A carrier for transmission electron microscopy

Abstract

A carrier for use in transmission electron microscopy, designed to receive a sample. The carrier comprises a membrane layer consisting of a material of a first type, wherein the membrane layer has a membrane layer thickness, a first membrane layer surface and a second membrane layer surface opposite to the first membrane layer surface, wherein the semiconductor material of the first type is at least partially transparent for electron beam irradiation, where at least part of the first membrane layer surface is designed as a membrane deposit area to receive a sample for electron microscopy. The carrier further comprises a substrate layer consisting of a material of a second type, different from the material of the first type, wherein the material of a second type is a semiconductor material. Said second layer has a first substrate layer surface and a second substrate layer surface opposite to the first substrate layer surface, wherein the material of the second type is at least partially transparent for electron beam irradiation. The membrane layer is mounted with its second membrane layer surface to the first substrate layer surface of the substrate layer, and the substrate layer is formed as a rigid body having a substrate layer thickness larger than the membrane layer thickness. The substrate layer further comprises at least one recess extending from the second substrate layer surface towards the first substrate layer surface, wherein the recess is located within or facing the membrane deposit area.

Description

[0001] TITLE

[0002] A carrier for transmission Electron Microscopy

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to a carrier designed to receive a sample for analysis using electron microscopy.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Electron microscopy (EM) is an invaluable tool for characterization due to its greater resolution compared to optical microscopy. More particularly, transmission electron microscopy can achieve atomic resolution, such that it has become the industry’s standard when determining the structures of small biomolecules, DNA, proteins, and viruses. In particular, frozen aqueous samples with analyte are analyzed, which technique is known as cryo-electron microscopy (cryoEM).

[0007] To determine biomolecular structures with cryoEM, a small amount of sample is deposited on a so-called TEM grid, whereafter the grid is rapidly cooled to form amorphous ice, also known as vitrification. Multiple grids with the same sample are prepared in this way and imaged subsequently, due to non-ideal conditions of the grids and the large amount of data needed for structural reconstruction.

[0008] The imaging of the samples is done in an EM, where robotic handlers are used to switch between the different TEM grids. In the field, this robotic handling system is known as an autoloader and comprises a robotic arm and a rack that hold multiple grids, which is known as a cassette, submerged in a cryogen. This robotic handling system allows for better sample preservation, a robust workflow, and reduced contamination.

[0009] Ever since the introduction of electron microscopes, these TEM grids have remained largely unchanged. Basically, EMs and the robotic handling systems have been designed to fit around the grids instead of the other way around.

[0010] This results in the problem that the TEM grids deform and / or tear when grabbed by the robotic handling system. Namely, typically, the TEM grids that are used are very thin (around 7 μm) and are made of soft metals, such as gold, molybdenum, or copper, making them flexible and sensitive to deformation. Therefore, robust frames have been introduced to aid the structural support of the TEM grids, when used in electron microscopy. The use of these robust frames, however, leads to additional costs, additional steps in the workflow, and chances to contaminate or unknowingly tamper with the sample.

[0011] Regarding the problem sketched from the background art, a purpose of the present disclosure is to provide a sample carrier that can be easily used in electron microscopy, in particular transmission electron microscopy, with reduced chance of deformation, tearing and / or bending, and with improved surface flatness resulting in improved data quality during acquisition in cryoEM. Furthermore, the new design of the sample carrier according to the disclosure allows for incorporating additional functionalities in the carrier, further improving its versality in cryoEM applications.

[0012] SUMMARY OF THE DISCLOSURE

[0013] According to the disclosure a carrier for use in electron microscopy, in particular transmission electron microscopy, TEM, is proposed. In a preferred example, the carrier comprises a membrane layer consisting of a material of a first type having a membrane layer thickness, a first membrane layer surface, and a second membrane layer surface opposite to the first membrane layer surface, wherein the material of the first type is at least partially transparent for electron beam irradiation, where at least part of the first membrane layer surface is designed as a membrane deposit area to receive a sample for electron microscopy in particular transmission electron microscopy, TEM. The carrier further comprises a substrate layer consisting of a material of a second type, different from the material of the first type. The material of a second type is a semiconductor material. The substrate layer has a first substrate layer surface and a second substrate layer surface, wherein the material of the second type is at least partially transparent for electron beam irradiation. The membrane layer is mounted with its second membrane layer surface to the first substrate layer surface of the substrate layer. The carrier forms a rigid body, due to the substrate layer, having a substrate thickness, larger than the membrane layer thickness. Microfabrication has made huge leaps in recent years due to the semiconductor industry, and reproducible manufacturing of structures based on semiconductor substrates can be done controllably to produce carriers that are rigid during EM usage, such that they are compatible with robotic handling systems. Additionally, these processes have higher controllability, thereby lower and much more accurate tolerances can be achieved in the manufacturing of these carriers compared to traditional EM carrier manufacturing, such that greater tunability of the carrier thickness and stiffness is possible. Lastly, these processes allow for the incorporation of additional functionalities into carrier.

[0014] In a further preferred example, the semiconductor material of the second type of the substrate layer is chosen from a list not limited to glass, quartz, borosilicate, calcium fluoride, germanium, silicon, silicon carbide, silicon dioxide, silicon on insulator material, silicon on sapphire material, sapphire, gallium nitride, gallium arsenide or indium phosphide.

[0015] This allows the manufacturing of the substrate layer using etching and deposition techniques on standardized semiconductor substrates in an optimized manner, such that higher accuracy and reproducibility during the manufacturing can be achieved. In particular, single atom layer techniques allow for atom precise manufacturing of thicknesses, width, lengths, and other dimensions.

