WORKSTATION, PREPARATION STATION AND METHOD FOR MANIPULATING AN ELECTRON MICROSCOPY GRID ASSEMBLY

Abstract

The invention relates to a workstation (1), a preparation station (2) and a method for manipulating an electron microscopy grid assembly (3). The workstation (1) comprises a first compartment (101), a first gas inlet (102) for generating an overpressure in the first compartment (101), a first glove (104) and a second glove (105), each being fixed in a respective opening (106, 107) of the workstation (1), wherein the first glove (104) and the second glove (105) are movable in the first compartment (101) to manipulate objects in the first compartment (101), wherein the workstation (1) comprises a port (109) for providing a transfer device (4) for an electron microscopy grid assembly (3) in the first compartment (101). The preparation station (2) comprises a coolant reservoir (201, 202), a first part (210) configured to hold a shuttle (6) for holding an electron microscopy grid assembly (3) in a fixed orientation, wherein the preparation station (2) is configured such that the first part (210) is submergable in the cryogenic coolant when the coolant reservoir (201, 202) contains the cryogenic coolant.

Claims

1. A workstation (1) for manipulating an electron microscopy grid assembly (3), comprising a first compartment (101), a first gas inlet (102) for providing a gas flow (G) of a dry gas, particularly dry nitrogen gas, from a gas reservoir (103) into the first compartment (101), wherein the workstation (1) is configured such that an overpressure can be generated in the first compartment (101) relative to an exterior of the workstation (1) by the gas flow (G), a first glove (104) and a second glove (105), each being fixed in a respective opening (106, 107) of the workstation (1) wherein the first glove (104) and the second glove (105) are configured such that a respective hand of a user may be inserted into the first glove (104) or second glove (105) from the exterior, wherein the first glove (104) and the second glove (105) are movable in the first compartment (101) to manipulate objects in the first compartment (101) using the respective hand of the user, characterized in that the workstation (1) comprises a port (109) for providing a transfer device (4) for an electron microscopy grid assembly (3) in the first compartment (101) from the exterior, to insert the electron microscopy grid assembly (3) into the transfer device (4) or remove the electron microscopy grid assembly (3) from the transfer device (4) in the first compartment (101).

2. The workstation (1) according to claim 1, characterized in that the first glove (104) and the second glove (105) each comprise a plurality of finger sections (110), wherein each finger section (110) of the first glove (104) and the second glove (105) is configured to receive a respective finger of a respective hand of the user, wherein each finger section (110) of the first glove (104) and the second glove (105) comprises a respective hole (112) at a tip (111) of the respective finger section (110) wherein the respective hole (112) is configured to allow the respective finger to extend into the first compartment (101) through the respective hole (112).

3. The workstation (1) according to claim 1, characterized in that the workstation (1) comprises a coolant tank (113) for storing a cryogenic coolant, particularly liquid nitrogen, wherein the workstation (1) comprises a dispensing mechanism (114) configured to dispense the cryogenic coolant from the coolant tank (113) through a coolant outlet (115) into the first compartment (101).

4. The workstation (1) according to claim 1, characterized in that the workstation (1) comprises a first guide rail (116) for slidably moving a preparation station (2) for manipulating an electron microscopy grid assembly (3), particularly the preparation station (2) in the first compartment (101) along a first direction, particularly wherein the workstation (1) comprises a second guide rail (117) for slidably moving the preparation station (2) in the first compartment (101) along a second direction non-parallel, particularly perpendicular, to the first direction.

5. The workstation (1) according to claim 1, characterized in that the workstation (1) comprises a loading lock (118) enclosing a second compartment (119), wherein the loading lock (118) comprises a first gate (120) connecting the second compartment (119) to the first compartment (101) of the workstation (1), and wherein the loading lock (118) comprises a second gate (121) connecting the second compartment (119) to the exterior, wherein the workstation (1) comprises a second gas inlet (122) configured to provide a gas flow (G) of the dry gas into the second compartment (119), such that an overpressure can be generated in the second compartment (119) relative to the exterior by the gas flow (G).

6. The workstation (1) according to claim 1, characterized in that the workstation (1) comprises at least one heating element (123) for heating the first compartment (101), particularly for heating a manipulation tool for manipulating an electron microcopy grid assembly (3) to evaporate residual water from the manipulation tool.

7. The workstation (1) according to claim 1, characterized in that the workstation (1) comprises a pump inlet (125), wherein the pump inlet (125) is configured to be connected to a vacuum pump (124), particularly wherein the vacuum pump (124) is configured to evacuate a chamber (41) of the transfer device (4) when the transfer device (4) is arranged in the port (109) of the workstation (1).

