Sample carrier for preparing a sample for electron cryo-microscopy analysis

Abstract

The present invention relates to a sample carrier for preparing a sample for electron cryo-microscopy analysis, said sample carrier comprising a base made of a first thermally conductive material and a surface layer made of a second thermally conductive material, said surface layer is configured to receive the sample, wherein the base is substantially planar having opposed faces substantially parallel to one another, wherein the base comprises an aperture connecting said faces, and wherein the surface layer is adhered to the base by adhering the surface layer to one of the faces of the base, and wherein the surface layer spans the aperture, and wherein the surface layer is substantially free of perforations.

Description

[0001] Title: Sample carrier for preparing a sample for electron cryo-microscopy analysis

[0002] Technical field

[0003] The present invention relates to a sample carrier for preparing a sample for electron cryo-microscopy analysis. The present invention further relates to a method of preparing a sample for electron cryo-microscopy analysis using the sample carrier of the present invention. The present invention also relates to the use of the sample carrier of the present invention and method for the manufacturing of the sample carrier of the present invention. Finally, the present invention relates to a surface layer for use in the sample carrier of the present invention.

[0004] Over the past decade, structure determination of biological molecules using electron cryo-microscopy has undergone a revolution, such that near-atomic resolutions are now possible. Improved, more stable electron microscopes and new computational methods have contributed to these advances. And a dramatic improvement has been afforded by the increased quantum efficiency of new highspeed direct electron detectors, resulting in improved images. It has long been known that cryo-specimens move when irradiated with the electron beam. This results in image blurring and loss of high-resolution information. Direct electron detectors permit tracking of specimen movement during irradiation, allowing realignment of movie frames to reduce (but not eliminate) image blurring. Importantly, these detectors also allow investigation of the origin of specimen movement and the development of new methods to reduce it.

[0005] There have been several recent attempts to design supports that reduce the movement of the specimen during irradiation, by changing either the geometry or the material composition of the suspended foil. It was shown that much of the particle motion in electron cryo-microscopy is due to movement of the support: upon irradiation, supports with a perforated carbon foil over a metal mesh grid move by an amount of 200 A to 400 A in the direction parallel to the electron beam.

[0006] In literature, several attempts have been made to design a support that reduces the movement of the supports. For example, Russo (RUSSO, Christopher J.; PASSMORE, Lori A. Ultrastable gold substrates for electron cryomicroscopy. Science, 2014, 346 (6215), 1377-1380) designed a support comprising a circular disk of gold, 3 mm in diameter, having a mesh pattern on which is suspended a thin, polycrystalline gold foil with a regular array of micrometer-sized holes. The design is commercially available under the tradename UltrAuFoil® (Quantifoil Micro Tools GmbH).

[0007] Despite recent developments in reducing the particle movement during imaging the supports do not provide for solutions to be used within electron cryo-microscopy analysis in cell research, e.g., by applying cryo-electron tomography techniques.

[0008] Detailed description of the invention

[0009] In order to provide a sample carrier for preparing a sample for electron cryo- microscopy analysis, in particular a sample for use in cell research, the present invention provides hereto a sample carrier for preparing a sample for electron cryo- microscopy analysis, wherein the sample carrier comprises: a base made of a first thermally conductive material, said base is substantially planar having opposed faces substantially parallel to one another, wherein the base is selected from the group consisting of one-hole grids, slot grids, gridless grids and aperture grids comprising an aperture connecting said faces; and a surface layer made of a second thermally conductive material, said surface layer is configured to receive the sample, wherein the surface layer is adhered to the base by adhering the surface layer to one of the faces of the base, and wherein the surface layer spans the aperture, and wherein the surface layer is substantially free of perforations.

