase the success rate, but the scope of this material's availability is limited. Furthermore, single-layer graphene may be preferred over multilayer graphene due to its stiffness and susceptibility to contamination from the preparation procedure in which it is deposited using polymer transfer. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] M. Texto and N. De Jonge, Nano Lett. 2018 (18): 3313 Summary of the Invention

[0011] In view of the foregoing, methods for fabricating two-dimensional membrane liquid cells, particularly GLCs, are presented herein.

[0012] Claim 1 defines a method for fabricating a thin film liquid cell according to the present invention.

[0013] Claim 12 defines a method for imaging a sample containing a thin film liquid cell obtained by such a method for making a thin film liquid cell.

[0014] Claim 15 defines a thin film liquid cell suitable for transmission electron microscopy at room temperature.

[0015] The thin film may be a substantially two-dimensional film such as a single molecular / atom-film, e.g., a graphene film, in particular a graphene liquid cell, and may further include, in particular, the methods described and / or specified elsewhere herein. In one embodiment, the method for fabricating a thin film liquid cell comprises: providing a support; providing a substantially two-dimensional thin film, in particular a graphene film; placing the thin film on a support having a liquid; forming one or more thin film liquid cells containing a volume of liquid; the method further comprising providing the liquid, preferably prior to forming the thin film liquid cell, with a luminescent substance, in particular a luminescent dye such as a fluorescent dye.

[0016] The luminescence may optionally be one or more of fluorescence, phosphorescence and / or multi-photon luminescence, preferably under light with a wavelength in the range of 5,000 to 200 nm, particularly in the range of 1,100 to 300 nm, more particularly in the range of 800 to 350 nm, for example in the range of 550 to 400 nm. The shorter the wavelength, the greater the intensity of the luminescence and / or the shorter the wavelength of the luminescence, the greater the intensity such that the luminescence may be easier to detect.

[0017] Suitable two-dimensional films may be single-molecule / atom thick films and / or single-layer or few-layer films, such as hexagonal boron nitride (h-BN) films, dichalcogenide films, and single-molecule / atom layer films, such as graphene films. The support may be or include any suitable support structure, such as an electron microscope support, e.g., a TEM grid (which may typically be gold), a SiN chip, etc. In the following, a graphene film-based liquid cell containing water is used only as an exemplary material to illustrate the concept, and the present disclosure does not limit the thin film material to graphene, the liquid contained in the liquid cell to water, or the support to a TEM grid.

[0018] The method may further include providing a target thin film, in particular a substantially two-dimensional film such as a single molecular / atomic film, e.g., a graphene film, on a support, and disposing the film on the target thin film with a liquid.

[0019] Thus, the thin film may be supported on a target thin film of the same material as the thin film, which is believed to improve the structural integrity of the thin film and the GLC formed therewith. In this way, the GLC can be formed between the thin film and the target thin film, facilitating the fabrication of a large number of GLCs.

[0020] The method may further include providing a sample including a substrate on which a thin film layer and / or thin film precursor is provided; supporting the sample on the surface of a liquid in a container; removing the substrate in and / or with the liquid (e.g., dissolving the substrate); and forming the thin film supported on the surface of the liquid. The thin film layer and thin film may preferably be identical and substantially unchanged except for the presence of the substrate or the absence of the substrate, such as when graphene on copper is etched. However, any suitable type of physical and / or chemical transformation, such as evaporating a substance from the thin film layer, reacting two or more components, and / or curing the thin film layer, may or may not convert the thin film layer to a thin film. Such an approach has been demonstrated to be a reliable method for forming substantially two-dimensional thin films.

[0021] A floating frame may be provided to stabilize the graphene during at least a portion of the step of removing the substrate in and / or with the liquid.

[0022] The method may further include transferring at least a portion of the thin film to a support using a loop that supports a droplet of liquid, which in turn supports the thin film. Such loop-assisted transfer ("LAT") has proven to be a reliable technique for transferring thin films from one configuration to another.

[0023] In the method, providing the luminescent material in the liquid may include providing the luminescent material in a liquid that supports the sample on its surface. Additionally or additionally, when LAT is used to transfer a thin film, the method may include providing the luminescent material in the liquid in a droplet and / or providing the droplet from the liquid in which the luminescent material has been provided.

[0024] In any of these options, the luminescent material may effectively comprise the volume of liquid contained in the liquid cell when forming the liquid cell.

[0025] There is also provided a method, which may be suitably combined with any other method described herein, comprising: acquiring optical image data of light associated with an optical aspect of at least a portion of a structure including a thin film, in particular at least one of transmittance, reflectance, reflectance, and luminescence; The method includes acquiring optical image data of light associated with an optical aspect of the electron beam of at least a portion of a structure including a thin film, and comparing at least a portion of the acquired optical image data of light with optical image data of electrons.

[0026] Typically, light optical image data can be obtained based on optical microscopy and can include transmitted light, reflected light, and luminescence such as fluorescence. Typically, electron optical image data can be obtained based on transmission electron microscopy and can include transmission and / or absorption data.

[0027] The method, and in particular the step of comparing the respective image data, may include one or more of the following steps: correlating the respective image data; mapping the respective image data to each other and / or to at least a portion of the structures, which may include identifying one or more corresponding structures in each image data; scaling at least a portion of the image data; providing an image that includes or represents both the light optical image data and the electron optical image data; comparing the image data as a function of time; identifying and / or locating one or more thin film liquid cells; and identifying one or more characteristics of the one or more thin film liquid cells.

[0028] The method may further include comparing at least a portion of the acquired light optical image data and electron optical image data and providing comparison data associated with using the comparison data to operate and / or analyze the thin film liquid cell.

[0029] There is also provided, in one aspect, a method suitably combinable with any other method described herein, including methods for making one or more substantially two-dimensional membrane liquid cells, in particular graphene liquid cells, said method comprising: providing a support; providing a substantially two-dimensional thin film, in particular a graphene film; placing the thin film on a support having a liquid thereon; The method further includes bending or folding a first portion of the thin film relative to a second portion of the thin film such that a volume containing a quantity of liquid is formed between the first and second portions of the thin film, enclosing the volume containing the quantity of liquid and forming a sealed thin film liquid cell.