8. A preparation station (2) for manipulating an electron microscopy grid assembly (3) comprising a coolant reservoir (201, 202) for receiving a cryogenic coolant, a first part (210) configured to be inserted into the coolant reservoir (201, 202, such that an upper surface (211) of the first part (210) is accessible from above the coolant reservoir (201, 202), wherein the first part (210) is configured to hold a shuttle (6) for holding an electron microscopy grid assembly (3) in a fixed orientation, particularly wherein the shuttle (6) is configured to be inserted into a preparation device, particularly a focused ion beam device for thinning of a sample (S) arranged on the electron microscopy grid assembly (3), wherein the preparation station (2) is configured such that the first part (210) is submergable in the cryogenic coolant when the coolant reservoir (201, 202) contains the cryogenic coolant.

9. The preparation station (2) according to claim 8, characterized in that the preparation station (2) comprises a removable holder (240) comprising at least one holding element (241a, 241b) for holding the shuttle (6) in a fixed orientation, wherein the first part (210) comprises a first recess (212) for receiving the removable holder (240).

10. The preparation station (2) according to claim 8, characterized in that the preparation station (2) comprises a second part (220) configured to be inserted into the coolant reservoir (201, 202), such that an upper surface (221) of the second part (220) is accessible from above the coolant reservoir (201, 202), wherein the second part (220) is configured to hold the shuttle (6) in a tilted orientation in respect of the upper surface (221) of the second part (220), wherein the preparation station (2) is configured such that the second part (220) is submergable in the cryogenic coolant when the coolant reservoir (201, 202) contains the cryogenic coolant, wherein particularly the second part (220) comprises a second recess (222) for receiving the removable holder (240), wherein the second recess (222) comprises a surface (223) which is tilted in respect of the upper surface (221) of the second part (220), such that the shuttle (6) is in said tilted orientation when the shuttle (6) is held by the at least one holding element (241a, 241b) of the holder (240).

11. The preparation station (2) according to claim 8, characterized in that the preparation station (2) comprises a third part (230) configured to be inserted in the coolant reservoir (201, 202), such that an upper surface (231) of the third part (230) is accessible from above the coolant reservoir (201, 202), wherein the third part (230) is configured to hold a cassette (7) for holding the electron microscopy grid assembly (3), wherein the cassette (7) is configured to be inserted into an imaging device, particularly a cryo-electron microscope, to image a sample on the electron microscopy grid assembly (3), wherein particularly the third part (230) comprises a third recess (232) for receiving the cassette (7), wherein more particularly the third recess (232) comprises a surface (233) which is tilted in respect of the upper surface (231) of the third part (230), such that the cassette (7) is in a tilted orientation with respect to the upper surface (231) of the third part (230) when the cassette (7) is received in the third recess (232).

12. The preparation station (2) according to claim 8, characterized in that the first part (210), the second part (220) and/or the third part (230) comprises a. at least one alignment groove (213) for receiving an electron microscopy grid assembly (3) in a tilted orientation with respect to an upper surface (211) of the respective first, second or third part (210, 220, 230) and aligning the electron microscopy grid assembly (3) in the alignment groove (213), wherein the alignment groove (213) is configured such that the electron microcopy grid assembly (3) protrudes above the upper surface (211) of the first part (210) from the alignment groove (213) when the electron microscopy grid assembly (3) is arranged in the alignment groove (213), or b. at least one alignment platform (235) for aligning the electron microscopy grid assembly (3) on the alignment platform (235), wherein particularly the alignment platform (235) comprises a surface (236) which is tilted in respect of the upper surface (211, 221, 231) of the respective first, second or third part (210, 220, 230), wherein particularly the surface (236) of the alignment platform (235) comprises a groove (237a) for receiving the electron microscopy grid assembly (3) and/or a slot (237) for receiving a manipulation tool, such that the electron microscopy grid assembly (3) can be aligned by the manipulation tool, when the electron microscopy grid assembly (3) is inserted in the groove (237a) and the manipulation tool is inserted in the slot (237).

13. The preparation station (2) according to claim 8, characterized in that the preparation station (2) comprises an adapter (250), particularly a removable adapter (250), for holding a transfer receptacle (8) for receiving a cassette (7) for holding the electron microscopy grid assembly (3), wherein the transfer receptacle (8) containing the cassette (7) is configured to be inserted into an imaging device, particularly a cryo-electron microscope, to image a sample on the electron microscopy grid assembly (3).

14. A method for manipulating an electron microscopy grid assembly (3) comprising the steps of a. providing a workstation (1) according to claim 1, b. providing a gas flow (G) of a dry gas to generate an overpressure in the first compartment (101) of the workstation (1) in respect of the exterior of the workstation (1), c. providing in the first compartment (101) a sample (S) arranged on a grid (31) of an electron microscopy grid assembly (3), d. arranging and fixing the electron microscopy grid assembly (3) on a shuttle (6) in a fixed orientation, particularly by means of a preparation station (2), e. providing a transfer device (4) for an electron microscopy grid assembly (3) in the first compartment (101) by means of the port (109) of the workstation (1), f. connecting the transfer device (4) to the shuttle (6) and/or inserting the shuttle (6) into a chamber (41) of the transfer device (4), g. inserting the shuttle (6) into a preparation device, particularly a focused ion beam device, by means of the transfer device (4), h. preparing the sample on the grid (31) of the electron microscopy grid assembly (3), particularly thinning the sample arranged on the grid (31) of the electron microscopy grid assembly (3) by a focused ion beam at cryogenic temperature by means of the focused ion beam device.