[0016] In an advantageous example, the material of the first type of the membrane layer is also a semiconductor material. In particular, the material of the first type is chosen from a list not limited to carbon, silicon, germanium, gallium nitride, gallium phosphide, titanium dioxide, silicon-germanium, silicon-tin, silicon-nitride, siliconoxide, aluminum gallium arsenide, aluminum gallium phosphide, indium gallium arsenide, indium gallium phosphide, or any doped variants.

[0017] This allows the deposition or growth of semiconductor or electrically conductive materials on top of another semiconductor material with relative ease. Additionally, these materials are chosen for their characteristics, such that they can be deposited with a thickness much smaller than the thickness of the substrate, while maintaining the carrier’s integrity during EM usage without ripping, bending, crackling or other deformations. Additionally, the material is chosen with specific characteristics to ensure that vitreous ice can be formed on the membrane layer when an aqueous sample is vitrified, rapidly cooled to form amorphous ice and sometimes even pressure is applied.

[0018] Making a material layer at least partially transparent for electron beam irradiation can be achieved by selecting the material of said layer based on its material properties. I.e. metals may be understood as opaque to electron beam irradiation and organic layers (like carbohydrates or carbon) as being transparent. Alternatively, transparency for electron beam irradiation can be achieved by implemented physical features in / on the material layer, i.e. local thinning of a material layer by means of at least one recess or even the formation of through holes in said material layer.

[0019] In another advantageous example of the carrier’s membrane layer, the material of the first type is an electrically conductive material. In particular, the material of the first type is chosen from a list not limited to graphene, carbon, silver, copper, gold, indium tin oxide, iron, zinc oxide or a 2D material, such as graphene, graphene oxide, hexagonal boron nitride or a monolayer of amorphous carbon.

[0020] Depositing or growing of electrically conductive materials on top of a semiconductor substate has the advantage of generating a better contrast when used in EM, especially in TEM, since the electrons not hitting the sample are conducted or diverted away and thus not appear on the detector. Additionally, most conductive materials are not crystalline aiding the formation of amorphous ice when a sample containing analytes is vitrified.

[0021] In a preferred example, the carrier has a thickness ratio defined between the membrane layer thickness and the substrate layer thickness. This thickness ratio is in the range of 1 :10 to 1 :1 ,000,000, in particular in the range of 1 : 100 to 1 :100,000, more in particular in the range of 1 :200 to 1 :10000 or 1 :1000 to 1 :5000.

[0022] Due to this thickness ratio, it is ensured that the rigidity of the carrier, is maintained, mostly due to the properties of the substrate layer. Whereas the thin membrane layer allows the carrier to have characteristics of improving the EM, especially TEM, imagery of the to be deposited sample on the carrier. These improvements can be of nature of improving amorphousness of the vitrified sample and / or conducting stray electrons and thereby improving contrast.

[0023] In a further preferred example, the carrier’s substrate layer thickness ranges from 10 micrometer to 500 micrometer, in particular from 50 micrometer to 300 micrometer, and / or the carrier has an outer peripheral configuration of an elliptical, circular, quasi-circular, or at least five-sided polygonal shape, when the first membrane surface is taken as cross-sectional plane. This particular outer peripheral shape is chosen such that the carrier is conformal to the inner peripheral dimensions of a carrier holder for electron microscopy, especially TEM. Such carrier holder can be a TEM sample holder rod, a robotic handling system, known as autoloader, a rotating TEM sample holder or a clip ring, known as autogrid, or the like.

[0024] Since the introduction of TEM and cryoEM, the sample carrier has remained largely unchanged. Standardized 3 mm grids with a thickness of 7 μm made of soft metals have been used ever since, and the electron microscopes have been designed around the sample carriers instead of the other way around. Moreover, the carrier holders of newer high-end electron microscopes comprise robotic handling systems to automize imagery and reduce the strain on samples and grids from loading and unloading into the vacuum chamber all the time. However, they are compatible with the standardized shapes and dimensions, but not with the flexible material, which the standardized sample carriers are made of. This disclosure offers a shape conformal rigid sample carrier for electron microscopy, especially TEM and cryoEM, that can be utilized with EM carrier holders, such as a robotic handling system, known as autoloader, a standard TEM sample holder rod, a rotating TEM grid holder or a clip ring, known as autogrid, or the like.

[0025] In yet another preferred example, the substrate layer of the carrier comprises at least one substrate recess extending from the second substrate layer surface towards the first substrate layer surface. In the example of various substrate recesses, the recesses may have identical substrate recess depths or alternatively various substrate recess depths. In a first embodiment the depth of the at least one substrate recess is smaller than the substrate layer thickness, and in another embodiment the depth of the at least one substrate recess is equal to the substrate layer thickness.

[0026] The at least one substrate recess improves the transparency for electron beam irradiation of the sample by thinning out the substrate layer at a specific area. As known from the Beer-Lambert Law the attenuation of transmission, in this case electron beam irradiation, is material thickness dependent. The reduction of the local substrate layer thickness in that area is at its maximum when the recess reaches towards or is equal to the substrate layer surface. In the latter example, the substrate layer is provided with a through opening. In another preferred example, wherein a plurality of substrate recesses are provided, wherein the plurality of substrate recesses are arranged in an array.

[0027] The substrate layer having a plurality of substrate recesses improves the substrate’s transparency for electron beam irradiation without jeopardizing the substrate’s, rigidity. Arranging the plurality of substrate recesses in an array, such as a rectangular, cubic, hexagonal, polar, or randomly distributed array or the like, offers local higher transmission, but the substrate ridges between adjacent substrate recesses underneath the membrane layer ensure or maintain carrier rigidity.

[0028] In another preferred example, the membrane layer of the carrier comprises at least one membrane recess extending from the first membrane layer surface towards the second membrane layer surface. The at least one membrane recess has a membrane recess depth. In a first embodiment, the membrane recess depth is smaller than the membrane layer thickness. In another embodiment the membrane recess depth is equal to the membrane layer thickness, thus forming a through opening.

