Sample holder for holding a sample carrier and corresponding arrangement

Abstract

Sample holder (100, 100a, 100b, 100c) configured for cryo-microscopy and used to hold a sample carrier (150, 150a, 150b, 150c), wherein the sample holder includes a receiving area (102) configured to receive the sample carrier (150, 150a, 150b, 150c), wherein the sample holder comprises two legs (106a, 106b), wherein the two legs (106a, 106b) are connected to each other and each leg has a free end (110a, 110b) and wherein the sample holder is configured to exert a clamping force (Fa, Fb, Fc, Fd) on the sample carrier via the two legs (106a, 106b) in order to clamp the sample carrier (150, 150a, 150b, 150c) between the two legs when the sample carrier is received in the receiving area (102), wherein the sample holder has a non-rotationally symmetrical shape with corners and a one-sided opening, wherein the sample holder is configured to be stackable.

Description

Technical field

[0001] The present invention relates essentially to a sample holder for holding a sample carrier and to a corresponding arrangement. background

[0002] In cryomicroscopy or cryo-electron microscopy, samples can be placed on sample carriers, which typically have a grid to hold the sample. The sample carrier containing the sample can then be flash-frozen to prepare the sample for microscopic examination. Handling such typically small sample carriers can be difficult. Summary

[0003] In view of the situation described above, there is a need for improvement in the handling of sample carriers. According to embodiments of the invention, a holder for receiving a sample carrier and an arrangement having the features of the independent claims are proposed. Advantageous further developments are the subject of the dependent claims and the following description.

[0004] The present invention relates to the sample carrier according to claim 1 and the arrangement according to claim 13. The dependent claims relate to preferred embodiments.

[0005] One embodiment of the invention relates to a sample holder for receiving a sample carrier; such a sample carrier can, for example, be a so-called autogrid, which will be described in more detail later. The sample holder has a receiving area configured to receive the sample carrier. This receiving area can be formed at least partially as a recess in the top side of the sample carrier. Furthermore, the sample holder comprises two legs that are connected to one another (e.g., at the corresponding ends), and each leg retains a free end. Furthermore, the sample holder is configured to exert a clamping force on the sample carrier via - and preferably also through - the two legs in order to clamp the sample carrier between the two legs when the sample carrier is received in the receiving area.This allows for easy fixation of the sample carrier in the sample holder without external force after insertion of the sample carrier into the sample holder, particularly through preloading that can be generated by the special shape or geometry of the sample holder. Furthermore, such a sample holder can inherently have a specific shape that allows for better orientation when handling the sample carrier (in the sample holder), which is particularly important for round-shaped sample carriers. The sample holder can, for example, have a square shape (in cross-section, e.g., parallel to the upper surface).

[0006] In one embodiment of the invention, a first minimum distance between the free ends of the two legs, when the sample carrier is not accommodated in the receiving area and when no external force is exerted on the sample holder, is smaller than a second minimum distance between the free ends of the two legs when the sample carrier is accommodated in the receiving area. In this way, the sample holder can exert a spring-like tensile effect to effectively clamp the sample carrier.

[0007] According to a further embodiment of the invention, the sample holder is designed to exert the clamping force on the sample carrier in such a way that the clamping force is evenly distributed between the two legs. In other words, both legs exert the same force on the sample carrier, with these forces generally being aligned with each other. This allows the sample carrier to be effectively fixed without the risk of deformation.

[0008] According to a further embodiment of the invention, the sample carrier comprises a plurality of different clamping surfaces arranged such that they contact the sample carrier or an outer edge of the sample carrier when the sample carrier is received in the receiving area. Each of the two legs comprises at least a portion of at least two of the different clamping surfaces. The sample holder is configured such that the clamping force is exerted across the different clamping surfaces. This enables an even better and more even distribution of the force acting on the sample carrier. The risk of deformation of the sample carrier is further reduced.

[0009] According to a further embodiment of the invention, the clamping surface or the various clamping surfaces are arranged such that they form part of a drum or a conical surface when the sample carrier is accommodated in the receiving area. This enables particularly effective fixation of round-shaped sample carriers such as the aforementioned so-called autogrids.

[0010] According to a further embodiment of the invention, the sample carrier comprises a plurality of different support surfaces configured to support the sample carrier (in particular perpendicular to the upper surface) when the sample carrier is received in the receiving area. At least a portion of each of at least two of the different support surfaces is arranged on each of the two legs and adjacent to the corresponding one of the different clamping surfaces. This enables better fixation of the sample carrier in the sample holder and, in particular, the determination of a specific position that the sample carrier must assume when received in the sample holder (specifically with respect to the height, perpendicular to the upper surface).

[0011] According to a further embodiment of the invention, the receiving area is at least partially formed as a recess in the top side of the sample holder. Furthermore, the sample holder is U-shaped when viewed in a plane parallel to the top side, and at least a portion of both legs forms at least a portion of the receiving area. Such a sample holder is very compact and easy to manufacture.

[0012] According to a further embodiment of the invention, the sample holder has a free space that is at least partially formed as a recess in the underside of the sample holder. The underside lies opposite the top side (and preferably also parallel to it). This free space is configured such that, when the sample carrier is received in the receiving area and clamped between the two legs, it can receive at least part of another sample carrier of the same type (and preferably also of the same size and / or shape) without clamping the other sample carrier when the other sample carrier is received in the receiving area and clamped between the two legs of another sample holder of the same type. This additional sample carrier can thus fit into the underside.This allows for easy stacking of multiple sample holders on top of each other, with the sample carriers being clamped into the sample holders and the clamped sample holders protruding from the respective top sides.