15. A method for manipulating an electron microscopy grid assembly (3) comprising the steps of a. providing a workstation (1) according to claim 1, b. providing a gas flow (G) of a dry gas to generate an overpressure in the first compartment (101) of the workstation (1) in respect of the exterior of the workstation (1), c. providing in the first compartment (101) of the workstation (1), a transfer device (4) comprising a shuttle (6) and an electron microscopy grid assembly (3) comprising a sample (S), the electron microscopy grid assembly (3) being held by the shuttle (6), wherein the transfer device (4) is provided in the first compartment (101) by means of the port (109) of the workstation (1), d. removing the electron microscopy grid assembly (3) from the shuttle (6) in the first compartment (101), particularly by the preparation station (2), e. inserting the electron microscopy grid assembly (3) into a cassette (7) for holding an electron microscopy grid assembly (3) in the first compartment (101), particularly by a preparation station (2), f. inserting the cassette (7) into a transfer receptacle (8) in the first compartment (101), g. removing the transfer receptacle (8) from the workstation (1), h. inserting the transfer receptacle (8) into an imaging device, particularly a cryo-electron microscope, i. imaging the sample (S) on the electron microscopy grid assembly (3) by means of the imaging device.

16. A method for transferring and handling of a cryo electron microscopy sample, wherein the cryo electron microscopy sample is handled in an anhydrous environment or in an environment with a humidity of less than 1%, wherein particularly the environment is dry nitrogen or a vacuum wherein all tools, particularly including a preparation station, used for handling the sample and/or preprocessing the sample, are placed in said anhydrous environment or said environment with a humidity of less than 1%; and wherein the cryo electron microscopy sample is transferred to a microscope in an anhydrous environment or in an environment with a humidity of less than 1%.

17. The method according to claim 16, wherein the preparation station and/or said tools are heated to a temperature of at least 40 degrees in order to dry the preparation station and/or said tools.

18. The method according to claim 16, wherein the sample is frozen to a vitreous state using a freezer that is placed in an anhydrous environment or in an environment with a humidity of less than 1%.

19. The method according to claim 16, wherein the sample is handled by a robotic arm.

Description

[0172] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0173] FIG. 1 shows a workstation according to the invention in a perspective view;

[0174] FIG. 2 shows a front view of the workstation according to FIG. 1, without the front panel or window;

[0175] FIG. 3 shows a further front view of the workstation according to FIGS. 1 and 2. Comprising the front panel or window and a schematic representation of a first glove and a second glove of the workstation;

[0176] FIG. 4 shows examples of an EM grid assembly (A), a shuttle for holding the EM grid assembly (B) and a cassette for holding the EM grid assembly (C), which may be used in the method according to the invention;

[0177] FIG. 5 shows an example of a transfer device for an EM grid assembly;

[0178] FIG. 6 shows a perspective view of a preparation station according to the invention;

[0179] FIG. 7 shows a perspective view of a preparation station according to the invention connected to guide rails of the workstation according to the invention;

[0180] FIG. 8 depicts a perspective view of a first coolant reservoir of a preparation station according to the invention;

[0181] FIG. 9 shows a top view (A) and a perspective view (B) of a second coolant reservoir of the preparation station according to the invention;

[0182] FIG. 10 depicts a perspective view of a removable holder for a shuttle for holding an EM grid assembly according to the invention;

[0183] FIG. 11 shows top views (A, B) and perspective views (C, D) of the first part of the preparation station according to the invention, with (B, D) and without (A, C) the removable holder according to FIG. 10;

[0184] FIG. 12 shows top views (A, B) and perspective views (C, D) of the second part of the preparation station according to the invention, with (B, D) and without (A, C) the removable holder according to FIG. 10;

[0185] FIG. 13 shows top views (A, B) and perspective views (C, D) of the third part of the preparation station according to the invention, with (B, D) and without (A, C) the removable holder according to FIG. 10, and FIG. 13 E shows a detail of the alignment platform of the third part;

[0186] FIG. 14 shows perspective views of an adapter of the preparation station according to the invention with (B) and without (A) a transfer receptacle held by the adapter.

[0187] FIG. 1-3 show an embodiment of the workstation 1 according to the invention. The workstation 1 is particularly suited for manipulation of an EM grid assembly 3 (see FIG. 4A), more particularly for placing an EM grid assembly 3 containing a vitrified sample S on a shuttle 6 (see FIG. 4B), transferring the EM grid assembly 3 to a transfer device 4 (see FIG. 5) for inserting the EM grid assembly 3 into a preparation device, such as a cryo-focused ion beam (FIB) device and/or for transferring the EM grid assembly 3 from the shuttle 6 to a cassette 7 (see FIG. 4C) to insert the cassette 7 into an imaging device, such as a cryo-electron microscope to image the sample S.