[0010] It was found that by providing the sample carrier of the present invention, a so- called griddles grid carrier can be provided solving several problems observed with sample carriers comprising a grid based carrier. By providing the sample carrier of the present invention, less thermal mass is present in the centre of the sample carrier, thus allowing for higher cooling potentials which result in better vitrification of the sample. In addition, or alternatively, the sample carrier of the present invention solves the problem that cells adhere to the grid bars of a grid based sample carrier. By having cells adhered to the grid bars, the milling angle that can be achieved is different (less favourable) compared to the sample carrier of the present invention wherein the cells do not adhere to grid bars (due to the lack of such grid bars in the sample carrier of the present invention). It is further noted that grid based sample carriers have been known for close to 40 years. Also the use of grid based sample carriers have been set as standards for application in electron cryo-microscopy. However, by providing the sample carrier of the present invention, the cooling potential significantly improves compared to grid based sample carriers. In addition, or alternatively, by providing the sample carrier of the present invention, the lack of grid bars allowing the sample carrier of the present invention to be milled. Also, the sample carrier of the present invention is in particular suitable for use with a vitrification jetting technique, which technique provides for a better performance over vitrification by plunging.

[0011] Furthermore, by providing the sample carrier of the present invention, the design of the sample carrier, i.e., the lack of grid bars, allows for more freedom in postprocessing methods, such as choosing an area for making lamellas.

[0012] In the sample carrier of the present invention, the surface layer spans the aperture of the base and is substantially free of perforations. In other words, the surface layer is produced as a continuous layer without comprising any intended perforations or through-holes. As such, the surface layer may also be defined as a continuous layer, in particular a closed continuous layer, i.e., a surface layer substantially free of perforations and / or through-holes. Such continuous layer may still comprise some perforations and / or through-holes due to inconsistencies in the process of manufacturing the surface layer. The present invention thus relates to a surface layer that spans the aperture of the base, wherein the surface layer is a perfect continuous layer or (at least) close to perfect continuous layer (comprising a negligible amount of incidental perforations).

[0013] In particular it is emphasized that the surface layer of the present invention spans 100% of the aperture of the base, i.e., completely spanning the aperture of the base. However, due to incidental perforations, the surface of the surface layer may cover less than 100% of the spanned aperture, albeit spanning 100% of the aperture at the same time. In an embodiment of the present invention, the surface layer of the present invention that is substantially free of perforation at least covers 85% of the spanned aperture. Preferably, the surface layer of the present invention covers at least 90%, preferably at least 95%, more preferably at least 98%, of the spanned aperture. It was found that by providing a surface layer that has a surface coverage of at least 85%, the surface layer was found to be suitable to receive a sample, such as a sample used in cell research. In other words, the surface layer of the sample carrier of the present invention is a continuous layer wherein the total surface area of any (incidental) perforations present in the continuous layer does not exceed 15% of the total area of the surface layer. Alternatively, the surface layer may also refer to a continuous non-permeable layer. Further alternatively, the surface layer may be defined as the contact surface for contacting the sample.

[0014] It is further noted that the base of the present invention is selected from the group consisting of one-hole grids, slot grids, gridless grids and aperture grids, wherein such grids comprise an aperture that is spanned by the surface layer of the present invention. It is noted that in this respect the aperture of the base referred to has to be interpreted as one single aperture. In other words, the surface layer of the present invention is adhered to the base and consequently covers one single aperture. Stated differently, the surface layer of the present invention is not adhered to a base wherein the surface layer covers multiple apertures (such as multiple apertures defined in a grid based carrier).

[0015] In the sample carrier of the present invention, the second thermally conductive material may be selected from the group consisting of materials having a relatively high thermal conductivity and a relatively low thermal capacity. Similarly, the first thermally conductive material may also be selected from the group consisting of materials having a relatively high thermal conductivity and a relatively low thermal capacity. In addition or alternatively, the second thermally conductive material and, optionally, the first thermally conductive material may be selected from the group consisting of biocompatible materials. Preferred materials may include copper, nickel, gold, aluminium, molybdenum, titanium, stainless steel, and combinations thereof. As such the second thermally conductive material and, optionally, the first thermally conductive material may be selected from the group consisting of copper, nickel, gold, aluminium, molybdenum, titanium, stainless steel and combinations thereof. Particular good results have been obtained by using a thermally conductive material made of gold.