[0030] The present method facilitates the preparation of two-dimensional thin-film liquid cells, particularly graphene liquid cells. By forming a volume for containing liquid by bending or folding, the amount of liquid can be easily and reliably obtained. Furthermore, the effort of maintaining a flat thin film can be eliminated. The present method can provide a relatively large number of liquid cells, and liquid cells fabricated in this manner have proven surprisingly robust and sized for many experiments. The bending or folding can include one or more of folding, pleating, and / or folding at least a portion of the thin film to provide a first portion and a second portion of the thin film.

[0031] The method may further include causing and / or establishing mutual contact between the first and second portions of the thin film around the volume and / or amount of liquid.

[0032] In this manner, a volume is defined that contains a quantity of liquid and that quantity of liquid is retained. The first and second portions of the membrane may be portions of different membranes. However, if the first and second portions of the membrane are portions on a membrane, processing of a single membrane may be sufficient to prepare the sample for analysis.

[0033] In connection with the above, in another aspect, there is provided a thin film liquid cell sample comprising a substrate, particularly a TEM grid, wherein the thin film may be particularly a substantially two-dimensional film, such as a single molecular / atomic film, e.g., a graphene film, and / or the thin film liquid cell may be particularly a graphene liquid cell containing a volume of liquid between a first portion and a second portion of the thin film, and wherein the volume of liquid in the thin film liquid cell may contain a luminescent material, thereby facilitating detection and analysis of the liquid cell.

[0034] The sample may include a support for supporting the thin film liquid cell, and the support may be luminescent and / or include a luminescent material. This may facilitate identifying and / or distinguishing the structure of the sample. It may be desirable for the luminescence of the support and liquid cell to have different characteristics with respect to one or more of brightness, wavelength (color), and luminescence (fluorescence vs. phosphorescence) to facilitate differentiation.

[0035] The sample may include a thin film liquid cell, with or without a luminescent material, formed by bending or folding a first portion of a thin film relative to a second portion of the thin film and sealing it to provide a volume. Such a sample may, in particular, provide one or more relatively large and / or robust liquid cells.

[0036] In connection with the above, in another aspect there is provided herewith a system for analyzing thin film liquid cells, particularly thin film liquid cells of samples as specified herein and / or thin film liquid cells made by any of the methods specified herein, the system comprising: a support provided with one or more thin films for providing samples for one or more thin film liquid cells, particularly the thin film liquid cells disclosed elsewhere herein; an optical imaging device for the light, such as a camera and / or a microscope; an electron optical imaging device, such as a transmission electron microscope; and one or more sources of luminescence in at least a portion of the support on which the one or more thin films are disposed.

[0037] The system facilitates the detection and analysis of thin film liquid cells.

[0038] To further facilitate detection and analysis, the system may include a controller for comparing light optical image data acquired using a light optical imaging device and associated with optical aspects of the light of at least a portion of the structure including the thin film with electron optical image data acquired using an electron optical imaging device and associated with optical aspects of the electron beam of at least a portion of the structure including the thin film.

[0039] In thin film liquid cell samples and / or systems, at least a portion of the thin film and / or support may include and / or be provided with markers that provide a reference to facilitate comparison of light optical image data with electron optical image data.

[0040] Furthermore, we present an efficient and reproducible method for fabricating GLCs by graphene loop-assisted transfer (LAT). Below, we compare the LAT method with two known GLC fabrication methods: touchdown and grid sandwich. These three methods (LAT, touchdown, and grid sandwich) require neither lithographic substrate preparation nor liquid handling equipment. However, at least the LAT method offers this potential. Furthermore, this method allows the sample liquid to be added as macroscopic droplets, eliminating the need for large-volume or minute sprays. However, at least the LAT method offers this potential. To broaden the applicability of GLCs to biomaterials and microbiology research, where microfabrication and nanofabrication are uncommon, the availability of GLC fabrication techniques is highly desirable. Furthermore, a light-electron correlative microscopy method is provided to obtain representative numbers of GLCs on each grid.

[0041] Herein, we provide a systematic comparison of preparation methods and a detailed description of the formation mechanism of GLCs to demonstrate improvements in GLC preparation that can, for example, facilitate their implementation in high-resolution TEM imaging of room-temperature samples.

[0042] Graphene liquid cells (GLCs) for transmission electron microscopy (TEM) enable high-resolution, real-time imaging of dynamic processes in water. However, large-scale implementation is hindered by significant difficulties in reproducing GLC fabrication. Here, we present a high-yield method for fabricating GLCs as an example. GLCs can be formed under graphene regions, potentially continuous, potentially millimeter-sized regions of graphene, facilitating efficient GLC formation on TEM grids as an exemplary support. Other supports may be used as well. Furthermore, we provide a method for localizing GLCs on grids or supports using correlative light-electron microscopy (CLEM), which limits electron exposure time and may reduce beam damage. CLEM has enabled us to obtain reliable statistics and surveys of the most common shapes of GLCs. In particular, we discovered a novel type of liquid cell formed solely from a single graphene film, greatly simplifying the fabrication process. The method presented in this study, particularly the reproducibility and simplicity of fabrication, will enable future applications of GLCs for high-resolution dynamic imaging of biomolecular systems.