[0029] In yet another embodiment, a plurality of membrane recesses are provided, which are arranged in an array. The array can be a rectangular, cubic, hexagonal, polar, or randomly distributed array or the like.

[0030] The membrane layer having at least one membrane recesses improves the membrane’s transparency for electron beam irradiation by thinning out the membrane layer. The transmission is known to be thickness dependent via the Beer-Lambert Law. The maximum depth of the at least one membrane recess is equal to the membrane layer thickness.

[0031] In an advantageous example, the at least one membrane recess and / or the at least one substrate recess are located within or facing the membrane deposit area.

[0032] The advantage of the at least one membrane recess and / or the at least one substrate recess being located within or facing the membrane deposit area is such that the transparency for electron beam irradiation is improved in the area where the sample is deposited onto the carrier. Accordingly, the remainder of the carrier surface that is not intended to accommodate the sample to be analyzed or is not to be actively irradiated with an electron beam is not thinned or is not provided with a recess and thus maintains the carrier’s overall rigidity.

[0033] In another advantageous example, an at least one membrane protrusion is located on the first membrane layer surface adjacent to the membrane deposit area. In a first embodiment said at least one membrane protrusion surrounds the membrane deposit area.

[0034] The at least one membrane protrusion is located on the first membrane layer surface to protect the deposited sample in the membrane deposit area on the carrier i.e. , from the pincers used in carrier holders, such as a robotic handling system, known as autoloader. This way the pincers will touch the membrane protrusion and therefore will not interfere with the sample.

[0035] In a preferred example, an at least one substrate protrusion is located on the second substrate layer surface adjacent to a substrate area on the second substrate layer surface facing the membrane deposit area. In another embodiment said at least one substrate protrusion surrounds the substrate area.

[0036] The at least one substrate protrusion is located on the second substrate layer surface in order to generate more stiffness of the substrate layer, and the carrier in general. Especially, in combination with the existence of substrate recesses that aid the carrier’s transparency for electron beam irradiation, the substrate protrusion can compensate for the loss of stiffness as it forms a stiffening structure or structures surrounding the substrate area. The substrate protrusions surround the substrate area, since this is the area which will mostly be irradiated with an electron beam, and additional material will only reduce the transparency in that area.

[0037] In another preferred example, the at least one membrane protrusion and / or the at least one substrate protrusion are formed by means of a depositing technique. Alternatively, in another example the at least one membrane protrusion and / or the at least one substrate protrusion are formed by means of an etching technique.

[0038] These semiconductor manufacturing techniques have the advantage of having an increased accuracy in the dimensions of manufacturing. Furthermore, the scalability provided by the standard microfabrication techniques makes it possible to produce the sample carriers across different wafer sizes (4 inch, 6 inch, 8 inch, 10 inch and 12 inch) so that the amount of chips produced per batch increases exponentially, while preserving the consistency in the architecture.

[0039] In yet another preferred example, the at least one substrate protrusion defines a circumferential space to accommodate a retention holding spring for use in a carrier holder for electron microscopy. Alternatively, the carrier further comprises a circumferential space to accommodate a retention holding spring, which circumferential space may be formed by the presence of a further substrate recess or by the above-mentioned at least one substrate protrusion.

[0040] Standardized TEM sample carriers, TEM grids are currently equipped with a thick robust frame and a retention holding spring to hold the 7 μm thick TEM grid in the robust frame, such that it can be used in carrier holders i.e. , a robotic handling system, known as autoloader. The embodiment of the present disclosure is designed in such a way that current working protocols can still be followed when utilizing the more rigid sample carrier of the present disclosure.

[0041] In another preferred example, the carrier comprises a coating layer deposited on the first membrane surface layer surface or on the second substrate layer surface. This coating layer consists of a material of a third type, which is at least partially transparent for electron beam irradiation. Preferably, the coating layer material is different to the material of the first type and different to the material of the second type and has an electrical conductivity greater than the electrical conductivity of the material of the first type and an electrical conductivity greater than the electrical conductivity of the material of a second type. The coating layer material may be chosen from a list not limited to graphene, carbon, silver, gold, indium tin oxide, iron, zinc oxide or a 2D material, such as graphene, graphene oxide, hexagonal boron nitride or a monolayer of amorphous carbon.

[0042] This coating layer is deposited on the carrier such that a thin layer with greater electrical conductivity is formed on the carrier improving the contrast during EM operation, wherein electrons hitting the carrier are conducted or diverted away from the detector.

[0043] In another preferred example, at least one electrode is mounted in or on the membrane layer, which can be used to detect characteristics during EM operation of the sample, such as temperature, conductivity, resistivity, etc.

[0044] The benefits of semiconductor manufacturing allow inclusion of at least one electrode on or in the carrier with high precision, and without jeopardizing the structural integrity and rigidity of the carrier itself. These electrodes can then be used to measure characteristics of the sample subjected to EM radiation, such as the temperature, conductivity, resistivity, voltage potential, or other valuable parameters of the sample during use in an EM. This allows the user to determine the status of the sample during use and would help to improve the data quality measured in cryoEM.

[0045] SHORT DESCRIPTION OF THE FIGURES

[0046] The disclosure will now be discussed with reference to the drawing, which show in:

[0047] Fig. 1 , a sample carrier for use in electron microscopy, EM.

[0048] Fig. 2a-2b and 3a-2b, various examples of carriers with membrane recesses.

[0049] Fig. 4a-4b and 5a-5b, various examples of carriers with substrate recesses.

[0050] Fig. 6a-6b, examples of carriers with membrane protrusions.

[0051] Fig. 7a-7b-7c, examples of carriers with substrate protrusions.