[0016] Although not considered necessary, in a preferred embodiment, the first and second thermally conductive materials may be made of the same thermally conductive material. It is noted that although it is preferred that both thermally conductive materials are selected from the group consisting of biocompatible materials having a relatively high thermal conductivity and a relatively low thermal capacity, the thermally conductive material forming the base (i.e. , the first thermally conductive material) may be selected from a group of thermally conductive materials that are not biocompatible and / or have different thermal conductivity and thermal capacity. As the surface layer forming thermally conductive material (i.e., the second thermally conductive material) is in direct contact with the sample, it is preferred that the second thermally conductive material is made of a material that has sample-friendly properties.

[0017] In the sample carrier of the present invention, the aperture comprised in the base may have a minimal width of at least 200 pm. However, the aperture may also have a minimal width of at least 300 pm or a minimal width of at least 400 pm. In a preferred embodiment the width of the aperture is defined by the base of the sample carrier and the circumferential edge enclosing the aperture. In other words, in an embodiment of the present invention the face of the base may have a circumferential width enclosing the aperture, and wherein the aperture having a maximum width depending on the width of the base minus the circumferential width of the face of the base.

[0018] The circumferential width of the face of the base (in other words the width of the edge of the base enclosing the aperture) may be at least 0.2 mm, preferably at least 0.5 mm. Preferably the circumferential width of the base is within the range of 0.2 mm to 0.8 mm, preferably within the range of 0.3 mm to 0.7 mm.

[0019] In order to provide a sample carrier wherein the surface layer is still able to span the aperture of the base, the aperture may have a maximum width of not more than 3 mm. Preferably the aperture may have a maximum width of not more than 2.5 mm, preferably a maximum width of not more than 2 mm.

[0020] In the sample carrier of the present invention, the surface layer is adhered to the base. Although this might imply that the surface layer is a part separated from the base, the base and surface layer may form an integral part of the sample carrier of the present invention. In the event the surface layer is adhered to the base as a separate part, the face of the base the surface layer is adhered to may have a circumferential width enclosing the aperture, and wherein the overlap between the surface layer and the circumferential width of the face of the base the surface layer is adhered to may be at least 0.2 mm, preferably at least 0.5 mm. The surface layer may have a thickness of at least 300 nm. Preferably the thickness of the surface layer may be at least 350 nm, preferably a thickness of at least 400 nm. Preferably, the surface layer may have a thickness that does not exceed 20 pm. Preferably, the surface layer may have a thickness of not more than 15 pm, preferably not more than 10 pm.

[0021] The surface layer may be adhered to the base by an adhesive, cold welding, diffusion bonding or by laser welding.

[0022] As already noted above, the sample carrier of the present invention relates to a so-called gridless grid. Synonyms for gridless grids may include one-hole grids, slot grids and aperture grids. It is noted that the present invention does not relate to a sample carrier comprising a base comprising a matrix based grid.

[0023] In a second aspect of the present invention, the invention relates to a method of preparing a sample for electron cryo-microscopy analysis, wherein the method comprises the steps of: a) providing the sample; b) providing the sample carrier according to the present invention; c) suspending the sample onto the surface layer of the sample carrier; and d) vitrifying the sample.

[0024] Given the method of preparing the for electron cryo-microscopy analysis, in a preferred embodiment the vitrification step d) is performed by jet vitrification, plunge vitrification, or combinations thereof.

[0025] The sample may comprise a medium, wherein said medium comprises an analyte. Preferably the analyte is selected from the group consisting of viruses, cells, proteins and combinations thereof.