[0013] According to a further embodiment of the invention, the sample holder is designed for use in cryo-microscopy, i.e., the sample holder (or the material from which it is made) must be suitable for low (cryo) temperatures of, for example, less than 77 K. Particularly suitable materials for such a sample holder are copper-beryllium, copper, or aluminum alloys from which the sample can be made.

[0014] According to a further embodiment of the invention, the sample holder is made from a single piece. The sample holder can be manufactured, for example, by machining from a solid piece of material or by 3D printing. This improves the stability of the sample holder and reduces its overall dimensions. The aforementioned tension effect can also be effectively achieved in this way.

[0015] According to a further embodiment of the invention, the sample holder comprises one or more channels, wherein the one channel or each of the various channels connects one lateral surface of the sample holder (e.g., the top side) to another lateral surface of the sample holder (e.g., the bottom side). Such channels can improve the cooling of the sample holder in that cooling fluid (e.g., during shock freezing, as mentioned above, or during high-pressure freezing) can flow through such channels.

[0016] According to a further embodiment of the invention, the sample holder has two or more working recesses (or handling recesses), wherein the two or more working recesses are arranged on the same first side (e.g., the top side) of the sample holder. Such working recesses can be used for handling the sample holder with the aid of a handling tool, which will be described in more detail later.

[0017] According to a further embodiment of the invention, at least one of the working recesses is formed by one or at least one of the different channels. This enables dual use of the channels.

[0018] According to a further embodiment of the invention, the sample holder has two or more pins arranged on the same second side of the sample holder, wherein the second side is opposite the first side, and wherein the two or more pins are arranged to align with the two or more working recesses of another sample holder. This allows for easy stacking of multiple sample holders.

[0019] According to a further embodiment of the invention, the distance between the top surface and at least one of the various (above-mentioned) support surfaces, viewed in a direction orthogonal to the top surface, is less than 0.4 mm or less than 0.35 mm. This results in typical sample supports such as autogrids, which have a thickness of 0.4 mm, protruding from the top surface when accommodated in the sample holder. This allows microscopes, especially light microscopes, to be brought into a very close distance from the sample located on the sample support.

[0020] A further embodiment of the invention relates to an arrangement comprising the sample holder in one of the above-mentioned embodiments with the support surfaces and a sample carrier. The sample holder is configured such that, when the sample carrier is received in the receiving area and clamped between the two legs and supported by the various support surfaces, the sample carrier protrudes from the top. This allows microscopes, especially light microscopes, to be brought into a very close proximity to the sample located on the sample carrier.

[0021] Another embodiment of the invention relates to a handling tool (or working tool) for use with the sample holder in one of the above-mentioned embodiments with the working recesses. The handling tool consists of two ends that fit into the two or at least two of the plurality of working recesses of the sample holder. The handling tool is configured such that, in the absence of external force on the working tool, the two ends are spaced less than the distance between the two respective working recesses, and the handling tool is configured as a reverse forceps such that a squeezing force applied to the handling areas of the handling tool pushes the two ends apart. Such a handling tool enables easy handling of the sample holder (since the sample carrier is accommodated in the sample holder, the sample holder, not the sample carrier itself, needs to be handled).The reversible tweezer mechanism keeps the sample carrier clamped (fixed) in the sample holder when the working tool is inserted into the working recess without external force being applied.

[0022] According to a further embodiment of the invention, both ends are designed as gripping pins, each of which engages in one of the working recesses. This enables simple manufacture of the working tool. According to a further embodiment of the invention, both ends have two gripping pins, which are designed to fit into the corresponding working recesses. This enables more secure handling of sample holders with four working recesses.

[0023] Another embodiment of the invention relates to a method for inserting a sample carrier into a sample holder using a handling tool. The method comprises the following steps: exerting a squeezing force on handling areas of the working tool; inserting the two ends of the handling tool into the corresponding working recesses of the sample holder when the squeezing force is exerted; inserting the sample carrier into the receiving area of ​​the sample holder while the squeezing force is still being exerted or is being exerted again; ceasing the application of the squeezing force to the handling tool; and removing the two ends of the handling tool from the corresponding working recesses of the sample holder. In one embodiment, the sample carrier is inserted into a sample holder of one of the above-mentioned embodiments. In another embodiment, the handling tool of one of the above-mentioned embodiments is used.

[0024] A further embodiment of the invention relates to a method for removing a sample carrier from a sample holder using a handling tool. The method comprises the following steps: applying a squeezing force to handling areas of the working tool; inserting the two ends of the handling tool into the corresponding working recesses of the sample holder when the squeezing force is applied; removing the sample carrier from the receiving area of ​​the sample holder while the squeezing force is still being applied or is being applied again; ceasing the application of the squeezing force to the handling tool; and removing the two ends of the handling tool from the corresponding working recesses of the sample holder. These are simple and effective ways of handling sample carriers in conjunction with sample holders. In one embodiment, the sample carrier is removed from a sample holder of one of the above-mentioned embodiments.In another embodiment, the handling tool of one of the above-mentioned embodiments is used.

[0025] Further advantages and embodiments of the invention will become apparent from the description and the accompanying graphics.

