Modular specimen holders for high pressure freezing and X-ray crystallography of a specimen

Abstract

A modular specimen holder (10) for high pressure freezing and/or X-ray crystallography of a specimen has a specimen holding element (100) and an extension element (200) connectable with and separable from each other; the specimen holding element (100) including a tubule (120) adapted to hold the specimen and a base element (110) adapted to hold the tubule (120), wherein a distance from a bottom of the base element (110) to a top of the tubule (120) is a first distance (d1); the extension element (200) being connectable with the base element (110), wherein, when the extension element (200) and the base element (110) are connected with each other, a second distance (d2) from a bottom of the extension element (200) to the top of the tubule (120) is larger than the first distance (d1).

Claims

1. A modular specimen holder (10, 10) for high pressure freezing and/or X-ray crystallography of a specimen comprising a specimen holding element (100, 100) and an extension element (200, 200) connectable with each other and separable from each other; the specimen holding element (100, 100) comprising a tubule (120, 120) and a base element (110, 110), wherein: the tubule (120, 120) is adapted to hold the specimen, the base element (110, 110) is adapted to hold the tubule (120, 120), a distance from a bottom of the base element (110, 110) to a top of the tubule (120, 120) is a first distance (d.sub.1); the extension element (200, 200) being adapted to be connected with the base element (110, 110), wherein, when the extension element (200, 200) and the base element (110, 110) are connected with each other, a distance from a bottom of the extension element (200, 200) to the top of the tubule (120, 120) is a second distance (d.sub.2); wherein the second distance (d.sub.2) is larger than the first distance (d.sub.1); wherein the first distance (d1) from the bottom of the base element (110, 110) to the top of the tubule (120, 120) is dimensioned to fit in a high pressure freezing unit.

2. The modular specimen holder (10, 10) according to claim 1, wherein the first distance (d.sub.1) is dimensioned to fit in a high pressure freezing unit for high pressure freezing of the specimen holding element (100, 100).

3. The modular specimen holder (10, 10) according to claim 1, wherein the second distance (d.sub.2) is dimensioned to fit in an X-ray crystallography unit for conducting an X-ray crystallography of the specimen.

4. The modular specimen holder (10, 10) according to claim 1, wherein at least a part of the tubule (120, 120) is made of polyimide.

5. The modular specimen holder (10, 10) according to claim 1, wherein the tubule (120, 120) comprises a pin (121, 121) and a holding element (122, 122), wherein the pin (121, 121) is made of a metal material and wherein the holding element (122, 122) is adapted to hold the specimen and is made of polyimide.

6. The modular specimen holder (10, 10) according to claim 1, wherein the base element (110, 110) is of cylindrical or essentially cylindrical shape comprising a lateral area (111, 111) and a top (112, 112) perpendicular to the lateral area (111, 111).

7. The modular specimen holder (10, 10) according to claim 6, the top (112, 112) of the base element (110, 110) being constructed as a plane or essentially plane sheet or as a plane or essentially plane sheet with a stepped outer rim (115, 115).

8. The modular specimen holder (10, 10) according to claim 1, the base element (110, 110) being hollow in its interior and the bottom of the base element comprising or forming an opening.

9. The modular specimen holder (10, 10) according to claim 8, wherein the extension element (200, 200) comprises connection means (202, 202) adapted to interact with the base element (110, 110), thus establishing the connection of the specimen holding element (100, 100) and the extension element (200, 200), the connection means (202, 202) of the extension element being adapted to be inserted into the interior of the base element (110, 110) through the opening in the bottom of the base element (110, 110).

10. The modular specimen holder (10, 10) according to claim 1, wherein the extension element (200, 200) comprises connection means (202, 202) adapted to interact with the base element (110, 110), thus establishing the connection of the specimen holding element (100, 100) and the extension element (200, 200).

11. The modular specimen holder (10, 10) according to claim 10, wherein the connection means (202, 202) are adapted to establish a magnetic and/or mechanical connection of the specimen holding element (100, 100) and the extension element (200, 200).

12. The modular specimen holder (10, 10) according to claim 1, wherein a diameter (d.sub.3) of the bottom of the extension element (200, 200) is essentially 12 mm.

13. The modular specimen holder (10, 10) according to claim 1, wherein the first distance (d.sub.1) from the bottom of the base element (110, 110) to the top of the tubule (120, 120) is in a range between 15 mm and 19 mm.

14. The modular specimen holder (10, 10) according to claim 1, wherein the second distance (d.sub.2) from the bottom of the extension element (200, 200) to the top of the tubule (120, 120) is in a range of 22 mm1.5 mm.