[0026] In a third aspect of the present invention, the invention relates to the use of the sample carrier of the present invention in the preparation of a sample for electron crymicroscopy analysis. The sample may comprise a medium, wherein said medium comprises an analyte. Preferably the analyte is selected from the group consisting of viruses, cells, proteins and combinations thereof. In a fourth aspect of the present invention, the invention relates to a method for the manufacturing of a sample carrier for preparing a sample for electron cryomicroscopy analysis, the method comprising the steps of: i) providing a base made of a first thermally conductive material, said base is substantially planar having opposed faces substantially parallel to one another, wherein the base comprises an aperture connecting said faces; ii) providing a surface layer made of a second thermally conductive material; iii) spanning the surface layer over the aperture; and iv) adhering the surface layer to one of the faces of the base.

[0027] In the method for the manufacturing of a sample carrier for preparing a sample for electron cryo-microscopy analysis, the surface layer may be adhered to one of the faces of the base by an adhesive, cold welding, diffusion bonding or by laser welding.

[0028] In a fifth aspect of the present invention, the invention relates to a surface layer for use in the sample carrier according to the present invention, wherein the surface layer is made of a thermally conductive material, wherein the surface layer is configured to receive a sample for electron cryo-microscopy analysis, wherein the surface layer is substantially free of perforations, and wherein the surface layer has a thickness of not more than 10 pm.

[0029] Alternatively, the surface layer in the fifth aspect of the present invention may be defined as a continuous layer, preferably a closed continuous layer. Although some inconsistencies and through-holes or perforations may be present in the surface layer, the surface layer is substantially free of perforations and / or through-holes. It is emphasized that although the surface layer of the present invention may be defined as a continuous layer, the surface layer, in case applied onto the base of the sample carrier of the present invention, does not necessarily have to cover 100% of the aperture of the base, i.e., sealing the aperture of the base. Due to incidental perforations, the surface coverage of the surface layer may be less than 100%, albeit covering the aperture of the base, when applied to the base, at the same time. In an embodiment of the present invention, the surface layer may have a surface coverage of at least 85%, more preferably of at least 90%, at least 95% or at least 98%. The thermally conductive material forming the surface layer is preferably selected from the group consisting of copper, nickel, gold, aluminium, molybdenum, titanium, stainless steel and combinations thereof. More preferably, the thermally conductive material of the surface layer is gold.

[0030] Brief description of the figures

[0031] The following figures are illustrative of the present invention, and are not considered to be limiting the present invention, wherein:

[0032] Figures 1 shows the sample carrier of the present invention;

[0033] Figure 2 shows the surface of the sample carrier of the present invention;

[0034] Figure 3 shows a first lamellae made on the sample carrier of the present invention;

[0035] Figure 4 shows the sample carrier of the present invention as well as the base and the surface layer;

[0036] Figure 5 shows the process for the manufacturing of the sample carrier of the present invention using cold welding;

[0037] Figure 6 shows the process for the manufacturing of the sample carrier of the present invention using adhesive bonding;

[0038] Figure 7 shows the process for the manufacturing of the sample carrier of the present invention using laser welding; and

[0039] Figure 8 shows the process for the manufacturing of the sample carrier of the present invention using diffusion bonding.

[0040] Detailed description of the figures

[0041] Figure 1 shows an electron beam image from a Scios dual beam microscope showing vitrified cells grown on a 2 pm gold surface layer (gold foil). The surface layer is glued onto a gold base (gold slot grid) with a 2000 pm aperture (slot) and clipped in an Autogrid before the vitrification step.

[0042] Figure 2 shows an ion beam image from a Scios dual beam microscope showing the monolayer of cells.

[0043] Figure 3 shows an ion beam image from a Scios dual beam microscope illustrating the FIB (Focused Ion Beam) milling procedure. This procedure demonstrates how thin slices of a monolayer of cells, referred to as lamellae, can be prepared using the dual beam microscope. The lamella is further thinned down to approximately 150 nm to enable visualization in a transmission electron microscope (TEM).