[0188] FIG. 1 is a perspective view of the workstation 1, where the front panel or window 108 (see FIG. 3) has been omitted for better visibility of the internal components of the workstation 1 The workstation 1 comprises a first compartment 101 enclosed by walls and said front panel 108 (see FIG. 3). The workstation 1 further comprises a first gas inlet 102 branching into the first compartment 101, wherein the first gas inlet 102 is connected to a gas reservoir 103, such that a gas flow G of a dry gas, particularly dry nitrogen gas, into the first compartment 101 can be provided to generate an overpressure in the first compartment 101 with respect to the exterior of the workstation 1. This overpressure of the dry gas prevents ice contamination of the sample S on the EM grid assembly 3, thereby allowing longer preparation times.

[0189] FIG. 1 further shows a port 109 for inserting at least a part of a transfer device 4 into the first compartment 101 and FIG. 1 shows a preparation station 2 according to the invention, which is arranged in the first compartment 101 (see FIG. 6-13 for details). The transfer device 4 can be arranged with respect to the preparation station 2, particularly to connect a shuttle 6 carrying the EM grid assembly 3 to the transfer device 4 and insert the shuttle 6 into the transfer device 4 or to remove the shuttle 6 from the transfer device 4. In the example shown in FIG. 1, the port 109 is configured such that the transfer device 4 extends from the port 109 downwards towards the preparation station 2 to conveniently connect the shuttle 6 to the transfer device 4.

[0190] FIG. 3 shows the workstation 1 in a front view depicting the front panel or window 108, which is particularly transparent, e.g., made from plexiglass or similar material, such that the first compartment 101 can be viewed from outside through the front panel or window 108. The front panel 108 comprises a first opening 106 and a second opening 107, wherein a first glove 104 is connected to the first opening 106, and a second glove 105 is connected to the second opening 107 in a similar manner to glove boxes known from the prior art, i.e. the respective glove 104, 105 is connected around the entire circumference of the first or second opening 106, 107 in a gas tight manner. The first glove 104 is configured for insertion of a left hand of a user and the second glove 105 is configured for insertion of a right hand of the user, wherein the fingers of the hand of the user are to be inserted in the fingers sections 110 of the first and second glove 104, 105. The first and second gloves 104, 105 may be moved around in the first compartment 101, e.g. to manipulate the EM grid assembly 3 in a similar manner to conventional glove boxes known from the prior art. However, according to an embodiment of the invention which is shown in FIG. 3, the finger sections 110 may comprise holes 112 at their tips 111, such that the first glove 104 and the second glove 105 are open towards the first compartment 101. Thereby, the fingers of the user can be extended through the holes 112 to manipulate objects in the first compartment 101 without being obstructed by the thick material which is commonly used for gloves of glove boxes according to the prior art.

[0191] As can be best observed in FIG. 2, the workstation 1 further comprises a coolant tank 113 for storing a cryogenic coolant such as liquid nitrogen, the coolant tank 113 being arranged within the first compartment 101 and comprising a coolant inlet 126 configured to fill the coolant tank 113 with cryogenic coolant from the exterior, wherein the coolant inlet 126 is arranged outside of the first compartment 101 on top of the workstation 1.

[0192] The coolant tank 113 comprises a coolant outlet 115 in the first compartment 101 and a dispensing mechanism 114 configured to open the coolant outlet 115 and dispense cryogenic coolant into the first compartment 101. In particular, the coolant tank 113 may be used to dispense cryogenic coolant into reservoirs of the preparation station 2, or transfer receptacle 8 (see FIG. 14) inside the first compartment 101.

[0193] Furthermore, as best illustrated in FIG. 2, the workstation 1 comprises a loading lock 118 comprising a second compartment 119 as well as a first gate 120 connecting the first compartment 101 and the second compartment 119 and a second gate 121 connecting the second compartment 119 to the exterior of the workstation 1. In the embodiment shown in FIG. 2, the first compartment 101 and the second compartment 119 are connected by a second gas inlet 122, which is used to provide an overpressure of the dry gas in the second compartment 119. Alternatively, the second compartment 119 may be directly connected to the gas reservoir 103 or another gas reservoir to provide the overpressure and the second compartment 119. The loading lock 118 may be used to transport components into the first compartment 101 or remove components from the first compartment 101 without releasing the overpressure and without transporting moisture into the dry atmosphere of the first compartment 101.

[0194] The workstation 1 may further comprise one or several heating elements 123 (see FIG. 1), which can be used to heat the first compartment 101 or specified areas of the first compartment 101, for instance, to evaporate residual moisture on manipulation tools or other components. The heating elements 123 are controlled by a control device 123a.

[0195] Referring to FIG. 2, the workstation 1 may further comprise a pump inlet 125 connected to a vacuum pump 124. For example, the pump inlet 125 may be connected to a valve 45 of the transfer device 4 to evacuate the chamber 41 of the transfer device 4 (see FIG. 5). The pump inlet 125 may be arranged outside of the first compartment 101 (as depicted in FIG. 2) or alternatively inside of the first compartment 101 to evacuate an alternative transfer device which is not provided in the first compartment through the port 109, but for example through the loading lock 118.