[0043] The presently presented aspects will be further explained in further detail and with further advantages hereinafter with reference to the drawings which show, by way of example, several embodiments. [Brief explanation of the drawings]

[0044] [Figure 1]Figure 1: Comparison of loop-assisted preparation of graphene liquid cells. a) Preparation of bottom membrane graphene supported by a porous polystyrene membrane on a TEM grid. The top left schematic shows membrane deposition. The top middle schematic shows copper etching followed by lifting onto the grid. The top right schematic shows drying. (b-d) Methods for preparing graphene-water-graphene stacks for liquid cell formation. b) Loop-assisted transfer (LAT) of graphene onto a graphene-coated TEM grid, where water is sandwiched between two graphene membranes to form a liquid cell. c) Touchdown liquid cell preparation, where a graphene-coated TEM grid is placed on an unsupported graphene membrane floating on water. d) Sandwich liquid cell preparation, where two graphene-coated TEM grids are placed on each other with a water droplet sandwiched between them. (f-h) Graphene transfer quality using methods described in (b-d). f) Optical microscope image showing graphene coverage against the paper background after LAT transfer as shown in (b). The drawn lines mark the edges of the graphene film. Scale bar: 1 mm. g) Optical microscope image showing graphene coverage after transfer by the touchdown method (c). The red lines mark the edges of the graphene film. Scale bar: 1 mm. h) Overlap of porous carbon support films occurring in a sample prepared by the sandwich method (d). The dark areas represent the support films obscuring most of the image, whereas the bright holes represent areas where only two graphene films are in the beam path and where graphene liquid cells could potentially form. Scale bar: 10 micrometers. [Figure 2]Figure 2: Correlative fluorescence electron microscopy. a) Overlay of a low-magnification TEM image with a correlated fluorescence microscope image in MAPS software. Shown is a TEM grid characterizing a graphene liquid cell fabricated by loop-assisted transfer. The white arrows indicate fluorescent spots that are confirmed to represent liquid cells at higher magnifications by transmission electron microscopy (b-d). Scale bar: 10 micrometers. b) Overlay of a fluorescence image and a high-magnification electron image of the white rectangle in (a), showing a single graphene liquid cell. Scale bar: 1 micrometer. c) Electron image of the area indicated by the white rectangle in b), showing the darker contrast of the liquid cell. Scale bar: 500 nm. d) The liquid cell in (c) after exposure to a focused electron beam. The contrast confirming the liquid content of the feature has disappeared. Scale bar: 500 nm. [Figure 3] Figure 3: Three types of graphene liquid cells. a) The total number of occurrences of liquid cells sorted by type as a function of their size. The equivalent cell size, defined as the square of the cell area, is plotted on the horizontal axis of the graph. The number of cells is plotted on the vertical axis of the graph. Cell types: "pocket," shown in the upper right image and in gray on the graph; "pleated," shown in the middle right image and in white on the graph; and "folded," shown in the lower right image and in dark gray on the graph. The white arrow in the bottom right image indicates the edge of the folded graphene film. b) TEM image of a graphene bilayer encapsulating several "pocket" and "pleated" type liquid cells. Scale bar: 500 nm. c) "Folded" type graphene liquid cells encapsulated in a single graphene film. Scale bar: 500 nm. [Figure 4]Figure 4: Gold nanoparticle formation in three cell types imaged by TEM. a) A folded cell containing an aqueous HAuCl4 solution at the beginning of beam exposure. Scale bar: 500 nm. b) A magnified view of the red rectangle in (a). Several nanoparticles have already formed during the initial beam exposure. Scale bar: 200 nm. c) The same area after 10 seconds of exposure to a beam of up to 100 e-Å-2 s-1, during which Au nanoparticles have formed in the liquid phase. Scale bar: 200 nm. d) A pleated and pocket cell containing an AuCl3 solution. Scale bar: 500 nm. e) A magnified view of the red rectangle in (d). Scale bar: 200 nm. e) The same area as in (e) after 4 seconds of exposure to the beam. Scale bar: 200 nm. [Figure 5] Figure 5: Bubble formation in bent and pocket-type cells imaged by TEM. (a) Bubble formation and expansion in a pocket-type cell. Within the first and second irradiation, a bubble appears and grows with longer exposure to the beam. Scale bar: 500 nm. (b) Bubble formation and movement in a bent-type cell, followed by cell collapse. Scale bar: 100 nm. (c) Graph of the percentage of pocket-type cells where the onset of bubble formation was observed, plotted on the vertical axis in correlation with the number of corners on the cell periphery, plotted on the horizontal axis. The number of corners is the degree of curvature of the graphene-top membrane. The cell thickness and / or volume can be derived from the shape and / or number of corner peripheries and membrane flexibility or from thickness measurements themselves, or alternatively. DETAILED DESCRIPTION OF THE INVENTION

[0045] [Detailed explanation, results and discussion] Low-Stress Graphene Transfer—Figure 1 summarizes the procedure for fabricating graphene liquid cells. As an example of thin-film deposition on a substrate, a single layer of graphene was grown on a copper substrate by chemical vapor deposition. For the LAT and touchdown methods, the GLC is assembled on a TEM grid covered with a porous polystyrene membrane that provides support for the bottom graphene membrane (Figure 1a, steps I-IV). However, while supports other than TEM grids and / or additional support membranes other than the porous polystyrene membrane may be used, porous and / or grid-like supports are preferred, e.g., to reduce or prevent interference with imaging. An exemplary description of the fabrication of polystyrene membranes is provided in the Methods section elsewhere in this disclosure. The pore-to-support ratio can be varied by changing fabrication parameters; in some cases, a 50:50 pore-to-support ratio may represent the largest area of open space while still providing a stable support membrane. It has been demonstrated that preparing grids on polystyrene supports is more reliable than direct transfer to a support film attached to the TEM grid. The success rate of coating TEM grids with polystyrene-supported graphene can exceed 98%. This high yield is due to the addition of the support film as a flexible film to the graphene-copper laminate, which results in very reliable attachment of the graphene to the film. Other types of support films may also be provided.

[0046] In Figure 1b, the left panel shows a preferred method for transferring a second graphene film: loop-assisted transfer, or LAT. The LAT method may be used for thin films other than graphene. In this method, a pristine graphene film is provided floating freely on a liquid surface, e.g., a water-air interface, before transfer. According to this concept, graphene is transferred onto the grid together with the floating graphene film using a loop holding a droplet of liquid, here water (Figure 1b). The loop may be of any suitable size and made of any suitable material. The loop's average diameter may be less than 5 mm. For use with standard-sized TEM grids with a diameter of approximately 3 mm, a loop with a diameter of 2 mm may be appropriate. The loop may be made of metal. In principle, the loop may be formed by any object with a hole adapted to hold a droplet of liquid, especially for LAT.