[0026] It is pointed out that the features mentioned above and those to be described further below can be used not only in the combination specified in each case, but also in further combinations or alone, without departing from the scope of the present invention. Short description of the figures Fig. schematically shows a sample holder according to an embodiment of the invention in a perspective view; Fig. shows schematically a sample carrier for use with a sample holder in a perspective view; Fig. shows schematically an arrangement of the sample holder of Fig. and the sample carrier of Fig. ; Fig. shows schematically the arrangement of Fig. in a side view; Fig. shows schematically several of the arrangements from Fig. stacked and in cross section; Fig. shows schematically a handling tool according to an embodiment of the invention; Fig. shows schematically an enlarged view of a part of the handling tool of Fig. ; Fig. shows schematically a handling tool of another embodiment of the invention; Fig. shows schematically an enlarged view of a part of the handling tool of Fig. ; Fig. shows schematically a method according to an embodiment of the invention in a flowchart; Fig. shows schematically a method according to an embodiment of the invention in a flow chart. Detailed description

[0027] Fig. schematically shows a sample holder 100 according to an embodiment of the invention in a perspective view. The sample holder 100 is designed to receive and hold a sample carrier. Fig. shows schematically such a sample carrier 150 in a perspective view. Fig. shows schematically an arrangement of the sample holder 100 from Fig. and the sample carrier of Fig. . The following are the Fig. described in combination. For simplicity, Fig. a Cartesian coordinate system with the axes x, y and z is shown, which is also used for the Fig. described.

[0028] The sample carrier 150 will be briefly described below using Fig. which shows the sample carrier 150 with a section. The Fig. The sample carrier 150 shown is also called an autogrid and is typically used in cryo-electron microscopy.

[0029] For cryo-electron microscopy, biological samples are shock-frozen (vitrified) on grids so that they can then be examined with cryo-electron microscopes. Such grids, such as the one in Fig. The grids 152 shown consist, for example, of a metal grid (e.g. made of copper or gold) onto which a very thin carrier grid (e.g. made of carbon) can be applied.

[0030] Due to their flat shape, these grids are suitable for rapid shock freezing by rapid immersion in a cryogenic liquid, such as a propane-ethane mixture cooled with liquid nitrogen, so that amorphous ice forms (vitrification). Relevant areas can then be localized and thinned, e.g., for cryogenic transmission electron microscopy (cryo-TEM). For this purpose, the grids are usually transferred between different instruments under cryogenic conditions (77 K), a so-called cryogenic workflow. High-end transmission electron microscopes, for example, may have an automatic loading mechanism (autoloader), in which the grids are typically clamped into round frames (e.g., metal frames), such as frame 154 in Fig. The combination of sample grid 152 and (metal) frame is a sample carrier, which is called an autogrid, as in Fig. shown. In addition, a ring 158 may be used to attach the grille 152 to or within the frame 154.

[0031] It is almost impossible to use a fragile grid (also called a bare grid) like grid 152 undamaged throughout the entire cryo workflow and then clamp it into a metal frame like frame 154 for TEM or another application at the end. Because autogrids like sample carrier 150 enable secure tweezing and thus reproducible sample transfer, they have become a standard for the entire cryo workflow in cryo-electron microscopy. In a modern cryo workflow, the grids are now clamped into the metal frames immediately after vitrification.

[0032] Such sample carriers, such as autogrids, typically need to be attached to different holders in the correct orientation for each individual instrument or microscope. Due to the round geometry dictated by the autoloader, precise alignment under cryogenic conditions has proven very difficult. Repeated tweezing and long transfer times inevitably lead to increased contamination on the samples, further complicating reproducibility and high sample throughput. For new and inexperienced users, this can even lead to complete sample loss.

[0033] The inventors have recognized that such problems can be overcome by the sample holder described below. Due to its preferably cuboidal or square shape (square in cross-section), such a sample holder can also be referred to as a "square grid" holder.

[0034] The sample holder 100 comprises a receiving area 102 which is configured to receive the sample carrier 150; while in Fig. the recording area 102 is shown without the sample carrier 150, is in Fig. the sample carrier 150 is received therein. The sample holder 100 may have an upper surface (or top side) 104a (here: parallel to the xy plane) and a lower surface (or bottom side) 104b (see later Fig. ), wherein the underside 104b is opposite the top side 104a (and thus also parallel to the xy plane). The top side 104a is the side of the sample holder 100 that is intended to receive the sample carrier 150, i.e., in one embodiment, the receiving area 102 is at least partially formed as a recess in the top side 104a. The sample holder 100 generally has the shape of a cuboid or block; however, the sample holder 100 can have additional circumferential sides.

[0035] In the embodiment in the Fig. The sample holder 100 is made from a single piece; a preferred material for the sample carrier is copper. Beryllium, copper, or aluminum alloys can also be used. These materials are particularly suitable for low or cryogenic temperatures.

[0036] Furthermore, the sample holder 100 comprises two legs 106a and 106b, wherein the two legs 106a and 106b are connected to each other and each leg has a free end 110a or 110b. In the embodiment shown, the two legs 106a and 106b are integrally connected to each other because they are made from the same piece or block of material, as the entire sample holder 100 is made from a single piece of material. In one embodiment, the sample holder 100 is U-shaped when viewed in a plane parallel to the top side 104, and at least a portion of the two legs 106a and 106b forms at least a portion of the receiving area 102.

[0037] It should be noted that, in general, two or more components can also be used to form the specimen holder. For example, the two legs can consist of separate blocks of material that are connected together, for example, by soldering or welding, a screw, or another fastening device.

