PREPARATION OF A SAMPLE FOR HIGH PRESSURE FREEZING

Abstract

A method including one or more steps of a procedure for preparing a microscopic sample (1) for a high-pressure freezing process is provided, wherein the sample (1) is provided using an arrangement comprising a middle plate (33) of a high-pressure freezing cartridge (30) and an incubation chamber (100), wherein the middle plate (33) is attached to a lower surface of a bottom (120) of the incubation chamber (100) by an adhesive and detachable from the incubation chamber (100) by effecting a relative movement between the middle plate (33) and the incubation chamber (100), wherein the sample (1) is provided on an enclosing element (37) which is fitted into an opening (36) of the middle plate (33), and wherein, when the sample (1) is to be subjected to the high-pressure freezing process, the relative movement between the middle plate (33) and the incubation chamber (100) is effected, thereby detaching the middle plate (33) with the sample (1) from the incubation chamber (100). Means to implement the method are also part of the present disclosure.

Claims

1. A method for preparing a microscopic sample (1) for a high-pressure freezing process, the method comprising: providing a middle plate (33) of a high-pressure freezing cartridge (30), the middle plate having an opening (36) and an enclosing element (37) fitted into the opening (36); arranging the sample (1) on the enclosing element (37) of the middle plate (33); providing an incubation chamber (100), the incubation chamber (100) including a bottom (120) having a lower surface; attaching the middle plate (33) to the lower surface of the bottom (120) of the incubation chamber (120) by an adhesive, wherein the middle plate is detachable from the incubation chamber (100) by effecting a relative movement between the middle plate (33) and the incubation chamber (100); and effecting the relative movement between the middle plate (33) and the incubation chamber (100) to detach the middle plate (33) with the sample (1) from the incubation chamber (100) when the sample (1) is to be subjected to the high-pressure freezing process.

2. The method according to claim 1, wherein the adhesive comprises an oxygen permeability less than 2 g.Math.m-1.Math.sec-1.Math.Pa-1×10-14 as determined by an oxygen transmission rate analyzer.

3. The method according to claim 1, wherein at least one of a paraffinic wax, a natural wax, an epoxy glue, a methacrylate glue, and an adhesive tape is used as the adhesive by which the middle plate (33) is attached to the lower surface of the bottom (120) of the incubation chamber (100).

4. The method according to claim 1, wherein said relative movement between the middle plate (33) and the incubation chamber (100) is effected using a device (10) comprising an engagement structure (11), the engagement structure (11) being adapted to engage with the middle plate (33) and to restrict a movement of the middle plate (33) while the incubation chamber (100) is moved.

5. The method according to claim 4, wherein the relative movement between the middle plate (33) and the incubation chamber (100) is at least one of: (i) a linear movement of the incubation chamber (100) in a plane corresponding to the lower surface of the bottom (120) of the incubation chamber (100), (ii) a rotational movement of the incubation chamber (100) in the plane corresponding to the lower surface of the bottom (120) of the incubation chamber (100), (iii) a tilting movement of the incubation chamber (100) around an axis which is parallel to the lower surface of the bottom (120) of the incubation chamber (100), and (iv) a lifting movement of the incubation chamber (100) in a direction at an angle to the lower surface of the bottom (120) of the incubation chamber (100) while the middle plate (33) engages with the engagement structure (11) of the device (10).

6. The method according to claim 4, wherein the middle plate (33) is attached to the bottom (120) of the incubation chamber (100) to protrude from the bottom (120) of the incubation chamber (100), and wherein the middle plate (33) is fully or partially inserted into the engagement structure (11) while the bottom (120) of the incubation chamber (100) rests on a surface (12) of the device (10) before said relative movement between the middle plate (33) and the incubation chamber (100) is effected.

7. The method according to claim 4, wherein the engagement structure (11) is provided as a recess matching a shape of an outer circumference of the middle plate (33) and/or wherein the engagement structure (11) is provided as a structure comprising at least one undercut matching at least one protrusion (13) provided by at least one edge of the middle plate (33).

8. The method according to claim 1, wherein the sample (1) is living matter, and the method further comprises exposing the sample (1) to defined environmental conditions provided in the incubation chamber (100).

9. An arrangement for incubating a microscopic sample (1) to be subjected to high-pressure freezing, the arrangement comprising: an incubation chamber (100) including a bottom (120), the bottom (120) having a lower surface and an opening (180); and a middle plate (33) of a high-pressure freezing cartridge (30), the middle plate having an opening (36); wherein the middle plate (33) is attached to the lower surface of the bottom (120) of the incubation chamber (100) by an adhesive and is detachable from the incubation chamber (100) by effecting a relative movement between the middle plate (33) and the incubation chamber (100); wherein the opening (180) of the bottom (120) of the incubation chamber (120) provides fluid access to the opening (36) of the middle plate (33).