15. The modular specimen holder (10, 10) according to claim 1, wherein the second distance (d.sub.2) from the bottom of the extension element (200, 200) to the top of the tubule (120, 120) is dimensioned according to the SPINE standard.

16. A modular specimen holder (10, 10) for high pressure freezing and/or X-ray crystallography of a specimen comprising a specimen holding element (100, 100) and an extension element (200, 200) connectable with each other and separable from each other; the specimen holding element (100, 100) comprising a tubule (120, 120) and a base element (110, 110), wherein: the tubule (120, 120) is adapted to hold the specimen, the base element (110, 110) is adapted to hold the tubule (120, 120), a distance from a bottom of the base element (110, 110) to a top of the tubule (120, 120) is a first distance (d.sub.1); the extension element (200, 200) being adapted to be connected with the base element (110, 110), wherein, when the extension element (200, 200) and the base element (110, 110) are connected with each other, a distance from a bottom of the extension element (200, 200) to the top of the tubule (120, 120) is a second distance (d.sub.2); wherein the second distance (d.sub.2) is larger than the first distance (d.sub.1); wherein the modular specimen holder (10, 10) further comprises a cartridge (300) connectable with the specimen holding element (100, 100) and separable from the specimen holding element (100, 100); the cartridge (300) comprising an encapsulating element (320) in its interior, wherein: the tubule (120, 120) and the encapsulating element (320) are formed such that the tubule (120, 120) can be placed inside the encapsulating element (320).

17. The modular specimen holder (10, 10) according to claim 16, wherein the encapsulating element (320) is constructed as a cavity inside the cartridge.

18. The modular specimen holder (10, 10) according to claim 16, wherein the cartridge (300) is constructed as a single piece.

19. The modular specimen holder (10, 10) according to claim 16, wherein the cartridge (300) comprises connection means (330) to connect the cartridge (300) with the base element (110, 110) of the specimen holding element.

20. The modular specimen holder (10, 10) according to claim 16, wherein the specimen holding element (100, 100) is designed to fit into the cartridge (300).

21. A method of high pressure freezing and X-ray crystallography of a specimen using a modular specimen holder (10, 10) according to claim 16, comprising the steps of: connecting the specimen holding element (100, 100) holding the specimen and the cartridge (300) with each other; placing the connected specimen holding element (100, 100) and the cartridge (300) in a high pressure freezing unit; conducting a high pressure freezing of the specimen by means of the high pressure freezing unit; separating the frozen specimen holding element (100, 100) and the cartridge (300) from each other; connecting the frozen specimen holding element (100, 100) with the extension element (200, 200); placing the connected specimen holding element (100, 100) and the extension element (200, 200) in an X-ray crystallography unit; and conducting an X-ray crystallography of the frozen specimen by means of the X-ray crystallography unit.

Description

BRIEF DESCRIPTION OF THE DRAWING VIEWS

(1) The present invention will now be described further, by way of example, with reference to the accompanying drawings, in which

(2) FIG. 1 schematically shows a preferred embodiment of a modular specimen holder with specimen holding element and extension element according to the invention in different perspective views,

(3) FIG. 2 schematically shows a preferred embodiment of a modular specimen holder with specimen holding element and extension element according to the invention in different perspective views,

(4) FIG. 3 schematically shows a preferred embodiment of a modular specimen holder with specimen holding element, extension element, and cartridge according to the invention in different perspective views, and

(5) FIG. 4 schematically shows a part of a high pressure freezing unit with a specimen holding element and a cartridge of a modular specimen holder according to the invention in a perspective view.

DETAILED DESCRIPTION

(6) In FIG. 1 a preferred embodiment of a modular specimen holder with specimen holding element and extension element according to the invention is schematically shown and labelled as 10. The modular specimen holder 10 is shown in FIG. 1a in a perspective exploded view and in FIG. 1b in its compound state in a perspective view. Identical reference signs in the figures refer to identical or structurally identical elements.

(7) In FIG. 1a, the two modules of the specimen holder 10, i.e. the specimen holding element 100 and the extension element 200 are shown separated from each other.

(8) The specimen holding element 100 comprises a base element 110 and a tubule 120. The tubule 120 comprises a pin 121 made of a metal material on which a holding element 122 made of polyimide, preferably Kapton, is mounted. A specimen can be placed in this holding element 122. The holding element 122 is particularly constructed as a small Kapton tube, inside which protein crystals can be placed as specimen in a specific solution, in which they are dissolved.