[0038] Furthermore, the sample holder 100 is configured to exert a clamping force on the sample carrier 150 via the two legs 106a and 106b in order to clamp the sample carrier 150 between the two legs when the sample carrier is received in the receiving area 102. This is shown in one embodiment in Fig. Four force components Fa, Fb, Fc, and Fd are shown by corresponding arrows. These force components together form the clamping force exerted by the sample holder 100 or its two legs on the sample carrier 150 or its outer edge. The outer edge 156 is in Fig. shown.

[0039] In a further embodiment, the sample holder 100 is designed to exert the clamping force on the sample carrier in such a way that the clamping force is evenly distributed between the two legs. This means that the force components Fa and Fb in combination (in vectorial and / or scalar addition) are equal in magnitude to the force components Fc and Fc in combination (in vectorial and / or scalar addition), but have the opposite orientation. This can be achieved by shaping the sample holder 100 such that the two legs 106a and 106b are mirror images of each other. The further distribution of the clamping force between the four Fig. The components shown are explained in more detail below.

[0040] In one embodiment, the sample holder 100 is designed such that a first minimum distance of d1 between the free ends 110a and 110b of the two legs 106a and 106b, when the sample carrier is not received in the receiving area 102 and when no external force is exerted on the sample holder (see Fig. , less than a second minimum distance d2 between the free ends 110a and 110b of the two legs 106a and 106b when the sample carrier 150 is received in the receiving area 102 (see Fig. ). This can be achieved by setting a diameter d3 of the receiving area 102 (which can generally be round or circular for the circular sample carrier 150, see Fig. is smaller than the (outer) diameter d4 of the sample carrier 150 (measured at the outer edge 156, see Fig. .

[0041] To do this, the two legs 106a and 106b—particularly at their respective free ends 110a and 110b—must be pulled apart slightly by an external force so that the sample carrier 150 can be placed in the receiving area 102. After the sample carrier 150 has been inserted and the external force has been removed, the sample holder 100 exerts the clamping force on the sample carrier 150 via and through the two legs 106a and 106b due to the preload and the special shape of the sample holder 100.

[0042] In one embodiment, the sample holder 100 comprises a plurality of different clamping surfaces 108a, 108b, 108c, and 108d, which are arranged to contact the sample carrier 150 or the outer edge 156 when the sample carrier 150 is received in the receiving area 102. Each of the two legs 106a and 106b comprises at least a portion of at least two of the different clamping surfaces 108a, 108b, 108c, and 108d. In the Fig. In the example shown, leg 106a includes clamping surfaces 108a and 108b, and leg 106b includes clamping surfaces 108c and 108d. Within leg 106a, clamping surfaces 108a and 108b are separated by a recess 116a, so that surfaces 108a and 108b are separated. Accordingly, clamping surfaces 108c and 108d within leg 106b are separated by a recess 116b, so that surfaces 108c and 108d are separated.

[0043] The sample holder 100 is configured so that the clamping force (with components Fa, Fb, Fc, and Fd) is exerted via the various clamping surfaces 108a, 108b, 108c, and 108d. In particular, each of the various clamping surfaces 108a, 108b, 108c, and 108d corresponds to one of the components Fa, Fb, Fc, and Fd and exerts force on one of them. These different clamping surfaces make it possible to distribute the total clamping force more evenly over the circumference or outer edge 156 of the sample carrier 150. It should be noted that more than four clamping surfaces can also be provided, e.g., three or four per leg. This can be achieved by providing the sample holder 100 with more recesses than the recesses 116a, 116b.

[0044] In one embodiment, the clamping surfaces 108a, 108b, 108c and 108d are arranged such that they form part of a drum or a conical surface when the sample carrier is received in the receiving area 102. This is particularly true for round-shaped sample carriers as in Fig. This is advantageous because the clamping force can then be distributed even better across the circumference. It should be noted that in this case, the clamping surfaces 108a, 108b, 108c, and 108d are not part of a drum or a conical surface if the sample holder is not received in the receiving area 102. This is due to the required preload and must be taken into account during the manufacture of the sample holder.

[0045] If the clamping surfaces form part of a drum surface, the total clamping force and also the individual components are (at least essentially) aligned in the xy-plane. If the clamping surfaces form part of a conical surface, the total clamping force and also the individual components also have a component in the z-direction. For example, a downward, i.e., in the negative z-direction (with the z-axis of Fig. directed component of the clamping force is exerted, which causes the sample carrier 150 to be pressed downwards.

[0046] In one embodiment, the sample holder 100 comprises a plurality of different support surfaces 112a, 112b, and 112d configured to support the sample carrier 150 when the sample carrier is received in the receiving area 102. At least a portion of each of at least two of the different support surfaces is arranged on each of the two legs 106a and 106b and adjacent to the corresponding one of the different clamping surfaces 108a, 108b, 108c, and 108d. In the Fig. In the example shown, one of the support surfaces is arranged next to one of the clamping surfaces (it should be noted that the support surface adjacent to the clamping surface 108c in Fig. is not visible). These support surfaces are in particular arranged and aligned so that they support the sample carrier 15 in a direction perpendicular to the upper side 104, as in Fig. In other words, the support surface is aligned parallel to the xy plane.

[0047] In this way, the specific position of the sample carrier 150, when it is received in the receiving area 102 and supported by the support surfaces (ie rests on it), can be arbitrarily selected in terms of height (ie in the z-direction) by selecting the position of the support surfaces accordingly (see also below Fig. ). It should be noted that the number of (different) support surfaces does not necessarily have to be identical to the number of clamping surfaces. Fewer support surfaces may also be sufficient to support the sample carrier 150. The size of the support surface may also vary, e.g., smaller than in Fig. shown.