[0196] FIG. 4A-C show different components used in the method according to the present invention. FIG. 4A depicts an EM grid assembly 3 comprising an electron microscopy grid 31, e.g., from copper coated by a carbon material, and a sample S disposed on the grid 31. FIG. 4B shows a shuttle 6 configured to be inserted in a preparation device, such as a cryogenic focused ion beam milling device. The shuttle 6 comprises a slot 61 for receiving an EM grid assembly 3, such as shown in FIG. 4A. FIG. 4C illustrates schematically a cassette 7 for insertion into an imaging device, particularly a cryo-electron microscope. The cassette 7 comprises several slots 71 for receiving an EM grid assembly 3, such as shown in FIG. 4A.

[0197] FIG. 5 shows an example of a transfer device 4 for an EM grid assembly 3 (such as shown in FIG. 4A). The transfer device 4 comprises an elongated outer rod 42, in which particularly an inner rod comprising a tip with a connection mechanism for shuttle 6 (see FIG. 4B) is slidably arranged (not shown). The transfer device 4 further comprises a chamber 41 for storing the shuttle 6 with the EM grid assembly 3 arranged thereon. In particular, a thermally conductive block, for example a copper block disposed in the chamber 41 is in thermal contact with the coolant reservoir 43, such that the shuttle 6 may be cooled to cryogenic temperatures when a cryogenic coolant is contained in the coolant reservoir 43 and the shuttle 6 is in a parking position in contact with the block. In addition, the chamber 41 comprises a window 44 for viewing the shuttle 6 in the chamber 41. A valve 45 of the transfer device 4 can be used to provide a vacuum in the chamber 41, e.g. by connecting a vacuum pump 124 to a port of the valve 45. Finally, the transfer device 4 comprises an adapter 46 for connecting the transfer device 4 to the port 109 of the workstation 1, and to a port of a preparation device, e.g., the cryo-FIB device.

[0198] FIGS. 6 to 14 depict different components of the preparation station 2 according to the invention. The preparation station 2 may be used in the first compartment 101 of the workstation 1 according to the invention or separately from the workstation 1.

[0199] FIG. 6 depicts a perspective view of the assembled preparation station 2, showing the first coolant reservoir 201 for containing a cryogenic coolant such as liquid nitrogen and tool holders 203 configured to hold respective manipulation tools, such that they are submerged in the cryogenic coolant to cool the manipulation tools prior to manipulating the EM grid assembly 3 at cryogenic temperatures. The first coolant reservoir 201 is resting on four feet 260, each being configured to be fixed to a first guide rail 116 and/or a second guide rail 117 of the workstation 1, when the preparation station 2 is arranged in the first compartment 101 of the workstation 1 (see FIG. 7).

[0200] A collar 201b is further arranged on top of the first coolant reservoir 201 to thermally isolate the first coolant reservoir 201 and protect the hands of a user manipulating the EM grid assembly 3 using the preparation station 2. In particular, an atmosphere of evaporated cryogenic coolant, which is almost free from contaminants forms below the collar 201b and protects the sample from contamination. In particular, the collar 201b may be applied when the preparation station 2 is used outside of the workstation 1.

[0201] FIG. 6 further shows the port 109 of the workstation 1 with a tube 47 extending from the opening of the port 109 towards the preparation station 2. The optional tube 47 additionally protects the sample from contamination during transfer to the transfer device 4. In particular, the tube 47 is constantly evacuated to provide an environment substantially free from contaminants. The tube 47 be applied when the preparation station 2 is used in the first compartment 101 of the workstation 1 (see FIG. 7) or outside of the workstation 1. An adapter 250 is attached to the preparation station 2 on one side of the first coolant reservoir 201, the adapter 250 holding a transfer receptacle 8 configured to receive a cassette 7 for holding the EM grid assembly 3 and configured to be inserted into an imaging device, such as a cryo-electron microscope.

[0202] FIG. 7 is a view of the assembled preparation station 2 arranged inside of the first compartment 101 of the workstation 1 according to the invention (see FIG. 1). The feet 260 of the preparation station 2 are slidably connected to two parallel first guide rails 116 of the workstation 1 which are fixed on a bottom plate of the workstation 1, such that the preparation station 2 can be moved parallel to the window 108 (see FIG. 3) in the first compartment 101 of the workstation 1. The first guide rails 116 are further slidably connected to two second guide rails 117 of the workstation 1, which are arranged perpendicular to the first guide rails 116. Thereby, the first guide rails 116 together with the preparation station 2 can be slidably moved along the second guide rails 117 perpendicular to the window 108. In this manner, the preparation station 2 can be conveniently moved to a desired location in the first compartment 101 of the workstation 1.

[0203] FIG. 7 further shows a first part 210, a second part 220 and a third part 230 of the preparation station 2 arranged in a second coolant reservoir 202 (see FIG. 9) disposed within the first coolant reservoir 201.