[0052] Fig. 8a-8b-8c, an example of a carrier compatible with a retention ring of a carrier holder.

[0053] Fig. 9a-9b, a sample carrier with a coating layer.

[0054] Fig. 10, a sample carrier with an at least one electrode.

[0055] Fig. 11a-11 b, various examples of carriers compatible with standardized carrier holders.

[0056] DETAILED DESCRIPTION OF THE DISCLOSURE

[0057] For a proper understanding of the disclosure, in the detailed description below corresponding elements or parts of the disclosure will be denoted with identical reference numerals as used in the drawings.

[0058] In this disclosure, the wording “transparency” and “transparent” either come from the physical characteristics of the material of the carrier 1000, such that electromagnetic radiation penetrates the material and might only be attenuated to such a degree that still a measurable amount of radiation passes through. Alternatively, the transparency could be achieved by geometric design of the object, where the material properties are such that attenuation is so big that effectively 0% of the electromagnetic radiation passed through the material, but due to its geometric design with for instance openings, holes, or recesses, becomes locally / partially transparent. Since the introduction of transmission electron microscopes, TEM, the sample carriers, known as TEM grids, have remained largely the same. These grids are typically made of thin, soft metals in the range of 7 μm thickness with a diameter of around 3 mm. Newer, high-end TEMs currently are being equipped with robotic handling systems, known as autoloaders, to streamline processes and reduce the chances of contamination. For these systems, however, the standard TEM grids usually are too flexible and fragile, such that they bend, break, or deform. Therefore, thick robust frames, known as clip rings, have been designed, which improve the stiffness and rigidity, but add additional steps to the preparation protocol, such that risk of sample contamination is increased.

[0059] In Fig. 1 a carrier for use in electron microscopy, EM, is shown, denoted with reference number 1000, and comprises a substate layer formed as a rigid body, denoted with reference number 2, that is at least partially transparent for electron beam irradiation. The rigid substrate layer 2 has a first substrate layer surface 2a and a second substrate layer surface 2b. The latter is oriented opposite of the first substrate layer surface 2a. The substrate layer 1 has a substrate layer thickness denoted with T2.

[0060] The carrier further comprises a membrane layer, denoted with reference number 1 , that is at least partially transparent for electron beam irradiation. Membrane layer 1 has a first membrane layer surface 1a and a second membrane layer surface 1 b, the latter being oriented opposite of the first membrane layer surface 1a.

[0061] The membrane 1 is placed with its second membrane layer surface 1 b on the first substrate layer surface 2a of the rigid substrate layer 2.

[0062] The first membrane layer surface 1a is provided with a membrane deposit area 1z designed to receive a sample 2000. Furthermore, the membrane layer 1 has a membrane layer thickness denoted with T1. In particular, a thickness ratio of the membrane layer thickness to the substrate layer is defined as T1 :T2, such that the substrate layer thickness is larger than the membrane layer thickness; T2>T1.

[0063] The sample carrier 1000 has improved rigidity over conventional soft metal transmission electron microscope, TEM, grids due to the substrate thickness being larger than the membrane thickness and due to the substrate's material of a second type being chosen due to its rigidity and stiffness. Especially, the substrate's material is chosen to be compatible with semiconductor manufacturing processes and may be chosen as a semiconductor material.

[0064] The traditional TEM grids tend to bend, fold, break or deform when used in carrier holders, in particular robotic handling systems, such as autoloaders. Therefore, thick robust frames, known as clip rings or autogrids, are used, which complicate the preparation protocol and increase the risk of sample contamination and damage to the traditional TEM grids. According to the present disclosure, the sample carrier 1000 in fig.1 allows for simple process workflows while maintaining rigidity and being compatible with various carrier holders and autogrids.

[0065] The sample for the carrier may be a liquid containing analytes to be analyzed. All types of biomolecules (e.g. proteins, DNA, viruses, exosomes, etc.) can be used for analyzing purposes, but also complete cells or tissue are feasible. These specimens can be used in standard TEM analysis, but also more specifically in cryo- electron microscopy, cryoEM, wherein the carrier plus sample are rapidly cooled sometimes with the application of pressure, known as vitrification, such that an amorphous material is formed of the liquid sample. The present disclosure is not just limited to biological samples, other scientific fields performing analysis on vitrified liquids can benefit from the technology of the current disclosure as well such as soft- matter and material science (i.e. , battery research).

[0066] In a preferred example, the substrate layer 2 is made of a semiconductor material, such that the sample carrier 1000 can be manufactured through semiconductor microfabrication. The semiconductor substrate can then be chosen for its characteristics for further manufacturing, crystallinity, rigidity, such that a rigid carrier can be formed, which is compatible with carrier holders e.g., robotic handling systems, and / or autogrids.

[0067] In another preferred example the membrane layer 1 is also made of a semiconductor material, such that it can be deposited or grown onto the first substrate layer 2a of the substrate layer 2. Alternatively, in another example the membrane layer 1 is made of a conductive material, such that it can be deposited onto the substrate layer 2 through semiconductor fabrication techniques such as sputtering, chemical vapor deposition, wet chemistry, etc. In both examples, these semiconductor manufacturing techniques allow for precise, sometimes even atomically precise control of the thicknesses of both the substrate layer 2 and the membrane layer 1 . In case the substrate material M2 is a semiconductor material, the material is chosen from a list not limited to glass, quartz, borosilicate, calcium fluoride, germanium, silicon, silicon carbide, silicon dioxide, silicon on insulator material, silicon on sapphire material, sapphire, gallium nitride, gallium arsenide or indium phosphide.