[0048] In one embodiment, the sample holder 100 comprises channels 114a, 114b, 114c, and 114d, wherein each of the channels connects a surface of one side of the sample holder 100 to a surface of another side of the sample holder 100. In the embodiment in the Fig. Each of the channels 114a, 114b, 114c, and 114d connects the top side 104a to the bottom side 104b. The channels can be positioned at the respective corners of the sample holder 100. Furthermore, these channels can be designed, for example, as through-holes or bores in the sample holder. Cooling fluid can flow through these channels. This leads to more efficient and faster cooling (or freezing), especially when freezing under high pressure.

[0049] In one embodiment, the sample holder 100 comprises two or more working recesses, all located on the same first side of the sample holder 100, for example, on the top side 104. In the Fig. In the embodiment shown, the sample holder 100 comprises four working recesses, wherein the working recesses are formed in particular by the channels 114a, 114b, 114c, and 114d. In this way, the channels have dual use for cooling and for handling the sample holder with the aid of a handling tool, which will be described in more detail later. It should be noted that in the embodiment shown in Fig. In the embodiment shown, working recesses are also provided on the underside 104b, since the channels forming the working recesses are through holes.

[0050] Furthermore, a distance d5a between the two working recesses 114b and 114d is specified. This distance d5a can be measured, for example, between the center axes of the working recesses. This distance d5a can be measured, for example, between the center axes of the working recesses. Furthermore, a distance d5b between the two working recesses 114a and 114c is specified. This distance d5b can be measured, for example, between the center axes of the working recesses.

[0051] The distance d5b can also be measured when there is no sample carrier in the sample holder 100. Similar to the distance d1 between the two free ends 110a and 110b, the distance d5b increases to the distance d5c when a sample carrier is accommodated in the sample holder 100 (see Fig. ). It should be noted that the distance d5a does not change, or does not change significantly, when the sample carrier is inserted into the sample holder 100. For example, the sample holder 100 can be configured so that the distance 5c is equal to the distance 5a.

[0052] In summary: In the embodiment from the Fig. The sample holder 100 has a slight recess 102 with a slightly smaller diameter (d3) than the sample carrier 150 (e.g., an autogrid, d4). Sample carriers such as autogrids can be clamped into the sample holder 100 at the beginning of the cryo workflow. The special square shape of the square grid from the Fig. is a possible and preferred implementation and allows the user to easily recognize the current orientation of the sample holder. The sample carrier can be clicked directly into the sample holder. The sample carrier is then held by the spring action resulting from the lateral opening and the smaller diameter of the recess 102 (“spring clip holding principle”). This holding principle is a particularly advantageous feature of the sample holder and can be easily transferred to differently shaped holders, i.e. to other holders with a different shape (than the square or cuboid shape from the Fig. ) can also be implemented using the same spring clip mounting principle.

[0053] With such a sample holder ("square grid"), direct contact between tweezers (or other handling tools) and the sample carrier is no longer necessary when handling the sample carrier or its grid. This makes transfer much safer and faster. Minimizing transfer time also minimizes ice contamination. The relatively large material volume of the sample holder also ensures that the sample remains sufficiently cold, and thus vitrified, longer without active cooling than a sample on a bare grid or a sample holder without the sample holder. Thanks to the advantageous non-rotationally symmetric shape with corners and the one-sided opening, constant orientation is guaranteed throughout the entire cryo workflow. This design allows the sample holder to be stacked and stored safely and space-savingly, and to be easily removed under cryo conditions.

[0054] This also facilitates the reuse of samples (or sample carriers) at a later time with precise knowledge of the positions of previous measurements (as is necessary, for example, for single-particle analyses). Furthermore, after modifying the corresponding microscope holder, the sample holders are compatible with already established technology (e.g., cryocorrelative light and electron microscopy (cryoCLEM), focused ion beam (FIB), cryoliftout).

[0055] Another area of ​​application is so-called high-pressure freezers. This technology allows biological samples to be frozen by applying liquid nitrogen under very high pressure. Previously, the cold samples had to be placed on the grids after high-pressure freezing and possibly also mechanically thinned. By modifying the existing holders in high-pressure freezers, it is possible to freeze samples directly on the grids using the sample holders described here. Optional channels can be inserted into the sample holder to channel the gases flowing during the high-pressure freezing process. The simple removal of the sample holder from the high-pressure freezer enables a faster and safer workflow.

[0056] Fig. shows schematically the arrangement of Fig. , ie the sample holder 100 and the sample carrier 150, in a side view. For simplicity, a Cartesian coordinate system with the axes x, y and z is shown, which corresponds to the Fig. corresponds.

[0057] In this view, the top side 104a and the opposite bottom side 104b are shown. Furthermore, the sample carrier 150 protrudes from the top side 104a of the sample holder 100 by a distance of d6. This can be achieved by a targeted arrangement of the Fig. The distance d5 can be achieved in the z-direction by the support surfaces shown, such as 112a, 112b, and 112d. The distance d5 can be, for example, 0.1 mm or more. This arrangement makes it possible, for example, to bring light microscope objectives with a very short working distance very close to the samples.