[0047] The droplet is then blotted with filter paper or another method to remove excess liquid. Optionally, the grid is dried, bringing the top and bottom graphene films into contact and forming a liquid cell between them. This technique is believed to reduce stress on the graphene film and result in a larger area of intact graphene after transfer (Figure 1f). A small amount of liquid may remain on the sample. In one example using water as the liquid, the drying of the last remaining water was recorded using an optical microscope. The results showed that a thin water film may hang over the pores in the support film, separating the two graphene films until the final moment when the liquid cell formed. Note that the optional polystyrene support film may otherwise have little effect on one or more of the liquid cell formation, size, or distribution observed in the experiment, and is considered to have little effect on all of these features here.

[0048] The bottom graphene film can preferably be a flat graphene film without noticeable wrinkles, and the top graphene film can be flexible, at least at or near the moment of contact with the bottom film, and can bend or wrinkle to form liquid cells (Figure 3b).

[0049] Thus, a method of making a thin film liquid cell can include contacting a thin film in combination with a liquid with a target substrate, providing a curve, wrinkle, and / or fold in the thin film, and trapping a quantity of liquid in the curve, wrinkle, or fold. The target substrate can be provided with a target thin film, and at least a portion of the liquid can be trapped between the thin film and the target thin film. Additionally or alternatively, at least a portion of the liquid can be trapped between opposing portions of the thin film associated with the curve, wrinkle, and / or fold.

[0050] Table 1 below shows the number of GLCs produced by various methods in a representative example comparing the efficiency of GLC formation and a comparative example using two commonly reported graphene liquid cell fabrication methods.

[0051] [Table 1]

[0052] Table 1: GLC formation efficiency for the three fabrication procedures. The total number of liquid cells (column 1) was confirmed by high-magnification TEM imaging. The number of GLCs per grid (column 3) is an average value, extrapolated to account for the grid area investigated for each prepared grid.

[0053] The GLC numbers listed in the table were obtained as described in the Methods section, and Figure 1c shows the results of the touchdown method. We found that this method resulted in patchy, torn graphene and poor coverage of the TEM grid. The second graphene film is subjected to mechanical stress during deposition. Furthermore, the surface tension interaction between the two aqueous phases (the droplet on the grid and the water beneath the second graphene film) creates localized water turbulence. For these reasons, this method is highly susceptible to tearing and shattering of the second graphene film, even when using multilayer or single-crystalline graphene.

[0054] Figures 1f and 1g are microscopic images of graphene deposited on white paper substrates by the LAT and touchdown methods, respectively. A comparison reveals the difference in surface coverage between the two methods. Because graphene films absorb very little light and cannot be visualized against the polystyrene film on the TEM grid, white paper was chosen as the background substrate to visualize the graphene coverage under an optical microscope. The coverage and integrity of the graphene after LAT and touchdown transfer were also investigated on silicon oxide wafers.

[0055] Figure 1d shows a further method used for comparative purposes. In this method, two graphene-coated TEM grids are sandwiched with a droplet of water between them, thus eliminating the entire graphene film transfer process. While this "grid-sandwich" method ensures mechanical support for both graphene films, the results are unsuccessful. It turns out that the formation of GLCs is hindered by the lack of flexibility caused by the porous support film, which rigidly suspends both graphene films. Three liquid cells were observed across the six grids successfully sandwiched in the experiment. In addition to the low number of GLCs, the two porous support layers were found to overlap with random orientations, resulting in most of the grid area being obscured by at least one of the support layers (Figure 1h). Any GLCs that do form are subject to reduced contrast due to the support layers in the beam path.

[0056] Therefore, loop-assisted transfer of graphene films was found to be more likely to keep the graphene intact.

[0057] [Fluorescent labeling for screening low-dose samples] GLCs tend to be very small compared to the size of TEM grids. Therefore, GLCs and their contents are typically exposed to the TEM electron beam while screening the grid to locate the GLCs. This is undesirable because GLCs and their aqueous contents are generally sensitive to the electron beam. Furthermore, because liquid cells can be sparsely distributed on the grid, locating the liquid cells at high magnification is a time-consuming task faced in all GLC experiments, regardless of the GLC preparation method. The same is true for other small objects and samples in TEM microscopy, especially liquid cells.

[0058] Thus, a method for locating a liquid cell using optical microscopy is provided herein. The method involves adding a low concentration of a fluorescent dye, such as a high quantum yield fluorescent dye (e.g., Atto 488, which can be used at a concentration of about 5-20 micromolar, e.g., 10 μM), to the liquid, which is added to water in this experiment. To separate the location and / or identification of the liquid cell, on the one hand, from one or more substances and / or processes within or otherwise associated with the liquid cell, on the other hand, it may be preferable for the dye or other luminescent material to be unreactive with and / or otherwise affect one or more substances and / or processes within or otherwise associated with the liquid cell, which may include being unreactive with and / or otherwise affecting portions of the thin film and / or support associated with the liquid cell. Figure 2a shows an optical microscope image of a TEM grid featuring fluorescently tagged GLC. In Figure 2a, some background fluorescence representing the contours of the polystyrene support film is visible due to polystyrene's weak fluorescence in the green spectral region. Other support films and / or structures may exhibit various types of fluorescence and / or other wavelengths. In some cases, luminescence effects may occur at various wavelengths, depending on illumination intensity and / or time scales (phosphorescence being a prominent example).