[0068] In case the membrane material M1 is a semiconductor material, the material is chosen from a list not limited to carbon, silicon, germanium, gallium nitride, gallium phosphide, titanium dioxide, silicon-germanium, silicon-tin, silicon-nitride, siliconoxide, aluminum gallium arsenide, aluminum gallium phosphide, indium gallium arsenide, indium gallium phosphide, or any doped variants.

[0069] In case the membrane material M1 is an electrically conductive material is chosen from a list not limited to graphene, carbon, silver, gold, indium tin oxide, iron, zinc oxide or a 2D material, such as graphene, graphene oxide, hexagonal boron nitride or a monolayer of amorphous carbon.

[0070] The substrate material of the substrate layer 2 may be chosen in such a way that the material is rigid, yet at least partially transparent to electron beam irradiation. Additionally, the substrate material may be chosen for its ease of manufacturing, the ease of membrane application, and its overall cost.

[0071] The membrane material may be chosen in such a way that vitrification of the liquid sample improves the sample's amorphousness when deposited on a specific membrane material. This in term improves the image quality of the EM examination. Additionally, the membrane material is chosen such that stray electrons are conducted or diverted away from the detectors to improve image contrast. Moreover, the fact that the membrane material is manufactured through semiconductor fabrication ensures almost atomic flatness and high mechanical stability of the first membrane layer surface 1a upon which the sample 2000 is deposited in the membrane deposit area 1z.

[0072] Additionally, during the micro-fabrication process the inventors have shown that they can control the internal stress level of the membrane layer 1 , which they orient in any direction in the membrane layer 1. For instance, they can define tensile stress to ensure that the membrane remains stretched to improve the surface flatness, which in turn improves the image quality.

[0073] Various examples of carriers provided with membrane recesses, denoted with 10, 10', ... , 10" are shown in figs. 2a-2b and 3a-3b. The membrane recesses 10, 10', 10" are positioned in the membrane deposit area 1z, the latter being meant for receiving the sample 2000 to be analyzed. These membrane recess(es) have a membrane recess depth TR1. In one example, the membrane recesses 10, 10', ... , 10" have identical membrane recess depths TR1 , in another example the membrane recess depths TR1 of the membrane recesses 10, 10', ... , 10" can be different for every individual membrane recess. Additionally, the membrane recess(es) 10, in general, improve the transparency of the sample carrier 1000 for electron beam irradiation by thinning out the membrane. The transmission of the material is known through the Beer-Lambert Law to depend on the thickness of the layer.

[0074] More specifically in the example of fig. 2a the membrane recess has a membrane recess depth TR1 equal to the membrane thickness T1 , thus forming a through opening reaching from the first membrane surface 1a to the second membrane surface 1b and has a tapered recess shape.

[0075] The example in fig. 2b shows a plurality of membrane recesses 10, 10, ... , 10" distributed over the membrane deposit area 1z. These recesses are arranged in an array, which may be cubic, rectangular, hexagonal, or randomly distributed. Moreover, the membrane recess depth TR1 in fig. 2b is also equal to the membrane layer thickness T1 , also forming through openings reaching from the first membrane surface 1a to the second membrane surface 1 b. In the case of a plurality of membrane recesses being distributed over the membrane deposit area, the recesses form ridges in between them, referenced to as intermediate membrane ridges.

[0076] The figs. 3a and 3b show similar examples of membrane recesses 10, 10', ... , 10" as in figs. 2a and 2b. However, in these figures, the membrane recess depth(s) TR1 is smaller than the membrane layer thickness T1 , such that a thin part 1y of material of the membrane layer 1 is present between the bottom surface side of the membrane recesses 10, 10', ... , 10" and the first substrate surface 2a of the substrate layer 2 of the carrier.

[0077] The membrane recess(es) from the figs. 2a-2b and 3a-3b ensure that the carrier has an improved electron beam transmission. Additionally, they can have any cross-sectional shape such as a circular, elliptical, rectangular, square, or polygonal shape, dependent on the crystal structure of the membrane layer 1 , the cross-sectional size of the membrane recess(es), and the method used to make the recess(es). Moreover, the shape, seen in a cross-sectional side view of the carrier 1000 can be straight, tapered or variable.

[0078] Moreover, the typical cross-sectional size of the recess(es) in the membrane is in between 200 nm and 50 μm. Additionally, the membrane recess(es) may be oriented in an m x n array, where m and n can be different integers. An example of an array of a minimalistic configuration exhibits two recesses positioned side by side, hence as a 2x1 array. However, more versatile, enlarged carriers may implement 3x1 , 2x2, 2x3, 2x4, 3x3 or larger arrays allowing multiple sample droplets 2000, each contained in a membrane recess 10 of the array to be analyzed. For instance, in a preferred example the recesses are distributed in a hexagonal array with a cross- sectional size of 2.5 μm spaced 5 pm apart.

[0079] The figs. 4a-4b and 5a-5b show various examples of carriers with substrate recesses, being distributed over a substrate area 2z. The substrate area 2z is located on the second substrate layer surface 2b and is oriented relative to or is facing the membrane deposit area 1z. These substrate recesses 20, 20’, ... , 20" are designed to improve the transparency of the sample carrier 1000 for electron beam irradiation. In the case of a plurality of substrate recesses, ridges are formed in between the recesses, known as intermediate substrate ridges.

[0080] The substrate thickness of the substrate layer is chosen in such a way that it has a reduced thermal mass, which is advantageous during the vitrification process. With low thermal mass, the received sample on the carrier crystallizes with an amorphous crystallinity, which offers reduced scattering during imaging. Additionally, the substrate recesses also help reducing the thermal mass, and they have a second functionality, such that greater illumination angles can be accessed when the carrier is rotated along a direction in the plane of the first membrane surface.

[0081] The example in fig. 4a shows a substrate recess 20 having a tapered shaped and a substrate recess depth TR2 being equal the substrate thickness T2. Whereas the example in fig. 5a comprises a substrate recess 20 with a tapered shape, where the substrate recess depth TR2 is smaller than the substrate thickness T2.