[0044] Figure 4 shows the sample carrier 10 of the present invention comprising a surface layer 30 adhered to a base 20 comprising an aperture 26. The base 20 comprises a first face 22 and an opposed second face 24 connected to each other by aperture 26 and edge 28.

[0045] Figure 5 shows the process for the manufacturing of the sample carrier of the present invention using cold welding. In this process the surface layer is placed on a polished hardened surface (step A). The surface layer is subsequently flattened (step B) by applying a pressure or force onto the surface layer using a stamp or the like. Optionally, ethanol may be applied to the surface layer in order to facilitate the detaching of the force applying stamp (step C). After removal of the force applying stamp, the base is placed onto the surface layer (step C). Subsequently a force is applied onto the assembly of surface layer and base in order to obtain a cold welded or diffusion bonded assembly (step D). Figure 5 further shows the perspective views of the resulting product including a top-view (upper part), side-view (middle part) and bottom-view (lower part).

[0046] Figure 6 shows the process for the manufacturing of the sample carrier of the present invention using adhesive bonding. Here (step A), the surface layer (left) is placed onto a PTFE base together with the base (right). The base is further provided on its edges with a thin adhesive layer (indicated by the arrow). Subsequently (step B), the base is placed onto the surface layer to let the adhesive cure and form an adhesive bonding. Optionally, pressure may be applied to further facilitate the adhesive bonding of the base to the surface layer. Figure 6 further shows the perspective views of the resulting product including a top-view (upper part), side-view (middle part) and bottom-view (lower part).

[0047] Figure 7 shows the process for the manufacturing of the sample carrier of the present invention using laser welding. In this process, the base is placed onto a glass slide (step A). Subsequently the surface layer is placed onto the base (step B). Another glass slide is placed on top of the assembly (step C) in order to prevent movement of the base and / or surface layer and to assure flatness of the foil. In step D laser welding is performed onto the edges of the surface layer in order to adhere the surface layer to the base. Figure 7 further shows the perspective views of the resulting product including a top-view (upper part), side-view (middle part) and bottom-view (lower part). It is noted that in the top-view, the weld spots are visible.

[0048] Figure 8 shows the process for the manufacturing of the sample carrier of the present invention using diffusion bonding. Here, the surface layer is placed onto a heat resistant substrate with capillary channels (step A). After applying a vacuum over the capillary channels in order to suck the surface layer flat to the substrate, the base is placed onto the foil together with a heat resistance clamping substrate (step B). Subsequently, a vacuum is applied to the assembly in combination with applying a pressure (indicated by the arrow) to the assembly (step C). The surface layer and base are simultaneously heated to a temperature just below the melting temperatures of the materials of the surface layer and base. The assembly is subsequently allowed to cool down (step D). Figure 8 further shows the perspective views of the resulting product including a top-view (upper part), side-view (middle part) and bottom-view (lower part). data

[0049] To prepare the sample carrier of the present invention, several techniques have been applied, including cold welding, gluing, and laser welding.

[0050] Cold

[0051] For the cold welding method the following materials were used: a slot grid type “Aperture Grids for TEM, 1000 pm Hole, 3 mm Diameter, Gold” was selected as the base carrier; a gold leaf material type “NGR075-080080L18” was selected as base material for the continuous layer; two custom polished Stainless-steel bases were selected as mould material; a hydraulic Press; and a razor blade.

[0052] Figure 5 depicts the cold welding method described here (method 1). The stainless-steel base was polished and cleaned properly. A small amount of isopropyl was applied to prevent adhesion of the gold to the base. The gold leaf was placed on the base. The second base was placed on top of the foil and pressure was applied to flatten the foil. The press was opened again, the slot grid was placed on the flattened foil. The second stainless steel base was placed on top of the assembly. Both bases were pressed against each other by means of loads of 200kg reaching to 2000kg. The pressure was kept like this for several minutes. The press was opened and the top base removed. The slot grid and gold leaf are bonded due to the high pressure, the remaining geometry was the slot grid with the gold leaf cold welded on top of it. Removal was done with a Razor blade. The resulting carrier was ready for direct use.