(9) The holding element 122 can e.g. also be constructed as a loop. The tubule 120 can especially be constructed as a so called Hampton pin.

(10) The base element 110 is adapted to hold the tubule 120 and is of cylindrical or essentially cylindrical shape comprising a lateral area 111 and a top 112 perpendicular to the lateral area 111. The interior of the base element 110 is particularly hollow. Guiding means can be provided on the inner wall of the lateral area 111 in order to insert the specimen holding element 100 in a cartridge as will be explained with reference to FIG. 3 and FIG. 4.

(11) The top 112 can be constructed as a plane or essentially plane sheet, particularly with a stepped outer rim 115. This stepped outer rim 115 is especially provided in order to enable a centred positioning of the specimen holding element 100 inside the cartridge, as will be explained with reference to FIG. 3.

(12) There is no sheet at the bottom of the base element 110. Thus, the bottom of the base element 110 forms an opening. The base element 110, i.e. the lateral area 111 and the top 112, are made of metal, e.g. ferromagnetic stainless steel.

(13) The base element 110 particularly comprises a holding element for the tubule 120 in form of a borehole 113 in the centre of the sheet 113, into which the tubule 120 can be inserted. The tubule 120 is thus arranged perpendicular or essentially perpendicular to the top 112 of the base element.

(14) There are several more boreholes 114 circumferential to the centre of the sheet 112 for conducting liquid nitrogen in the course of the high pressure freezing.

(15) The extension element 200 comprises a base 201 on which connection means 202 are arranged. These connection means 202 are adapted to connect the specimen holding element 100 with the extension element 200.

(16) The connection means 202 can be inserted into the hollow interior of the base element 110 through the opening in the bottom of the base element 110. In this particular example, the connection means 202 are embodied as clamping elements, establishing a force-fitted connection between specimen holding element 100 and extension element 200 by pressing or forcing against the interior wall of the base element 110.

(17) The modular specimen holder 10 is shown in FIG. 1b in its compound state, i.e. with the specimen holding element 100 and the extension element 200 connected with each other. In FIG. 1b, a distance from a bottom of the extension element 200 to the top of the tubule 120 is d.sub.2. This distance d.sub.2 is 22 mm1.5 mm and is hence dimensioned according to the SPINE standard. Particularly, the set distance of 22 mm of the SPINE standard refers to the distance from the bottom of the extension element 200 to the specimen. Thus, if the specimen is not placed exactly at the edge of the holding element 122, the distance d.sub.2 can be slightly larger than 22 mm.

(18) Thus, the distance d.sub.2 is dimensioned to fit in an X-ray crystallography unit for conducting an X-ray crystallography of the specimen constructed according to the SPINE standard. Moreover, a diameter d.sub.3 of a bottom of the extension element 200 is essentially or precisely 12 mm and thus also dimensioned according to the SPINE standard.

(19) In its compound state, the modular specimen holder 10 thus fulfils the requirements of the SPINE standard. However, the compound modular specimen holder 10 cannot be used for specific types of high pressure freezing units, particularly Leica EM ICE or of similar design.

(20) When separated from each other, however, the specimen holder can be used in the Leica EM ICE or a device designed in that way. This device allows a maximum height of 19 mm of the corresponding specimen holding element. Thus, a distance d.sub.1 from a bottom of the base element 110 to the top of the tubule 120 is at most 19 mm, particularly in the range between 15 mm and 19 mm, and is thus dimensioned to fit in a high pressure freezing unit in form of the Leica EM ICE.

(21) In FIG. 2 another preferred embodiment of a modular specimen holder according to the invention is schematically shown and labelled as 10 with specimen holding element 100 and extension element 200.

(22) FIG. 2a shows the modular specimen holder 10 in a perspective sectional view with specimen holding element 100 and extension element 200 separated from each other. FIG. 2b is a perspective a sectional view of specimen holding element 100 and extension element 200 connected with each other. FIG. 2c is a perspective view analogously to FIG. 2b.

(23) Analogously, to the modular specimen holder 10 of FIG. 1, also the modular specimen holder 10 of FIG. 2 comprises a base element 110 adapted to hold the tubule 120 comprising a metal pin 121 and a Kapton holding element 122. A holding element 113 for the tubule 120 is constructed as a cylinder.

(24) The base element 110 is of cylindrical or essentially cylindrical shape. In contrast to the base element 110 of FIG. 1, this base element 110 is not hollow in its interior but an essentially solid cylinder with lateral area 111 and top 112. Boreholes 114 inside the base element 110 are provided for conducting liquid nitrogen in the course of the high pressure freezing. The cylindrical base element 110 can in this case have a height from the bottom to the top surface 112 in the range between e.g. 1 mm and 3 mm.