[0082] The example in fig. 4b shows a carrier having a plurality of substrate recesses, distributed on the substrate area 1z having a straight shape and a substrate recess depth equal to the substrate thickness. Similarly, in fig. 5b, a carrier is shown having a plurality of substrate recesses, distributed on the substrate area 1z having a straight shape, but the substrate recess depth is smaller than the substrate thickness.

[0083] In one example, the substrate recesses 20, 20', ... , 20" have identical membrane recess depths TR1 , in another example (not shown) the membrane recess depths TR1 of the membrane recesses 20, 20', ... , 20" can be different for every individual membrane recess.

[0084] In both configurations of fig. 5a (and 5b), a thin part 2y of substrate layer material is present between substrate recesses 20, 20’ and the membrane layer 1 of the carrier.

[0085] These substrate recesses 20, 20',... , 20" may be arranged in an array or in an overlapping orientation in the substrate layer 2, conformal or corresponding to the array or orientation of the membrane recesses 10, 10',... , 10" in the membrane layer 1. These arrays may be cubic, rectangular, hexagonal, or randomly distributed. Additionally, the substrate recess(es) may be oriented in an k x I array, where k and I can be different integers. Similar k x I array configurations can be envisaged as the m x n arrays of the membrane recesses 10.

[0086] The substrate recess(es) from the figs. 4a-4b and 5a-5b ensure that the carrier 1000 has an improved electron beam transmission. Additionally, they can have a cross-sectional shape which is circular, elliptical, rectangular, square, or polygonal, dependent on the crystal structure of the membrane, the cross-sectional size of the substrate recess(es), and the method used to make the recess(es). Moreover, the shape, seen in a cross-sectional side view of the carrier 1000, can be straight, tapered or variable.

[0087] The typical recess size may be between 10 μm and 1.5 mm. In the case of only one recess, the recess size may be as large as 1.5 mm. Whenever a plurality of recesses is used this maximum recess size becomes less than 1 mm. Importantly, the substrate recess cross-sectional size is always larger than the membrane recess cross-sectional size.

[0088] Figs. 6a-6b shows examples of carriers with membrane protrusions, located adjacent to the membrane deposit area. In fig. 6a, a side view is shown with two membrane protrusions 11 and 11' located at adjacent sides of the membrane deposit area 1z. These two membrane protrusions 11 and 11' protect the sample 2000, when the sample carrier 1000 is pinched with grippers or pincers from the side during handling. Similarly, as shown in fig. 6b, membrane protrusion 11 can be formed as a ring-shaped membrane protrusion, surrounding the membrane deposit area 1z, and also this configuration likewise protects the sample 2000 from disturbances during the automated handling of the carrier 1000. The membrane protrusion 11 , 11' ... , 11" may thus form a protective structure with multiple protrusions or one single protrusion positioned around or circumventing the membrane deposit area 1z.

[0089] The figs. 7a-7b-7c show examples of carriers with substrate protrusions 21 , 21',... ,21" likewise located adjacent to the substrate area 2z. These substrate protrusions 21 , 21',... ,21" serve to improve the rigidity of the sample carrier 1000, especially when substate recess(es) 20 are present. A side view of a sample carrier with two substrate protrusions 21 and 2T is shown in fig. 7a, and a 3D view of a sample carrier is shown in fig. 7b having one ring-shaped substrate protrusion, surrounding the substrate area 2z.

[0090] The carrier in fig. 7c comprises a substrate protrusion 21 having a ring shape surrounding the substate area 2z. Additionally, the carrier comprises five substrate recesses 20, each having a squared shape arranged in an array similar as the 5-side of a dice. Additionally, the carrier comprises membrane recesses 10 having a circular shape arranged in a different array. The membrane recesses 10 have a smaller cross- sectional size than the substrate recesses 20 and in this particular example the recesses 10 corresponding with each substrate recess 20 are arranged in a 4x4 array. This design is shown as an example. Thanks to microfabrication techniques, the inventors have the flexibility to arbitrarily change the number of recesses, shapes, and sizes with ease and high controllability.

[0091] Moreover, figure 7c shows that the features of earlier described figures can be selectively combined due to the flexible nature of the microfabrication process. For instance, a carrier with membrane recesses and substrate recesses can be manufactured. Alternatively, a carrier with membrane recesses, substrate recesses, and substrate protrusions. Or even further, a carrier with membrane recesses, substrate recesses, substrate protrusions, and membrane protrusions. However, one could also simply remove one of these features if they are not needed for the examination of a particular sample. Therefore, the above-mentioned configurations are purely exemplary and highly depend on what sample one is to study. Fig. 8a-8b-8c show a sample carrier that is compatible with a retention ring of a carrier holder. Automatic carrier holders, such as robotic handling systems, known as autoloaders, need an intermediate component to ensure that the carrier plus the intermediate component is stiff enough for proper handling by the pincers of the robot. The example shown in figs. 8a-8b is compatible with such systems since these are currently used in the standardized workflows and processes of laboratory personnel using these carriers. The at least one substrate protrusion 21 may define a circumferential space 23 adjacent to the outer circumference 1000c of the carrier 1000. Alternatively, this circumferential space 23 adjacent to the outer circumference 1000c of the carrier 1000 may be formed by a further substrate recess. In this circumferential space 23 a retention holding spring 4 can be accommodated, for use in a carrier holder for electron microscopy 3000, see fig. 11.

[0092] The carrier in fig. 8c also comprises five substrate recesses 20 having a square cross-sectional shape arranged in an array. Moreover, the carrier comprises membrane recesses 10 having a circular cross-sectional shape arranged in another array.