[0204] FIG. 8 is a perspective detailed view of the first coolant reservoir 201 without the other components of the preparation station 2. A first slot 204a and a second slot 204b for receiving the adapter 250 for holding a transfer receptacle 8 (see FIG. 14) are arranged on opposite sides of the first coolant reservoir 201. In this manner, the adapter 250 may be arranged on either side of the first coolant reservoir 201, particularly as required by left and right-handed operators. The slots 204a, 204b are each formed by opposing latches 205. FIG. 8 further shows an opening 201a for providing cryogenic coolant in the first coolant reservoir 201.

[0205] In particular, the first reservoir 201 comprises an inner part consisting of a thermally isolating material and an outer part (e.g. an outer frame), the outer part being particularly formed from a plastic material (the inner part is not shown). In particular, the inner part is configured to contain the cryogenic coolant and thermally isolate the outer part from the cryogenic coolant contained in the inner part, more particularly such that the outer part remains at ambient temperature when the cryogenic coolant is contained in the inner part.

[0206] FIG. 9 depicts in a top view (A) and a perspective view (B) a second coolant reservoir 202 configured to be inserted in the first coolant reservoir 201. The second coolant reservoir 202 comprises a through-hole 209 configured such that cryogenic coolant filled into the second coolant reservoir 202 enters the first coolant reservoir 201 via the through-hole 209, when the second coolant reservoir 202 is placed within the first coolant reservoir 201. As shown in FIG. 9, the second coolant reservoir 202 further comprises a first slot 207a, a second slot 207b and a third slot 207c, configured to receive the first part 210, the second part 220 and the third part 230, respectively, particularly in any desired order or arrangement. The slots 207a, 207b and 207c may optionally contain fourth slots 208, particularly four fourth slots 208 each, which are arranged in a circle (see FIG. 9A). The fourth slots 208 are particularly configured to receive an additional module such as a clipping station configured for clipping of electron microscopy grids. Furthermore, FIG. 9 shows tool slots 206 configured to receive respective manipulation tools when the manipulation tools are received by the tool holders 203 of the first coolant reservoir 201.

[0207] FIG. 10 depicts a removable holder 240 configured to receive the shuttle 6 (see FIG. 4B). The removable holder 240 comprises a first holding element 241a and a second holding element 241b for holding the shuttle 6 in a fixed orientation. In addition, the holder 240 comprises a first groove 242 comprising a protrusion 243 arranged in the first groove 242. The protrusion 243 is configured such that the removable holder 240 can be gripped by a gripping tool by inserting the gripping tool into the first groove 242 and gripping the protrusion 243.

[0208] FIG. 11A-D show a first part 210 of the preparation station 2 according to the invention as top views (A-B) and perspective views (C-D). The first part 210 comprises an upper surface 211 and a first recess 212 extending parallel to the upper surface 211, wherein the first recess 212 is configured to receive the removable holder 240 (FIGS. 11A and C show the first part 210 without the removable holder 240, and FIGS. 11 B and D show the first part 210 with the removable holder 240 arranged in the first recess 212).

[0209] The first part 210 further comprises a first hole 214a and a second hole 214b configured to each receive a respective screw to fix the removable holder 240 in the first recess 212. Furthermore, the first part 210 comprises alignment grooves 213a, 213b on either side of the first recess 212. The alignment grooves 213a, 213b are particularly V-shaped in cross-section, and their depth is dimensioned such that an EM grid assembly 3 inserted into a respective alignment groove 213a, 213b protrudes with its upper edge from the alignment groove 213a, 213b and is in a tilted orientation with respect to the upper surface 211. In this manner, the EM grid assembly 3 can be easily rotated around its central axis to align the EM grid assembly 3 using a manipulation tool. Since an alignment groove 213a, 213b is provided on either side of the first recess 212, users may choose the desired alignment groove 213a, 213b according to their preferences, for instance, depending on if they are left-handed or right-handed. The first part 210 further comprises second recesses 215a, 215b arranged on either side of the first recess 212 above the alignment grooves 213a, 213b. The second recesses 215a, 215b are each configured to receive a respective container for storing one or several EM grid assemblies 3. Left and right-handed users may choose the respective second groove 215a, 215b according to their preferences or may use both second grooves.

[0210] FIG. 12A-D show a second part 220 of the preparation station 2 according to the invention as top views (A-B) and perspective views (C-D). The second part 220 comprises an upper surface 221 and a second recess 222 comprising a surface 223 which is tilted in respect of the first surface 221. The second recess 222 of the second part 220 is configured to receive the removable holder 240 in a tilted orientation with respect to the upper surface 221 (FIGS. 12A and C show the second part 220 without the removable holder 240, and FIGS. 12 B and D show the second part 220 with the removable holder 240 arranged in the second recess 222).

[0211] The second part 220 further comprises a first hole 225a and a second hole 225b configured to each receive a respective screw to fix the removable holder 240 in the second recess 222. Moreover, the second part 220 comprises an edge 224 below the second recess 222, wherein the edge 224 comprises a rounded cut-out 224a configured such that the removable holder 240 can be easily accessed from below and particularly be removed from the second part 220 from the rounded cut-out 224a.