[0058] Fig. shows schematically several of the arrangements from Fig. stacked and in cross-section. For simplicity, a Cartesian coordinate system with the axes x, y and z is shown, which corresponds to the Fig. In particular, three sample holders 100a, 100b and 100c are shown with the corresponding sample carriers 150a, 150b and 150c. Each of these sample holders 100a, 100b and 100c may correspond to the sample holder 100 shown in Figures 1a, 1c and 2, and each of these sample carriers 150a, 150b and 150c may correspond to the sample holder shown in Figures Fig. shown sample carrier 150.

[0059] For the sample holder 100a, the top side 104a and, as an example, the support surface 112d are shown. The distance between these two surfaces in the z-direction is indicated by d8. Furthermore, the thickness (or height) of the sample carrier 150 in the z-direction is indicated by d7. It can be seen that in this embodiment, the distance (thickness) d7 is smaller than the thickness d8. In particular, the sum of the distances d6 (see Fig. ) and d8 is equal to the height or thickness d7. This shows that the arrangement of the support surface 112d (and also the other support surfaces) at a certain height in the z-direction results in the sample carrier 150 protruding from the upper surface 104a when the sample carrier 150 is received in the receiving area, clamped between the two legs and supported by the various support surfaces, as in Fig. shown. The distance d6 can, for example, be selected to be less than 0.4 mm or less than 0.35 mm. A typical value for the thickness d7 is 0.4 mm. In this way, the sample carrier 150 protrudes from the upper surface 104a, as shown in Fig. shown.

[0060] In one embodiment, the sample holder comprises a free space 318 which is Fig. shown as an example for the sample holders 100a and 100c. The free space 318 is at least partially formed as a recess in the underside of the sample holder 100a. This free space 318 is configured such that when the sample carrier 150a is received in the receiving area 102 and clamped between the two legs (i.e., when the distance between the free ends of the two legs is d2, see Fig. ), can accommodate at least part of another sample carrier of the same type (and preferably also the same size and / or shape) without clamping the other sample carrier. In Fig. This other sample carrier corresponds to the sample carrier 150, which is clamped between the two legs of the sample holder 100b.

[0061] This allows the stacking of sample holders on top of each other, with the sample carriers being clamped in each of these sample holders, as in Fig. To ensure that the sample carrier clamped in the receiving area of ​​the other sample holder is not clamped in the free space of the stacked sample holder, the diameter of the free space, as shown in Fig. 3, the diameter d9 can be selected slightly larger than the diameter d3 of the receiving area. It should be noted that the free space 318 can, for example, be designed similarly to the receiving area, but on the underside instead of the top. As with the receiving area, it should also be noted here that the diameter of the free space is larger when a sample carrier is clamped in the sample holder than when it is not.

[0062] The height (or depth) of the free space 318 in the z-direction is Fig. with the distance d10. To allow stacking of sample holders, the distance d10 should be greater than the distance d6 (see Fig. ), so that the sample carrier of the lower sample holder (e.g. sample carrier 150b in Fig. ) the inner surface of the free space of the upper sample holder (e.g. sample holder 100a in Fig. ) is not touched. Note that the distance d10 can also be chosen so that it has, for example, the same value as the distance d8.

[0063] Fig. schematically shows a handling tool 400 according to an embodiment of the invention. Fig. shows schematically an enlarged view of a part of the handling tool 400 from Fig. . Fig. are described together below.

[0064] The handling tool 400 is intended for use with a sample holder such as the sample holder 100 shown in Figures 1a and 1c, having working recesses such as 114a, 114b, 114c, and 114d. The handling tool 400 consists of two ends 460 and 462 that fit into the two or at least two of the numerous working recesses of the sample holder. In one embodiment, each of the two ends 460, 462 is configured as a gripping pin 464 or 466, respectively, that fits into one of the respective working recesses. Such gripping pins can have a cylindrical shape and a diameter slightly smaller than the diameter of a working recess. This allows a gripping pin to be inserted into a corresponding recess.

[0065] The handling tool 400 is configured such that in the absence of an external force on the handling tool 400, the two ends 460 and 462 are separated by a distance which is Fig. 4b as d11. This distance d11 can be measured, for example, between the center axes of the gripping pins 464 and 466. The distance d11 is smaller than a distance between two corresponding working recesses, e.g., the one shown in Fig. shown distance d5a and / or the distance 5b. Furthermore, the handling tool 400 is configured as a reverse tweezers, so that a squeezing force such as F1 and F2 in Fig. , which is exerted on the handling areas 472 and 474 of the handling tool 400, pushes the two ends 460 and 462 away from each other.

[0066] The handling tool 400 is thus compressed to push the two ends 460 and 462 apart so that the ends or gripping pins can be inserted into the respective working recesses. The handling will be described in more detail later.

[0067] Fig. schematically shows a handling tool 500 according to another embodiment of the invention. Fig. shows schematically an enlarged view of a part of the handling tool 500 from Fig. . Fig. are described together below.

[0068] The handling tool 500 is intended for use with a sample holder such as the sample holder 100 shown in Figures 1a and 1c, having working recesses such as 114a, 114b, 114c, and 114d. The handling tool 500 consists of two ends 560 and 562 that fit into the two or at least two of the numerous working recesses of the sample holder. In one embodiment, the two ends 560 and 562 each have two gripping pins 564 and 568, and 566 and 570. Each gripping pin is designed to fit into one of the working recesses. Such gripping pins may have a cylindrical shape and a diameter slightly smaller than the diameter of a working recess. This allows a gripping pin to be inserted into a corresponding recess.