(25) The extension element 200 comprises a base 201 and connection means 202. In this example the connection means 202 are constructed as magnetic connection means in order to establish a force-fitted connection between specimen holding element 100 and extension element 200. For this purpose one or several magnets 202 are provided which can interact with the base element 110 made of metal.

(26) Analogously to FIG. 1b, the first distance d.sub.1 from the bottom of the base element 110 to the top of the tubule 120 is in the range between 15 mm and 19 mm, and is thus dimensioned to fit in a high pressure freezing unit in form of the Leica EM ICE.

(27) The second distance from the bottom of the extension element 200 to the top of the tubule 120 is d.sub.2 and is in the range 22 mm1.5 mm and is hence dimensioned according to the SPINE standard.

(28) Moreover, the diameter d.sub.3 of the bottom of the extension element 200 is essentially or precisely 12 mm and thus also dimensioned according to the SPINE standard.

(29) According to the invention, the specimen holding element is also connectable with a cartridge for high pressure freezing, as will now be explained in reference to FIG. 3.

(30) FIG. 3 shows the specimen holding element 100 and the extension element 200 of FIG. 2 as well as a corresponding cartridge 300.

(31) FIG. 3a shows the three modules 100, 200, and 300 separated from each other in a perspective sectional view. These modules 100, 200, and 300 are shown connected with each other in a perspective sectional view in FIG. 3b and in a perspective view in FIG. 3c

(32) The cartridge 300 has an encapsulating element 320 in its interior in the form of a tubular cavity. This tubular cavity 320 is located inside a casing 310 of the cartridge 300. The tubule 120 is adapted to be placed inside this tubular cavity 320. A diameter of said tubular cavity 320 is particularly only slightly larger than a diameter of the tubule 120.

(33) Moreover, connection means 330 are provided inside the casing 310 in order to connect the cartridge 300 with the specimen holding element 100. These connection means 330 are preferably constructed as magnetic connection means 330, e.g. one or several magnets. These magnets 330 can interact with the base element 110 in order to establish a force-fitted connection between cartridge 300 and the metal base element 110.

(34) In FIG. 4, a part of a high pressure freezing unit in form of the Leica EM ICE is schematically shown in a perspective view. Particularly, a loading station 400 of the Leica EM ICE is shown, into which the specimen is loaded for high pressure freezing.

(35) The loading station 400 comprises a chamber 401, into which firstly the cartridge 300 is loaded by a mechanical guiding element 402, e.g. a rod. Afterwards the specimen holding element 100 is positioned in the chamber by the mechanical guiding element 402 and is thus inserted into the cartridge 300.

(36) For loading the specimen holding element 100 into the chamber 401, the mechanical guiding element 402 is connected with guiding means in the interior of the base element 110 of the specimen holding element 100. The mechanical guiding element 402 enables precise insertion of the specimen holding element 100 in the cartridge 300 without damaging the specimen.

(37) It is also possible to provide two different mechanical guiding elements; a first one for loading the cartridge into the chamber 401 and a second one for the specimen holder.

(38) When both the cartridge 300 and the specimen holding element 100 are loaded into the chamber 401, a lid 404 with a handle 404 is closed. Closing this lid 403 triggers the high pressure freezing of the specimen. For this purpose, the cartridge 300 and the specimen holding element 100 are connected with each other and transported through a conduit 405 into the interior of the Leica EM ICE, where the high pressure freezing process is conducted.

(39) After the freezing process, the combination of cartridge 300 and specimen holding element 100 is automatically ejected by the Leica EM ICE into a cooling bath of a cryogenic medium, especially liquid nitrogen, e.g. in a Dewar vessel.

REFERENCE SIGNS

(40) 10 modular specimen holder 100 specimen holding element 110 base element 111 lateral area of the base element 112 top of the base element, sheet 113 holding element for the tubule 120, borehole 114 boreholes 120 tubule 121 pin 122 holding element 200 extension element 201 base 202 connection means, clamping elements 10 modular specimen holder 100 specimen holding element 110 base element 111 lateral area of the base element 112 top of the base element, sheet 113 holding element for the tubule 120 114 boreholes 120 tubule 121 pin 122 holding element 200 extension element 201 base 202 connection means, magnet 300 cartridge 310 casing of the cartridge 320 encapsulating element, tubular cavity 330 connection means, magnets 400 loading station of a high pressure freezing unit 401 chamber 402 mechanical guiding element, rod 403 lid 404 handle 405 conduit