[0053] For the gluing (method 2) the following materials were used: a slot grid type “Aperture Grids for TEM, 1000 pm Hole, 3 mm Diameter, Gold” was selected as the base carrier: a gold leaf material type “NGR075-080080L18” was selected as base material for the continuous layer; from a wide selection of adhesives, a 2-component epoxy glue type “Loctite EA9483” showed best adhesive results and was used in the experiment; and a Duran bottle with a custom cap including a PTFE microporous membrane was assembled, wherein the used PTFE microporous membrane dimensions were 47 mm diameter with 1 .5 pm pore size holes.

[0054] Figure 6 depicts the gluing method described here (method 2). The gold leaf was placed on top of the PTFE microporous membrane, applying a rough vacuum on the inside of the bottle which resulted in flattening the gold leaf. By means of a sewing needle, a very small amount of properly mixed epoxy glue was applied to the ring of the slot grid. After application, the ring was placed on top of the gold leaf. The assembled grid was allowed to cure for 24 hours. The gold leaf was larger than the grid and by means of a scalpel, the outer rim of the sample carrier was cleaned to obtain the remaining geometry; the slot grid with the gold lead glued on top of it. The resulting carrier was ready for direct use.

[0055] For the gluing (method 3) the following materials were used: a slot grid type “Aperture Grids for TEM, 1000 pm Hole, 3 mm Diameter, Gold” was selected as the base carrier; a gold leaf material type Goodfellow: AU00-FL-000170 Gold discs Au 99.9% was selected as base material for the continuous layer (with a diameter of 3 mm and a thickness of 1 and 2 pm); from a wide selection of adhesives, a 2-component epoxy glue type “Loctite EA9483” showed best adhesive results and was used in the experiment; and a PTFE flat disk was used as a gluing base.

[0056] Figure 6 depicts the gluing method described here (method 3). The gold leaf was placed on top of the PTFE disk. By means of a sewing needle, a very small amount of properly mixed epoxy glue was applied to the ring of the slot grid. After application, the ring was placed on top of the gold leaf. The assembled grid was allowed to cure for 24 hours. The gold leaf was of the same size as the grid, and no cleaning was needed. The remaining geometry resulted in the slot grid with the gold leaf glued on top of it. The resulting carrier was ready for direct use.

[0057] Laser

[0058] For the laser welding (method 4) the following materials were used: a slot grid type “Aperture Grids for TEM, 1000 pm Hole, 3 mm Diameter, Gold” was selected as the base carrier; a gold leaf material type Goodfellow: AU00-FL-000170 Gold discs Au 99.9% was selected as base material for the continuous layer (having a thickness of 1 and 2 pm); two standard microscope slides were used as a base for the assembly; and one brass cylindrical bushing was used as a handling tool for welding.

[0059] Figure 7 depicts the laser welding method described here (method 4). The microscope slide was placed on top of the brass ring. The gold leaf was placed on the microscope slide, and centered in the brass ring. The slot grid was placed on top of the gold leaf (centered). Another microscope slide was put on top of this assembly, and the gold leaf and ring were flattened by means of surface pressure. By means of laser spot welding, the gold leaf was welded to the ring while the assembly was sandwiched between the microscope slides. The ring and the gold leaf were attached by 20 or more laser welded spots roughly divided over the outer ring of the grid. The resulting carrier was ready for direct use.

Claims

CLAIMS1. Sample carrier (10) for preparing a sample for electron cryo-microscopy analysis, wherein the sample carrier (10) comprises: a base (20) made of a first thermally conductive material, said base (20) is substantially planar having opposed faces (22, 24) substantially parallel to one another, wherein the base (20) is selected from the group consisting of one-hole grids, slot grids, gridless grids and aperture grids comprising an aperture (26) connecting said faces (22, 24); and a surface layer (30) made of a second thermally conductive material, said surface layer (30) is configured to receive the sample, wherein the surface layer (30) is adhered to the base (20) by adhering the surface layer (30) to one of the faces (22, 24) of the base, and wherein the surface layer (30) spans the aperture (26), characterised in that the surface layer (30) is substantially free of perforations.