[0212] FIG. 13A-D show a third part 230 of the preparation station 2 according to the invention as top views (A-B) and perspective views (C-D). The depicted embodiment of the third part 230 is particularly designed to be used by right-handed users. Particularly, a mirrored version of the third part 230 specifically designed for left-handed users is also envisioned within the scope of the present invention. The third part 230 comprises an upper surface 231, a third recess 232 for receiving a cassette 7, a fourth recess 234 (with a surface extending parallel to the upper surface 231) for receiving the removable holder 240, and a fifth recess 238 for receiving a cassette 7. FIGS. 13 A and C show the third part 230 without the removable holder 240, and FIGS. 13 B and D show the third part 230 with the removable holder 240 arranged in the fourth recess 234.

[0213] As best seen in FIGS. 13A and B, the third recess 232 comprises a first section 232a and a second section 232b arranged along a first direction D1, wherein the first section 232a has a first width W1, and wherein the second section 232b has a second width W2 perpendicular to the first direction D1, wherein the second width W2 is greater than the first width W1. As apparent from the perspective view in FIGS. 13 C and D, the third recess 232 has a bottom surface 233 which is tilted in respect of the upper surface 231 of the third part 230, and wherein the depth of the third recess 232 increases in the first direction D1, that is towards the second section 232b. The fifth recess 238 has a bottom surface that is parallel to the upper surface 231 and comprises a uniform width along its length.

[0214] The third part 230 further comprises a first hole 234a and a second hole 234b on either side of the fourth recess 234 configured to each receive a respective screw to fix the removable holder 240 in the fourth recess 234.

[0215] An alignment platform 235 for aligning an EM grid assembly 3 is arranged between the third recess 232 and the fourth recess 234. A detailed perspective view of the alignment platform 235 is provided in FIG. 13 E. The alignment platform 235 comprises a surface 236 which is tilted in respect of the upper surface 231, a slot 237 for inserting a manipulation tool and a groove 237a, particularly a circular groove 237a, for receiving the EM grid assembly 3, wherein the groove 237a is arranged adjacent to the slot 237 and is connected to the slot 237. The slot 237 comprises a bottom surface which is tilted in respect of the upper surface 231 of the third part 230 and tilted in respect of the surface 236 of the alignment platform 235, wherein the angle between the bottom surface of the slot 237 and the upper surface 231 is smaller than the angle between the surface 236 and the upper surface 231. In other words, the inclination of the slot 237 is less than the inclination of the surface 236 of the alignment platform 235. The slot 237 and the groove 237a are configured (that is dimensioned and arranged in respect of each other) that the EM grid assembly 3 can be inserted into the groove 237a using a manipulation tool, such as tweezers, and aligned rotationally around its central axis by a manipulation tool, particularly tweezers, to obtain a desired orientation of the EM grid assembly 3, while the manipulation tool is inserted into the slot 237.

[0216] Moreover, the third part 230 comprises a sixth recess 239 configured to receive a container for one or several EM grid assemblies 3.

[0217] In particular, during use of the preparation station 2, the first part 210, the second part 220 and the third part 230 are arranged in the second coolant reservoir 202 of the preparation station 2, which in turn is placed in the first coolant reservoir 201, and thus the first part 210, the second part 220 and the third part 230 are submerged in cryogenic coolant. In particular, the preparation station 2 is provided in the first compartment 101 of the workstation 1.

[0218] The shuttle 6 is then received in the holding elements 241a, 241b of the removable holder 240, and the holder 240 is placed in the first recess 212 of the first part 210 and optionally fixed by screws placed in the holes 213a, 213b. An EM grid assembly 3 with a vitrified sample S thereon is subsequently supplied, e.g. in a storage container which is placed in one of the second recesses 215a, 215b of the first part 210. The EM grid assembly 3 of interest is then removed from the container using a manipulation tool and placed in one of the alignment grooves 213a, 213b for rotational alignment by the manipulation tool. After the desired orientation of the EM grid assembly 3 is obtained, the EM grid assembly 3 is placed in the shuttle 6 held by the removable holder 240 in the first recess 212 of the first part 210.

[0219] Subsequently, the removable holder 240 with the shuttle 6 and the EM grid assembly 3 therein is placed in the second recess 222 of the second part 220, and optionally fixed by screws using the holes 225a, 225b. A transfer device 4 is then advanced with the tip of its inner rod to the shuttle 6, connected to the shuttle 6, and the shuttle 6 is inserted into the chamber 41 of the transfer device 4. Thereafter the shuttle 6 is transported to the preparation device, particularly the cryo-FIB device by the transfer device 4, wherein the shuttle 6, which is arranged in the chamber 41 is cooled to cryogenic temperatures and the chamber 41 evacuated. The shuttle 6 is inserted into the preparation device, and preparation of the sample S, particularly thinning by FIB, is initiated. When preparation is completed, the shuttle 6 is inserted back into the transfer device 4 and particularly removed from the transfer device 4 back into the removable holder 240 of the preparation station 2, e.g. in the first compartment 101 of the workstation 1.