[0069] The handling tool 500 is configured such that in the absence of an external force on the handling tool 500, the two ends 560 and 562 are separated by a distance which is Fig. 5b as d12. The distance d12 can be measured, for example, between the center axes of two opposing gripping pins 568 and 570 (one at each end). Typically, this distance is the same for the other two gripping pins. The distance d12 is smaller than a distance between two corresponding opposing working recesses, e.g., the one shown in Fig. The distance d5a is, for example, equal to the distance d5c in Fig. . Furthermore, the handling tool 500 is configured as a reverse tweezers, so that a squeezing force such as F2 and F3 in Fig. , which is exerted on the handling areas 572 and 574 of the handling tool 500, pushes the two ends 560 and 562 away from each other.

[0070] The handling tool 500 is thus compressed to push the two ends 560 and 562 apart so that the ends or gripping pins can be inserted into the respective working recesses. The handling will be described in more detail later.

[0071] Fig. schematically shows a method according to an embodiment of the invention in a flow chart. The method relates to inserting a sample carrier, such as the sample carrier 150, into a sample holder, such as the sample holder 100, using a handling tool, such as the handling tool 400 or 500 described above. In a step 600, a squeezing force such as the force F1 and F2 in Fig. or the force F3 and F4 in Fig. on the handling areas of the handling tool, as described in the Fig. described. The squeezing force pushes the two ends away from each other.

[0072] In step 602, the two ends of the handling tool are inserted into the respective working recesses of the sample holder while the squeezing force is (still) being applied. Depending on the handling tool used, either the two gripping pins of the handling tool 400 or the four gripping pins of the handling tool 500 are to be inserted into the respective working recesses. Care must be taken to ensure that the squeezing force is selected such that the two ends are spaced apart enough for the gripping pins to easily fit into the working recesses. Particularly in the case of the handling tool 500, a simple way to insert the gripping pins into the working recesses is to first insert two gripping pins into the working recesses 114a and 114c, apply slightly more squeezing force, and then insert the other two gripping pins into the working recesses 114b and 114d.

[0073] In step 604, the sample carrier is then placed in the receiving area while the squeezing force is still applied or reapplied. The squeezing force should be selected so that the two legs of the sample holder, or their free ends, are sufficiently spaced apart. After inserting the sample carrier into the sample holder, the squeezing force of the handling tool is partially removed in step 606. In this way, the external force on the sample holder can be canceled. However, a certain squeezing force is still required for the next step. In step 608, the two ends of the handling tool are then removed from the respective working recesses of the sample holder.

[0074] This clamps the sample carrier into the sample holder. Further handling of the sample holder (with the sample carrier clamped in place) may involve inserting the ends of the handling tool into the respective working recesses, ensuring that the two legs of the sample holder, or their free ends, are not too far apart. This is supported by the reverse tweezer style of the processing tool.

[0075] Fig. schematically shows a method according to an embodiment of the invention in a flow chart. The method relates to the removal of a sample carrier, such as the sample carrier 150, from a sample holder, such as the sample holder 100, using a handling tool, such as the handling tool 400 or 500 described above. In a step 700, a squeezing force such as the force F1, F2 in Fig. or the force F3, F4 in Fig. on the handling areas of the handling tool, as described in the Fig. described. The squeezing force pushes the two ends away from each other.

[0076] In step 702, the two ends of the handling tool are inserted into the respective working recesses of the sample holder while the squeezing force is (still) being applied. Depending on the handling tool used, either the two gripping pins of handling tool 400 or the four gripping pins of handling tool 500 are to be inserted into the respective working recesses. Care must be taken to select the squeezing force so that the two ends are spaced apart enough for the gripping pins to easily fit into the working recesses.

[0077] In step 704, the sample carrier is then removed from the receiving area while the squeezing force is still applied or reapplied. The squeezing force should be selected so that the two legs of the sample holder, or their free ends, are sufficiently spaced apart. After the sample carrier has been removed into the sample holder, the squeezing force of the handling tool is removed in step 706. In this way, the external force on the sample holder can be canceled. However, a certain squeezing force is still required for the next step. In step 678, the two ends of the handling tool are then removed from the respective working recesses of the sample holder.

[0078] The term “and / or” as used herein includes all combinations of one or more of the items listed and may be abbreviated to “ / ”.

[0079] Although some aspects have been described in the context of a device, it is clear that these aspects also represent a description of the corresponding method, with a block or device corresponding to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block, element, or feature of a corresponding device. List of reference symbols 100, 100a, 100b, 100c sample holders 102 Recording area 104a top 104b bottom 106a, 106b Legs 108a, 108b, 108c, 108d clamping surfaces 110a, 110b free ends 112a, 112b, 112d support surfaces 114a, 114b, 114c, 114d Working recesses, channels 116a, 116b recesses 150, 150a, 150b, 150c sample carriers 152 grids 154 frames 156 outer edge 158 rings 318 open space 400, 500 handling tool 460, 462, 560, 562 endings 464, 466, 564, 566, 568, 570 gripping pins 472, 474, 572, 574 Handling areas 600-608, 700-708 procedural steps d1, d2, d5-d6, d8, d10-d12 distances d3, d4, d9 diameter d7 Thickness of the sample carrier Fa, Fb, Fc, Fd clamping force F1, F2, F3, F4 crushing force x, y, z directions