[0059] Figure 2b shows an overlay of a fluorescence image with a low-magnification electron image of the same grid. White arrows indicate liquid cells, an example of which is shown in Figure 2c. It can be demonstrated that confined liquid water loses its signature and appears as a small dot on the fluorescence image when exposed to high electron doses. Note that when using dyes, the dye solution can dry on the TEM grid outside the thin-film containment that provides the thin-film liquid cell. This can result in bright spots on the fluorescence image that may be large compared to the thin-film liquid cell. TEM images reveal dried deposits of the dye in these locations. Therefore, this type of dried dye deposit is clearly distinguishable from dyes that are encapsulated and dissolved in GLC. While the method effectively involves relying on capturing light optical image data with a camera and capturing electron optical image data with a TEM, capturing light optical image data relating to the light optical aspects of at least a portion of a structure including a thin film and capturing electron optical image data relating to the electron beam optical aspects of at least a portion of a structure including a thin film, and comparing the captured light optical image data and electron optical image data may suitably be performed using, for example, a transmission optical microscope, a phase contrast microscope, and the like, and / or a reflected optical microscope. Comparing the types of image data may include overlaying and / or scaling and / or otherwise correlating the various types of image data. As an example consistent with the above, fluorescence electron correlation microscopy can be used to rapidly screen an overall standard size circular TEM grid, approximately 3 mm in diameter, at the level of individual grid squares, where the individual grid squares are (100 x 100 μm 2 ), which allows direct identification of GLCs on the grid.

[0060] [Characteristics of the graphene liquid cell] GLCs produced by the loop-assisted fabrication method exhibit dense GLC domains (Figure 3b). In a series of examples of the type described above, graphene-based thin-film liquid cells were prepared on TEM grids. On each grid, 100 × 100 μm 2 Eight window areas were imaged to obtain a count of GLCs on the grid. For the 21 grids prepared and investigated in this way, the average density of GLCs, extrapolated to the total grid area, was 300 liquid GLCs per grid, equivalent to one GLC per two grid squares. The size distribution of the identified GLCs is presented in Figure 3a, and it can be seen that the occurrence rate decreases with cell size. Cell sizes range up to 700 nm in lateral dimension, with the total number being relatively large at smaller lateral dimensions.

[0061] To form a thin-film liquid cell, for the amount of liquid, here water, that is confined in a thin film, here graphene, the thin film or membrane should provide a continuous seal around the periphery of the liquid cell. The likelihood of a leaky seal increases as the cell periphery increases, which may explain the general trend toward a lower incidence of GLCs with increasing periphery. Thus, this concept facilitates the formation of liquid cells of various sizes.

[0062] In the examples shown here, with a bottom membrane that is generally stretched flat, the volume of the liquid cell is primarily determined by the shape of the top thin film. The thin film should be most flexible during the final stages of deposition, where liquid cell formation occurs. This is particularly the case for graphene, and for other atomic and molecular monolayers.

[0063] In this example, it was demonstrated that the size of the small liquid cell can be reduced to a stage where the liquid cell can no longer be accurately distinguished from the irregularities of the top graphene film (some examples can be seen in Figure 3b). For simplicity and considering the need for a specific volume so that the liquid cell is suitable for further use and / or investigation, a lateral size of 200 nm was chosen. 2GLCs less than this will be ignored in the following discussion.

[0064] In total, 200 nm 2 More than 90 cells were observed, yielding an average of one GLC per two grid squares. Various types of thin-film liquid cells were fabricated. In particular, three types of graphene liquid cells are presented in panels 3a, 3b, and 3c. The three types of graphene liquid cells include: I. "pocket"-type cells, in which the liquid volume is enclosed in approximately equilateral sides by an upper film portion on a bottom substrate or a bottom thin film; II. "pleated"-type cells, in which the liquid volume is trapped in possibly long folds in at least one of the thin-film portions that confine the liquid volume; and III. "folded"-type cells, in which a single film is folded back on itself, here on the graphene thin film. The pleated and / or folded-type cells can be easily fabricated by tearing a portion of the thin film.

[0065] In some embodiments, pocket-type cells may constitute the majority (e.g., about 70-80%) of a GLC. However, the relative number of cells may vary depending on the manufacturing method, particularly the quality and / or flexibility of the thin films. The flexibility of the upper thin film (or upper film portion) can play an important role in accommodating the liquid volume or amount in forming pocket-type cells, and the flexible upper film can be curved or formed to curve around that volume. This typically involves folding at or near the edge of the liquid cell to form a discrete number of corners. Note that such forming and / or folding can occur during deposition or other removal of liquid between the thin films, which may involve blotting. The top center panel of Figure 3a shows a pocket-type cell with five corners. In TEM images, the relative thickness of the liquid cells can be roughly predicted by comparing the contrast of the liquid cells on a single TEM image. It should be noted that such comparisons are only relative and valid under equivalent beam settings, and that various measurement and / or image processing methods and / or related equipment may alternatively or additionally be used as appropriate. In this example, various liquid cells identified in the same image were compared. A greater number of corners around a pocket-type cell generally provides a thicker pocket or provides greater separation between the top and bottom membrane portions, which may generally correlate (or be fabricated to correlate) with a greater confined volume and / or amount of liquid. Liquid pockets confined by van der Waals surfaces, such as graphene or other thin films, generally demonstrate a correlation between pocket size and height. In this example, cells with three or four corners generally appear thicker than cells with five or more corners of the same lateral dimension. The volume of a pocket-type cell, therefore, does not strictly correlate to its projected size on a TEM image. Alternatively, the number of corners has been found to determine the curvature of the top membrane and, therefore, the volume of a pocket-type GLC.Thus, a method for determining the volume of a liquid cell may include identifying the liquid cell, determining the number of perimeters and corners of the liquid cell, and determining the volume of the liquid cell and / or the amount of liquid in the cell based on the perimeter and number of corners.

[0066] The pleated liquid cells can be formed by forming long bends or long folds in two opposing thin films, possibly in at least one of the upper graphene films. In some experiments, the pleated liquid cells are found to be a minority of the liquid cells, for example, about 10-15% of the liquid cells, particularly in the 200 nm range. 2 The pleated cells can be fabricated in a range of sizes exceeding 100 μm. The structural differences between pleated and pocket-type cells are highlighted in Figures 3b and 3c. As can be seen from the distribution in Figure 3a, pleated cells are larger on average than pocket-type cells, and the nature, size, and distribution of any pleats can result in relatively long, straight walls in the pleated membrane. Therefore, a pleated membrane can more easily form a longer, liquid-tight seal than a pocket-type cell, all else being equal. Depending on how the membrane is transferred and / or the structure of the support on which the membrane is deposited, the membrane, e.g., the top graphene membrane, can have a large or very small number of pleats. In the latter case, the pleated cells may account for only a small percentage of the liquid cell.