[0220] The removable holder 240 is then inserted into the fourth recess 234 of the third part 230, and the holder 240 is optionally fixed with screws using the holes 234a, 234b. Subsequently, a cassette 7 is particularly placed in the third recess 232 of the third part 230. The EM grid assembly 3 is removed from the shuttle 6, optionally aligned on the alignment platform 235, and placed in the cassette 7 in the correct orientation. The cassette 7 may then be transferred from the third recess 232 to the fifth recess 238, and moved into the transfer receptacle 8, particularly containing a cryogenic coolant. Alternatively, the cassette 7 may be directly transferred from the third recess 232 to the transfer receptacle 8. The transfer receptacle 8 is then inserted into an imaging device, e.g. a cryo-electron microscope, and the sample S is imaged. The transfer receptacle 8 is optionally held by the adapter 250 during insertion of the cassette 7 into the transfer receptacle 8. Subsequently, the transfer receptacle 8 can be inserted into a cryo-electron microscope, particularly for cryo-electron tomography.

[0221] In an alternative method that can be performed using the preparation station 2 according to the invention, an EM grid assembly 3, particularly containing a vitrified sample S for single particle analysis by cryo-electron microscopy, is provided, particularly in a container inserted into the sixth recess 239 of the third part 230. The EM grid assembly 3 is then directly transferred from the container into a cassette 7, particularly inserted into the fifth recess 238. Subsequently, the cassette 7 is transferred from the fifth recess 238 to the transfer receptacle 8, and the transfer receptacle 8 is inserted into a cryo-electron microscope for single particle analysis.

[0222] FIGS. 14A and B show details of the adapter 250 that can be attached to either side of the first coolant reservoir 201 of the preparation station 2, as shown in FIG. 6. FIG. 14A depicts the adapter 250 without the transfer receptacle 8, and FIG. 14B depicts the adapter 250 with the transfer receptacle 8 held by the adapter 250. The adapter 250 comprises a fixing plate 252 and a supporting plate 253, wherein the fixing plate 252 is attached to the supporting plate 253, particularly by means of screws inserted through the bores 255 of the supporting plate 253 into the connecting sections 254 of the fixing plate 252, and wherein the supporting plate 253 is tilted in respect of the fixing plate 252. The supporting plate 253 comprises a through-hole 251 with a tapered rim 251a, wherein the through-hole 251 is configured to be aligned with an opening of the transfer receptacle 8. The adapter 250 further comprises an edge 256 with a cut-out 257 adjacent to the through-hole 251.

TABLE-US-00001 List of reference numerals Workstation 1 Preparation station 2 Electron microscopy grid assembly 3 Transfer device 4 Shuttle 6 Cassette 7 Transfer receptacle 8 Grid 31 Chamber 41 Outer rod 42 Coolant reservoir 43 Window of transfer device 44 Valve 45 Adapter 46 Tube 47 Slot of shuttle 61 Slot of cassette 71 First compartment 101 First gas inlet 102 Gas reservoir 103 First glove 104 Second glove 105 Opening 106, 107 Window 108 Port 109 Finger section 110 Tip 111 Hole 112 Coolant tank 113 Dispensing mechanism 114 Coolant outlet 115 First guide rail 116 Second guide rail 117 Loading lock 118 Second compartment 119 First gate 120 Second gate 121 Second gas inlet 122 Heating element 123 Control device  123a Vacuum pump 124 Pump inlet 125 Coolant inlet 126 First coolant reservoir 201 Opening  201a Collar  201b Second coolant reservoir 202 Tool holder 203 First slot  204a Second slot  204b Latch 205 Tool slot 206 First slot of second coolant reservoir  207a Second slot of second coolant reservoir  207b Third slot of second coolant reservoir  207c Fourth slot of second coolant reservoir 208 Through-hole 209 First part 210 Upper surface of first part 211 First recess 212 Alignment groove 213, 213a, 213b First hole  214a Second hole  214b Second recess 215, 215a, 215b Second part 220 Upper surface of second part 221 Second recess 222 Surface of second recess 223 Edge 224 Rounded cut-out  224a First hole  225a Second hole  225b Third part 230 Upper surface of third part 231 Third recess 232 First section  232a Second section  232b Surface of third recess 233 Fourth recess 234 First hole  234a Second hole  234b Alignment platform 235 Surface of alignment platform 236 Slot 237 Groove  237a Fifth recess 238 Sixth recess 239 Removable holder 240 First holding element  241a Second holding element  241b First groove 242 Protrusion 243 Adapter 250 Through-hole 251 Tapered rim  251a Fixing plate 252 Supporting plate 253 Connecting section 254 Bore 255 Edge 256 Cut-out 257 Foot 260 First direction on third part D1 Gas flow G Sample S First width W1 Second width W2