[0093] Similarly to fig. 7c, fig 8c comprises multiple features of the earlier described figures 1 through 7 to indicate that the microfabrication process is highly flexible with its choice of features, which mostly depend on the type of sample to be examined.

[0094] To accommodate the retention holding ring, also known as C-clip, for autogrids, the dimensions of the circumferential space are less than 300 μm in height, more than 200 μm in width, and it should have an outer circumferential diameter when the first membrane layer surface 1a is taken as a cross-section smaller than 3.1 mm. These dimensions are defined from standardized autogrid rings and C-clips, which are commercially available, and commonly used in robotic handling systems, such as autoloaders.

[0095] Fig. 9a shows a carrier comprising a coating layer 3 deposited on the first membrane surface layer 1a the coating layer 3 consisting of a material of a third type M3 at least partially transparent for electron beam irradiation. Alternatively, a carrier comprising a coating layer 3 deposited on the second substrate surface layer 2b is shown in Fig. 9b. The coating layer material M3 is different to the semiconductor material of the first type M1 and different to the semiconductor material M1 of the second type and has an electrical conductivity greater than the electrical conductivity of the material of the first type M1 and greater than the electrical conductivity of the material of a second type M2. The coating layer material is chosen from a list not limited to graphene, carbon, silver, gold, indium tin oxide, iron, zinc oxide or a 2D material, such as graphene, graphene oxide, hexagonal boron nitride or a monolayer of amorphous carbon. The coating material ensures that a better contrast can be achieved during EM examination of the sample 2000 due to it conducting electrons not hitting the sample away from their path towards the detectors.

[0096] The coating layer 3 has a first coating layer surface 3a designed to receive a sample for electron microscopy on an area projected from the membrane deposit area 1z of the first membrane layer surface 1a onto the first coating layer surface 3a of the third layer 3.

[0097] In a first example, the coating layer also has a second coating layer surface 3b adhering to the first membrane surface 1a of the membrane layer 1 wherein the material of a third type M3 is at least partially transparent for electron beam irradiation.

[0098] In a second example, the coating layer also has a first coating layer surface 3a adhering to the second substrate surface 2b of the substrate layer 2 wherein the material of a third type M3 is at least partially transparent for electron beam irradiation.

[0099] Moreover, the coating layer can also aid the formation of amorphous ice due to its surface chemistry or crystal structure for instance or its thermal mass.

[0100] A carrier 1000 wherein at least one electrode 5 is mounted in or on the membrane layer 1 is shown in fig. 10. The inclusion of an at least one electrode onto the carrier can be done with high precision thanks to semiconductor manufacturing processes without jeopardizing the structural integrity and rigidity of the carrier itself. Then, these electrodes can then be used to measure characteristics of the sample subjected to EM radiation, such as the temperature, conductivity, resistivity, potential, light, or other valuable parameters of the sample to be studied under EM radiation. For example, such design may be considered a micro-electromechanical systems, MEMS, device.

[0101] Fig. 11 shows various examples of carriers 1000 compatible with standardized carrier holders 3000. The carrier holder 3000 has a mounting area 3000z in which any example of a carrier 1000 as disclosed in the above detailed description, can be accommodated. When placed in mounting area 3000z, the carrier 1000 can be clamped in the mounting area 3000z using the retention holding spring 4 which is pressed against the second surface side 2b of the substrate layer 2 and the inner circumferential edge 3001 of the mounting area 3000z. The carrier has an outer peripheral configuration of an elliptical, circular, quasi-circular, or at least five-sided polygonal shape, when the first membrane surface 1a is taken as cross-sectional plane. The circumferential diameter of the carrier 1000, when the first membrane layer surface, 1a, is taken as a cross-section, are conformal to the inner diameter of the carrier holder, preferably between 2.4 to 3.6 mm in diameter. Additionally, the combined thickness of all the including layers is at a maximum of 10 to 300 μm, when a direction perpendicular is taken to the first membrane surface 1a.

[0102] Fig. 11 b shows a complete assembled carrier 1000 in a standardized carrier holder 3000, which is held together with the retention holding spring 4. Moreso, fig. 11b shows that the carrier 1000 has a circumferential space for a retention holding spring, such that the second substrate layer surface 2b is flush or lower than the top of the carrier holder 3000.

[0103] The rigid sample carriers for electron microscopy disclosed in the above- mentioned figures may all be shape conformal to EM carrier holders. These holders can have slightly different dimensions, such that the carrier can be utilized for example with a robotic handling system, known as autoloader, or a clip ring with retention ring, known as autogrid, or the like. Alternatively, standard TEM sample holder rod or a rotating TEM grid holder can be used.

[0104] Reference list:

[0105] 1000 carrier

[0106] 2000 sample

[0107] 3000 carrier holder or autogrid

[0108] 3000z mounting area of carrier holder

[0109] 3001 inner circumferential edge of mounting area

[0110] 1 membrane layer

[0111] 2 substrate layer

[0112] 3 coating layer

[0113] 4 retention holding spring for electron microscopy

[0114] 5 at least one electrode

[0115] 1a first membrane layer surface

[0116] 1 b second membrane layer surface

[0117] 1y thin part of material of the membrane layer

[0118] 1z membrane deposit area

[0119] 2a first substrate layer surface

[0120] 2b second substrate layer surface

[0121] 2y thin part of material of the substrate layer

[0122] 2z substrate area (facing membrane deposit area)

[0123] M1 material of a first type

[0124] M2 material of a second type

[0125] M3 coating layer material

[0126] T1 membrane layer thickness

[0127] T2 substrate layer thickness

[0128] TR1 at least one membrane recess depth

[0129] TR2 at least one substrate recess depth

[0130] 10 at least one membrane recess

[0131] 11 at least one membrane protrusion

[0132] 12 intermediate membrane ridge between membrane recesses

[0133] 20 at least one substrate recess

[0134] 21 at least one substrate protrusion

[0135] 22 intermediate substrate ridge between substrate recesses 23 circumferential space for retention holding spring for electron microscopy