[0067] Folded-type cells are provided here as a third type of thin-film liquid cell, again designated GLC-type. In some embodiments, folded-type cells may constitute a minority of liquid cells, particularly in one or more size ranges, e.g., 25-40% of the cells in a size range of interest. Folded-type cells can be formed by folding a portion of a thin film into a first thin-film portion and a second thin-film portion, creating a volume between the first and second thin-film portions to accommodate a certain amount of liquid. For example, folded-type cells can be formed where the bottom graphene film is torn and folded back on itself (Figure 3c). The formation of folded-type cells can eliminate the need for a second thin film. For example, in some experiments, folded-type cells were fabricated during the deposition of the first graphene film on a TEM grid. In other experiments, folded-type cells were fabricated on a grid supporting only a single graphene film (i.e., after step III of Figure 1a).

[0068] Folded-type cells can easily contain large volumes of liquid compared to other cell types, and folded-type GLCs may be most prevalent in the size range of 250–400 nm (Figure 3a). Furthermore, folded-type cells can be formed from a single thin film, here a graphene film, in case of (partial) rupture, folded or wound up or down against a support structure, such as a polymer support layer. One side of a folded-type cell can consist of a single, continuous piece of thin film, so that the first and second thin film portions can be sealed or closed along a portion of the cell's periphery, or on the remaining side of the cell opposite the folded side. Thus, folded-type cells may have a cylindrical cross-sectional shape and can be thicker on average than pocket-type or pleated-type cells, potentially resulting in a larger total volume.

[0069] The liquid cells provided by the presently presented methods and discussions can be suitably used for a variety of targets, particularly for the study of nanoparticles and / or submicron-sized biological samples. As an example of the presently presented techniques, we investigated the effectiveness of each liquid cell type for imaging dynamic processes. In particular, nanoparticle formation (here, gold nanoparticles under electron irradiation) was investigated for each liquid cell type. For some experiments, one or more precursors, here, an aqueous solution of HAuCl4, were encapsulated in GLC and exposed to an electron beam, reducing gold(IV) ions to metallic gold nanoparticles. In the experiments, GLCs were fabricated using the LAT, which primarily fabricates pocket-cell and pleated-cell types of GLCs, all as described above, to transfer a second thin film onto a TEM grid provided with a polymer support layer and a graphene target thin film.

[0070] To fabricate pocket-type and pleated-type GLCs, a droplet of 10 mM HAuCl4 aqueous solution was placed on the thin graphene film supporting the TEM grid by the LAT method before the transfer of the graphene top film. The excess water was then blotted away and the remaining water was allowed to evaporate. The formation of Au nanoparticles from HAuCl4 aqueous solution was chosen as the test reaction because it is a well-documented procedure that has been extensively characterized in the literature. After a 10-second beam exposure, nanoparticles of various sizes were formed in all liquid cells, and the high-contrast regions were the actual pockets containing the liquid (Figures 4a–c). In a similar experiment, as shown above, a pleated-type GLC was fabricated without the transfer of the graphene top film. Instead, after step II in Figure 1a, the above-mentioned HAuCl4 aqueous solution was flowed under the polymer layer that supported the graphene film. The polymer-graphene stack was then lifted from the liquid surface onto a TEM grid (see step III in Figure 1a), forming folded-type cells that encapsulated the HAuCl4 solution. The latter is evident from the formation of gold nanoparticles (Figures 4d-f). By forming and mounting graphene liquid cells from a single thin film, specifically a single graphene film, the fabrication and use of thin-film liquid cells is significantly easier than previously known techniques, in which graphene liquid cells always required the assembly of two graphene films. The size and number of folded-type cells can be adjusted to some extent by varying the speed and / or lateral movement of the TEM grid during lifting of the polymer-graphene stack. Similarly, one or more other supports may be used instead of and / or in addition to the polymer layer, but preferably a porous and / or alternative base support than a similarly porous and / or grid-like TEM grid.

[0071] [GLC under electron beam] The thin-film liquid cells provided herein can be used for various studies relating to both the cell itself and the liquid contained therein. When irradiated with an electron beam, such as in an electron microscope like a TEM, gas bubbles can form in the liquid within the thin-film liquid cell. In Figure 3b, which shows a GLC, some GLCs have a low-contrast region in their center, where liquid water has been displaced due to a gas bubble. In a thin-film liquid cell containing water, the appearance of gas bubbles can be due to radiolytic splitting of the water. For example, gas bubbles form when the hydrogen concentration in the liquid reaches a critical value, which can be essentially instantaneous. The formation of gas bubbles within a liquid cell (or within the liquid) increases the volume of material contained within and in the cell, increasing pressure on the cell walls, so the occurrence of gas bubbles in a GLC is related to the stability of the seal formed by the thin film, in this example, graphene. This allows the stability of various cell types to be examined in terms of the formation of gas bubbles observed under exposure to a relatively intense beam.

[0072] Figures 5a to 5c show the 144e accelerated to 120 keV. - Å -2 s -1 Selected frames from a series of images of a folded-type cell under exposure to an electron beam of 10 e (typically, high-resolution imaging of single particles requires exposure times of 1–2 seconds). - Å -2 s -1It should be noted that, depending on the electron beam parameters, a higher or lower beam exposure (dose and / or acceleration energy) may be used, depending, for example, on the properties of the liquid and / or thin film material in the cell. In a series of frames, a faint contrast area representing a bubble appears already on the first frame. During live observation of many liquid cells, the liquid cell generally does not contain a bubble at the moment of the first beam exposure. However, under strong beam conditions, it can be observed that bubble formation progresses rapidly, sometimes too rapidly to capture a bubble-free GLC. From the first frame, one or more bubbles can be seen moving around the available space of the liquid cell in which they form until the cell contents are substantially completely lost. This can be understood as the liquid being expelled into the vacuum within the electron beam microscope, possibly due to damage inflicted on the graphene film by the electron beam. Such behavior is also likely to occur in thin-film liquid cells, such as folded-type cells, with other thin films.