2. Sample carrier (10) according to claim 1 , wherein the second thermally conductive material and, optionally, the first thermally conductive material are selected from the group consisting of: materials having a relatively high thermal conductivity and a relatively low thermal capacity; and / or biocompatible materials.

3. Sample carrier (10) according to claim 1 or 2, wherein the first thermally conductive material and / or the second thermally conductive material is selected from the group consisting of copper, nickel, gold, aluminium, molybdenum, titanium, stainless steel, and combinations thereof.

4. Sample carrier (10) according to any of the preceding claims, wherein the first and second thermally conductive materials are made of the same thermally conductive material.

5. Sample carrier (10) according to any of the preceding claims, wherein the aperture (26) having: a minimal width of at least 200 pm, preferably a minimal width of at least 300 pm, preferably a minimal width of at least 400 pm; and / ora maximum width of not more than 3 mm, preferably a maximum width of not more than 2.5 mm, preferably a maximum width of not more than 2 mm.

6. Sample carrier (10) according to any of the preceding claims, wherein the face (22, 24) of the base (20) the surface layer (30) is adhered to has a circumferential width enclosing the aperture (26), and wherein the overlap between the surface layer (30) and the circumferential width of the face (22, 24) of the base (20) the surface layer (30) is adhered to is at least 0.2 mm, preferably at least 0.5 mm.

7. Sample carrier (10) according to any of the preceding claims, wherein the surface layer (30) has a thickness: of at least 300 nm, preferably a thickness of at least 350 nm, preferably a thickness of at least 400 nm; and / or not more than 20 pm, preferably not more than 15 pm, preferably not more than 10 pm.

8. Sample carrier (10) according to any of the preceding claims, wherein the surface layer (30) is adhered to the base (20) by an adhesive, cold welding, diffusion bonding or by laser welding.

9. Method of preparing a sample for electron cryo-microscopy analysis, the method comprising the steps of: a) providing the sample; b) providing the sample carrier (10) according to any of the preceding claims; c) suspending the sample onto the surface layer (30) of the sample carrier (10); and d) vitrifying the sample, preferably wherein the vitrification step d) is performed by jet vitrification, plunge vitrification, or combinations thereof.

10. Use of the sample carrier (10) according to any of claims 1-8 in the preparation of a sample for electron cryo-microscopy analysis.

11. Method according to claim 9 or use according to claim 10, wherein the sample comprises a medium, said medium comprises an analyte, preferably wherein the analyte is selected from the group consisting of viruses, cells, proteins and combinations thereof.

12. Method for the manufacturing of a sample carrier (10) for preparing a sample for electron cryo-microscopy analysis, the method comprising the steps of:i) providing a base (20) made of a first thermally conductive material, said base (20) is substantially planar having opposed faces (22, 24) substantially parallel to one another, wherein the base (20) comprises an aperture (26) connecting said faces (22, 24); ii) providing a surface layer (30) made of a second thermally conductive material; iii) spanning the surface layer (30) over the aperture (26); and iv) adhering the surface layer (30) to one of the faces (22, 24) of the base (20).

13. Method according to claim 12, wherein the surface layer (30) is adhered to one of the faces (22, 24) of the base (20) by an adhesive, cold welding, diffusion bonding or by laser welding.

14. Surface layer (30) for use in the sample carrier (10) according to any of claims 1-8, wherein the surface layer (30) is made of a thermally conductive material, and wherein the surface layer (30) is configured to receive a sample for electron cryomicroscopy analysis, characterised in that the surface layer (30) is substantially free of perforations, and in that the surface layer (30) has a thickness of not more than 10 pm.

15. Surface layer (30) according to claim 14, wherein the thermally conductive material is gold.

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