Claims

CLAIMS1. A carrier for use in transmission electron microscopy, designed to receive a sample, the carrier comprising: a membrane layer consisting of a material of a first type, the membrane layer having a membrane layer thickness, a first membrane layer surface and a second membrane layer surface opposite to the first membrane layer surface, wherein the material of the first type is at least partially transparent for electron beam irradiation, where at least part of the first membrane layer surface is designed as a membrane deposit area to receive a sample for electron microscopy; a substrate layer consisting of a material of a second type, different from the material of the first type, the material of a second type being a semiconductor material, the second layer having a first substrate layer surface and a second substrate layer surface opposite to the first substrate layer surface, wherein the material of the second type is at least partially transparent for electron beam irradiation, where the membrane layer is mounted with its second membrane layer surface to the first substrate layer surface of the substrate layer, and wherein the substrate layer is formed as a rigid body having a substrate layer thickness larger than the membrane layer thickness, the substrate layer further comprising at least one substrate recess, extending from the second substrate layer surface towards the first substrate layer surface, wherein the at least one substrate recess has a substrate recess depth and is located within or facing the membrane deposit area, and wherein the carrier has an outer peripheral dimension, when the first membrane layer is taken as cross-sectional plane, conformal to an inner peripheral dimension of a carrier holder or an autogrid for electron microscopy.

2. The carrier according to claim 1 , wherein the material of the first type is a semiconductor material, preferably wherein the semiconductor material of the first type is chosen from the list not limited to carbon, silicon, germanium, gallium nitride, gallium phosphide, titanium dioxide, silicon-germanium, silicon-tin, silicon-nitride, silicon- oxide, aluminum gallium arsenide, aluminum gallium phosphide, indium gallium arsenide, indium gallium phosphide, or any doped variants.

3. The carrier according to claim 1 , wherein the material of the first type is a conductive material, preferably wherein the conductive material of the first type is chosen from a list not limited to graphene, carbon, silver, copper, gold, indium tin oxide, iron, zinc oxide or a 2D material, such as graphene, graphene oxide, hexagonal boron nitride or a monolayer of amorphous carbon.

4. The carrier according to any of the previous claims, wherein the semiconductor material of the second type is chosen from the list not limited to germanium, silicon, silicon carbide, silicon dioxide, silicon on insulator material, silicon on sapphire material, gallium nitride, gallium arsenide or indium phosphide.

5. The carrier according to one or more of the preceding claims, wherein a thickness ratio is defined between the membrane layer thickness and the substrate layer thickness, being in the range of 1 :10 to 1 : 1 ,000,000, in particular in the range of 1 : 100 to 1 :100,000, more in particular in the range of 1 :200 to 1 :10000.

6. The carrier according to one or more of the preceding claims, wherein a maximum thickness of the substrate layer thickness ranges from 10 micrometer to 500 micrometer, in particular from 50 micrometer to 300 micrometer.

7. The carrier according to any of the preceding claims, wherein at least one substrate recess depth is smaller than or equal to the substrate layer thickness.

8. The carrier according to any of the preceding claims, wherein said at least one substrate recess comprises a plurality of substrate recesses, the plurality of substrate recesses being arranged in an array.

9. The carrier according to one or more of the preceding claims, wherein the membrane layer comprises at least one membrane recess, extending from the first membrane layer surface towards the second membrane layer surface, the at least one membrane recess having a membrane recess depth.

10. The carrier according to claim 9, wherein the membrane recess depth is smaller than or equal to the membrane layer thickness.

11. The carrier according to claim 10, wherein said at least one membrane recesses comprises a plurality of membrane recesses, the plurality of membrane recesses being arranged in an array.

12. The carrier according to any one of the claims 9-11 , wherein the at least one membrane recess is located within or facing the membrane deposit area.

13. The carrier according to one or more of the preceding claims, wherein at least one membrane protrusion is located on the first membrane layer surface adjacent to the membrane deposit area.

14. The carrier according to claim 13, wherein said at least one the membrane protrusion surrounds the membrane deposit area.

15. The carrier according to one or more of the preceding claims, wherein at least one substrate protrusion is located on the second substrate layer surface, adjacent to a substrate area on the second substrate layer surface facing the membrane deposit area.

16. The carrier according to claim 15, wherein said at least one substrate protrusion surrounds the substrate area.

17. The carrier according to any one of the claims 13-15, wherein the at least one membrane protrusion and / or the at least one substrate protrusion are formed by means of a depositing or an etching technique.

18. The carrier according to one or more of the preceding claims, further comprising a circumferential space to accommodate a retention holding spring.

19. The carrier according to one or more of the preceding claims, wherein the carrier has an outer peripheral configuration of an elliptical, circular, quasi-circular, or at least five-sided polygonal shape, when the first membrane surface is taken as cross- sectional plane.

20. The carrier according to one or more of the preceding claims, comprising a coating layer, deposited on the first membrane layer surface or on the second substrate layer surface, the coating layer consisting of a material of a third type at least partially transparent for electron beam irradiation, the coating layer material being different to the material of the first type and different to the material of the second type, and having an electrical conductivity greater than the electrical conductivity of the material of a second type.

21. The carrier according to claim 20, wherein the coating layer material being chosen from a list not limited to graphene, carbon, silver, gold, indium tin oxide, iron, zinc oxide or a 2D material, such as graphene, graphene oxide, hexagonal boron nitride or a monolayer of amorphous carbon.

22. The carrier according to one or more of the preceding claims, wherein at least one electrode is mounted in or on the membrane layer.

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