[0073] Similarly, Figures 5d-5f show selected frames of a GLC from a pocket-type cell. Again, although more bubbles may occasionally form, bubbles are formed immediately upon the first electron beam exposure. However, unlike the folded-type cell, the dark contrast remains present in the pocket-type cell even after prolonged exposure to a relatively powerful electron beam. It is clear that two graphene membranes are sealed around the pocket-type cell, creating a highly leak-resistant containment, allowing bubbles to aggregate and equilibrate with the remaining liquid water. Figure 3b shows that various pocket-type cells experience bubble formation, while other cells maintain a uniform dark contrast, indicating no bubble formation, even with prolonged beam exposure. This was a general trend observed across all pocket-type cells. Figure 5g plots the percentage of pocket-type cells that collapsed under beam exposure, revealing a strong correlation with the number of corners and, therefore, cell curvature. A possible reason is that in cells with fewer corners, the graphene film is nearly flat, leaving no room for the increased volume required for bubble formation, making the GLC extremely stable.

[0074] Overall, the stability and leak resistance of the pocket-type cell make it a favorable candidate for further applications. It is clear that the bent-type cell is less easily sealed than the pocket-type cell. Nevertheless, its larger volume and undoubtedly dynamic liquid volume lends itself very well to containing more polymer samples. Furthermore, the ease of fabricating a single graphene film required to form the bent-type cell compared to the need for two thin films is a clear advantage.

[0075] [method] Below, various method steps are described in further detail as examples and / or provide further information to the above example descriptions. In any example, even if not explicitly stated, numerous options and / or modifications may be suitably selected and used to advantage within the scope of the concepts presented herein and / or claimed subject matter.

[0076] [Graphene preparation and processing for GLC fabrication] Other methods can be used and / or provided for the graphene liquid cell example shown here; graphene is grown on copper flakes by chemical vapor deposition in a tube furnace, although other substrates can be used. Here, one side of the copper foil was protected by taping a glass slide around the copper foil etchant, so that only one side of the foil featured graphene. As an option, the graphene on the other side of the copper was then removed. A suitable technique was demonstrated: exposure to oxygen plasma (e.g., 160 watts for 2 minutes), which provided a single graphene film on the copper foil.

[0077] The optional support membrane may be porous and made from any suitable material. For the examples shown and described above, an example porous polymer support membrane was prepared. These membranes were prepared from a 0.3-1%, e.g., approximately 0.5%, solution of polystyrene (average Mw ∼192,000) in ethyl acetate; various concentrations can be used. Glycerol was added at 10 volume percent (≥99.0%) to form a two-phase mixture. The mixture was thoroughly shaken for 1 minute to disperse the glycerol in the polystyrene solution. Among other things, the duration and / or force of shaking can determine one or more dispersion characteristics, thereby affecting the pore size and / or density of the polymer membrane. The dispersion was applied to a substrate, e.g., a glass slide, by dipping it into the dispersion and drawing it out to form a porous polystyrene membrane on the glass surface. Alternatively, the membrane was wiped off one side of the glass slide. At least a portion of the film, for example, the film remaining on the other side of the glass substrate, was peeled off from the glass slide by gently immersing the glass slide in ultrapure water, and the polystyrene film was floated on the water. Thus, the prepared polystyrene porous film can be made thin, for example, to a thickness well below 1 micrometer, for example, 20 to 50 nm, particularly 30±5 nm, or less than 100 nm, and this thickness can be confirmed by atomic force microscopy on a film deposited on a silicon wafer if desired.

[0078] For the samples, for example, graphene on copper prepared as described above was cut to form 3 mm circular pieces, although other shapes and / or sizes can be used. For samples including a (porous) support layer, the thus-formed pieces were placed on the aforementioned polymer film on water, preferably with the graphene side in contact with the polymer film. The pieces were then lifted from the water surface, leaving the polystyrene film attached to the graphene-copper flakes. After optional air drying, the pieces were placed copper-side down on an etching solution, e.g., 0.1 M aqueous ammonium persulfate (APS, ≥98%), to etch the copper. Once the copper was removed, the APS solution was replaced with ultrapure water by gently pumping to prevent surface vibration, which could damage the graphene film. The graphene-polystyrene laminate was then scooped onto a gold TEM grid (optionally made hydrophilic, e.g., by oxygen plasma exposure for 2 minutes), yielding a porous polystyrene-supported graphene TEM substrate.

[0079] In particular, to form pocket and / or pleated cell-type liquid cells, a second graphene film was transferred to a graphene-polystyrene-coated TEM substrate. To obtain free-floating graphene for transfer, a 3 mm diameter piece of graphene-copper was placed on the surface of, for example, a 0.1 M APS etching solution, as described above, without the additional support layer described above. A floating plastic frame, optionally with round or other shaped holes, was used to stabilize the graphene during copper etching. The copper was then etched, for example, overnight at 4 °C. The etching solution was then replaced with ultrapure water by gently pumping it out, and the graphene was then transferred onto a TEM grid substrate using one of the transfer methods described above.

[0080] [Number of graphene liquid cells] To obtain a representative number of GLCs on the grids fabricated by the three transfer methods (grid sandwich method, touchdown method, and LAT), the exemplary grids were examined using transmission electron microscopy. 2 For grids with a density of >1 GLC per grid square, at least eight grid squares were surveyed to obtain an estimate of the GLC density for each grid. 2 For grids with <1 GLC per grid square, larger areas of the grid were investigated to obtain reliable estimates of GLC density.

[0081] For the grid-sandwich method, graphene on a support film was obtained from a commercial vendor instead of being fabricated as described above. A flat surface appears necessary for successful GLC formation using this grid-sandwich method. Of 20 attempts, six grid-sandwiches were successfully assembled (30%). Failed attempts were typically due to misalignment of the two grids at the moment they contacted each other. Misaligned stacks cannot be used because they do not fit into the electron microscope sample holder. Of the six successful stacks, liquid cells were observed in only one (~15%). Although the total number of liquid cells on these six grids was three, these cells were located partially or completely on the carbon support film rather than on free-standing graphene.