Claims

[1] Sample holder (100, 100a, 100b, 100c) configured for cryo-microscopy and serving to hold a sample carrier (150, 150a, 150b, 150c), wherein the sample holder comprises a receiving area (102) configured to receive the sample carrier (150, 150a, 150b, 150c), wherein the sample holder comprises two legs (106a, 106b), wherein the two legs (106a, 106b) are connected to each other and each leg has a free end (110a, 110b) and wherein the sample holder is configured to exert a clamping force (Fa, Fb, Fc, Fd) on the sample carrier via the two legs (106a, 106b) in order to clamp the sample carrier (150, 150a, 150b, 150c) between the two legs when the sample carrier is received in the receiving area (102), wherein the sample holder has a non-rotationally symmetric shape with corners and has a one-sided opening, wherein the sample holder is configured to be stackable. [2] The sample holder (100, 100a, 100b, 100c) of claim 1, wherein the first minimum distance (d1) between the free ends (110a, 110b) of the two legs (106a, 106b) when the sample carrier is not received in the receiving area (102) and when no external force is exerted on the sample holder is less than a second minimum distance (d2) between the free ends (110a, 110b) of the two legs (106a, 106b) when the sample carrier (150, 150a, 150b, 150c) is received in the receiving area (102). [3] The sample holder (100, 100a, 100b, 100c) according to claim 1 or 2, which is configured to exert the clamping force (Fa, Fb, Fc, Fd) on the sample carrier such that the clamping force (Fa, Fb, Fc, Fd) is evenly distributed between the two legs (106a, 106b). [4] The sample holder (100, 100a, 100b, 100c) according to one of the preceding claims, comprising a plurality of different clamping surfaces (108a, 108b, 108c, 108d) arranged to contact the sample carrier (150, 150a, 150b, 150c) or an outer edge (156) of the sample carrier (150, 150a, 150b, 150c) when the sample carrier (150, 150a, 150b, 150c) is received in the receiving area (102), wherein each of the two legs (106a, 106b) comprises at least a part of at least two of the different clamping surfaces (108a, 108b, 108c, 108d) and wherein the sample holder (100, 100a, 100b, 100c) is configured such that the clamping force (with the components Fa, Fb, Fc, Fd) is exerted via the various clamping surfaces (108a, 108b, 108c, 108d). [5] The sample holder (100, 100a, 100b, 100c) of claim 4, wherein the clamping surfaces 108a, 108b, 108c, 108d are arranged to form part of a drum or a conical surface when the sample carrier is received in the receiving area (102). [6] The sample holder (100, 100a, 100b, 100c) of claims 4 and 5 comprises various support surfaces (112a, 112b, 112d) configured to support the sample carrier (150, 150a, 150b, 150c) when the sample carrier is received in the receiving area (102), wherein at least a portion of each of at least two of the various support surfaces is arranged on each of the two legs (106a, 106b) and adjacent to the corresponding one of the various clamping surfaces (108a, 108b, 108c, 108d). [7] The sample holder (100, 100a, 100b, 100c) according to one of the preceding claims, wherein the receiving area (102) is at least partially formed as a recess in the upper side (104a) of the sample holder (100), wherein the sample holder (100, 100a, 100b, 100c) is U-shaped when viewed in a plane parallel to the upper surface (104), and wherein at least a part of each of the two legs (106a, 106b) forms at least a part of the receiving area (102). [8] The sample holder (100, 100a, 100b, 100c) according to claim 7, comprising a free space, wherein the free space is at least partially formed as a recess in the underside of the sample holder (100, 100a, 100b, 100c), wherein the underside is opposite the upper side (104a), wherein the free space is configured such that, when the sample carrier (150, 150a, 150b, 150c) is received in the receiving area (102) and clamped between the two legs (106a, 106b), it receives at least a part of another sample carrier of the same type without clamping the other sample carrier when the other sample carrier is received in the receiving area (102) and clamped between the two legs (106a, 106b) of another sample holder of the same type, wherein the Diameter of the free space (d9) is larger than the diameter (d3) of the receiving area. [9] The sample holders (100, 100a, 100b, 100c) of any preceding claim, comprising one or more channels (114a, 114b, 114c, 114d), wherein the one channel or each of the several channels connects one lateral surface of the sample holder (100, 100a, 100b, 100c) to another lateral surface of the sample holder (100, 100a, 100b, 100c). [10] The sample holder (100, 100a, 100b, 100c) according to any one of the preceding claims, comprising two or more working recesses (114a, 114b, 114c, 114d), wherein the two or more working recesses are arranged on the same first side of the sample holder (100, 100a, 100b, 100c). [11] The sample holders (100, 100a, 100b, 100c) according to claims 9 and 10, wherein at least one of the working recesses (114a, 114b, 114c, 114d) is formed by the one or at least one of the various channels (114a, 114b, 114c, 114d). [12] The sample holders (100, 100a, 100b, 100c) according to any one of the preceding claims, wherein reference is made at least to claims 6 and 7, wherein the distance (d8) between the top side (104a) and at least one of the different support surfaces (112a, 112b, 112d), seen in a direction (z) orthogonal to the top side (104a), is less than 0.4 mm or less than 0.35 mm. [13] The assembly comprising the sample holder (100, 100a, 100b, 100c) according to any one of the preceding claims, wherein reference is made to at least claims 6 and 7, and a sample carrier (150), wherein the sample holder (100, 100a, 100b, 100c) is designed such that when the sample carrier (150) is received in the receiving area (102), clamped between the two legs (106a, 106b) and supported by the various support surfaces (112a, 112b, 112d), the sample carrier protrudes from the top side (104a).

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