[0082] For the touchdown method, graphene deposition of the top film was successful in 8 out of 12 attempts (66%). Failure typically occurs when the graphene is washed away from the grid upon touchdown, causing the graphene film to crumble or shatter, necessitating repeated deposition attempts. Of the 8 grids examined, GLCs were observed in 2 grids (25%), with a total of 18 liquid cells observed on these 2 grids.

[0083] For the LAT method, 21 of 24 graphene deposition attempts were successful (88%). Failures were typically due to collapse of the water droplets in the loop. GLCs were observed on 19 of 21 grids (90%). The total number of GLCs on these 21 grids was 184.

[0084] [Fluorescent light microscopy] Fluorescent labeling of GLCs is achieved by depositing a small (e.g., approximately 5–15 μL, e.g., approximately 10 μL) droplet of a dye-containing liquid onto a TEM grid coated with graphene and a porous support polymer. For example, a 10 mM solution of "Atto488" fluorescent dye (≥98%) in water. Multiple dyes may be used. A top graphene film is then deposited. In the case of deposition using loop-assisted transfer, the dye solution can be mixed with a droplet of ultrapure water, e.g., up to 2 μL, that holds the graphene within the loop. Fluorescence microscopy was performed using an optical microscope to obtain optical image data. The prepared sample was then imaged with an electron microscope to obtain electron images (see below). The acquired images, including the camera image, and the data were correlated using image processing software.

[0085] [Electron microscope] In the examples, various electron microscopes were selected to obtain cell size statistics and to image single liquid cells. In each case, a GLC TEM grid was prepared and installed in each microscope 48 hours after preparation. Collection of cell size statistics data was performed at 100 kV. Low-dose, high-resolution imaging of single liquid cells was performed in a microscope operated at an accelerating voltage of 200 kV to minimize beam-sample interactions. Electron images were acquired as recorded images with a camera.

[0086] To summarize the above, preparing graphene liquid cells often requires relatively large amounts of intact single-layer graphene transferred without a support layer. Herein, we present a reproducible method for producing large, millimeter-scale single-layer graphene films on a support, which can be a TEM grid coated with conventional graphene. Because liquid cell formation tends to occur randomly on the grid, a related fluorescence electron microscopy technique is provided to identify the location of GLCs on the grid before exposure to the electron beam, which can be assisted by tagging the GLCs with fluorescent dyes. Three types of GLCs can be formed by the described technique, each with a typical size distribution and stability. The morphology of the seam between two opposing thin films can be a determining factor in the stability of the containment. In particular, folded-type cells can form where thin films, particularly cleaved graphene films, are folded and / or rolled up, and can trap water. This formation process requires only one graphene film instead of two, and these folded-type cells have a greater potential for containing relatively large volumes of liquid, typically in the 250-400 nm size range, compared to other types of liquid cells. This makes them suitable for containing macromolecular assemblies in the exploration and development of GLCs for real-time imaging of biological systems in the liquid phase. The implementation of correlative light-electron microscopy (CLEM) specifically allows for the development of automated data collection protocols that target fluorescence-identified grid locations for electronic image recording. This not only prevents beam damage prior to imaging, but also allows for automated data collection of liquid-phase samples, enhancing the potential for recording tomographic images, which are currently widely used to generate 3D reconstructions of cryogenically stored samples.

[0087] The present disclosure is not limited to the above-described embodiments, but can be modified in several ways within the scope of the claims. Furthermore, elements and features described with respect to or in connection with a particular embodiment may be combined with elements and features of other embodiments as appropriate, unless expressly stated otherwise.

Claims

1. 1. A method for making a thin film liquid cell suitable for transmission electron microscopy at room temperature, the method comprising: providing a thin film floating freely on a liquid containing a sample; a transfer step of transferring the droplet of liquid with the thin film floating freely thereon to a support by a loop, the loop holding the droplet, which in turn holds the thin film during the transfer step; and removing sufficient liquid from the droplet on the support to form the thin film liquid cell containing the sample.

2. 10. The method of claim 1, wherein the thin film liquid cell is formed by a portion of the thin film bending and contacting another portion of the thin film while containing the liquid from the droplet.

3. 2. The method of claim 1, wherein the support is provided with a further thin film, whereby the thin film liquid cell is formed by confining liquid from the droplet between the thin film floating over the droplet and the further thin film provided on the support.

4. The method of claim 3 , wherein the additional thin film is on a porous membrane disposed on the support.

5. The method of claim 4 , wherein the porous membrane comprises polystyrene.

6. The method according to any of claims 4 and 5, wherein the porous membrane is less than 100 nm thick.

7. The method of any one of claims 1 to 6, wherein the loop holding the droplet has an average diameter of less than 5 mm.

8. A method according to any preceding claim, wherein the support comprises a grid for transmission electron microscopy.

9. The step of preparing the thin film floating freely on the liquid containing the sample, comprising: forming the thin film on a substrate; placing the substrate having the thin film thereon on a surface of an etching solution, whereby the substrate comes into contact with the etching solution; removing the substrate with the etching solution; and replacing the etching liquid with the liquid containing the sample on which the thin film is to float.

10. The method according to any one of claims 1 to 9, wherein the thin film comprises at least one layer that is a two-dimensional structure of at least one of atoms and molecules.

11. The method of claim 10 , wherein the thin film comprises graphene to form a graphene liquid cell.

12. The method according to any one of claims 1 to 11, wherein the thin film liquid cell is fabricated to contain a luminescent material.

13. A method for imaging a sample comprising a thin film liquid cell obtained by the method of any one of claims 1 to 12, wherein the sample is imaged by a transmission electron microscope.

14. 14. A method for imaging a sample according to claim 13, wherein the thin film liquid cell is obtained by the method according to claim 12, the method comprising: optically imaging the sample to identify the location of the thin film liquid cell within the sample; and performing electronic imaging of the sample targeted at the identified location of the thin film liquid cell.

15. A thin film liquid cell suitable for transmission electron microscopy at room temperature, the thin film liquid cell being formed by a portion of a single thin film bending and contacting another portion of the single thin film, thereby containing a liquid.

Images