10. An incubation chamber (100) adapted for being used in an arrangement comprising the incubation chamber (100) and comprising a middle plate (33) of a high-pressure freezing cartridge (30), the incubation chamber (100) being adapted for attachment of the middle plate (33) to the incubation chamber (100) by an adhesive such that the middle plate (33) is detachable from the incubation chamber (100) by effecting a relative movement between the middle plate (33) and the incubation chamber (100), and a bottom (120) of the incubation chamber (100) comprising an opening (180) for providing fluid access to an opening (36) of the middle plate (33).

11. A middle plate (33) of a high-pressure freezing cartridge (30) adapted for being used in an arrangement comprising an incubation chamber (100) and comprising the middle plate (33), the middle plate (33) being adapted to be attached to the incubation chamber (100) by an adhesive such that the middle plate (33) is detachable from the incubation chamber (100) by effecting a relative movement between the middle plate (33) and the incubation chamber (100).

12. The middle plate (33) according to claim 11, comprising at least one protrusion (13) at least at one edge of the middle plate (33), the at least one protrusion (13) being adapted to engage with an engagement structure (11) comprising at least one undercut and being provided in a device (10) adapted to be used for effecting the relative movement between the middle plate (33) and the incubation chamber (100).

13. A device (10) for preparing a microscopic sample (1) for a high-pressure freezing process, wherein the sample (1) is provided using an arrangement comprising a middle plate (33) of a high-pressure freezing cartridge (30) and an incubation chamber (100), wherein the middle plate (33) is attached to a lower surface of a bottom (120) of the incubation chamber (100) by an adhesive and detachable from the incubation chamber (100) by effecting a relative movement between the middle plate (33) and the incubation chamber (100), wherein the sample (1) is provided on an enclosing element (37) which is fitted into an opening (36) of the middle plate (33), wherein, when the sample is to be subjected to the high-pressure freezing process, the relative movement between the middle plate (33) and the incubation chamber (100) is effected, thereby detaching the middle plate (33) with the sample (1) from the incubation chamber (100), and wherein the device (10) comprises an engagement structure (11), the engagement structure (11) being adapted to engage with the middle plate (33) and to restrict a movement of the middle plate (33) while the incubation chamber (100) is moved relative to the middle plate (33).

Description

BRIEF DESCRIPTION OF THE DRAWING VIEWS

[0047] FIG. 1 illustrates an incubation chamber for incubating a sample not forming part of the present disclosure in a simplified sectional view.

[0048] FIG. 2 illustrates a high-pressure freezing cartridge usable in connection with the present disclosure in a simplified exploded view.

[0049] FIG. 3 illustrates a middle plate of a high-pressure freezing cartridge usable in connection with the present disclosure in a simplified sectional view.

[0050] FIG. 4 illustrates an incubation chamber for incubating a sample according to an embodiment of the present disclosure in a simplified sectional view.

[0051] FIGS. 5A and 5B illustrate steps of detaching a middle plate from an incubation chamber by tilting according to an embodiment of the present disclosure.

[0052] FIGS. 6A and 6B illustrate steps of detaching a middle plate from an incubation chamber by lifting according to an embodiment of the present disclosure.

[0053] In the Figures, like elements are indicated with identical reference numerals. Repeated explanations thereof are omitted for reasons of conciseness only.

DETAILED DESCRIPTION

[0054] FIG. 1 shows an incubation chamber 900 for incubating a sample not forming part of the present disclosure in a simplified sectional view and was already explained at the outset. Reference is made to the explanations above. Again, it is to be pointed out that the present disclosure is not intended to be limited by the specific type of incubation chamber shown here.

[0055] FIG. 2 shows a high-pressure freezing cartridge 30, i.e. a sample holding means, which may be used in connection with the present disclosure in an exploded view. The cartridge 30 can be used in high-pressure freezing devices generally known from the art which were referred to above. Reference is made, for example, to the literature cited above for the “EM ICE”, “EM HPM100” and “EM PACT” systems by Leica Microsystems, e.g. to DE 10 2013 003 164 A1 which further describes details of a high pressure freezing system, and to scientific review literature such as Kaech & Ziegler (see above). The cartridge 30 can, in an assembled state, be transferred into the high-pressure freezing device by using a known holder. Handling of cartridges such as the cartridge 30 is extensively described elsewhere and will not be explained here for reasons of conciseness.

[0056] The cartridge 30 comprises two holding elements 31, 32, each essentially of a half cylindrical shape. A middle plate 33 is arranged between the holding elements 31, 32 of the cartridge 30. The holding elements 31, 32 provide refrigerant channels 34 therebetween, the refrigerant channels 34 being formed by grooves along the longitudinal direction on the inner flat surfaces of the holding elements 31, 32. Through the refrigerant channels 34, a cryogenic refrigerant can be passed to a sample received or contained in the middle plate 33 in an opening 36 forming a sample space, essentially along the longitudinal axis of the cartridge 30 and in the direction of the dotted arrow 35 as illustrated in FIG. 2.

[0057] FIG. 3 shows the middle plate 33 which is used in the cartridge 30 as shown in FIG. 2 in a longitudinal section along a vertical plane, so that the sample space formed in the opening 36 such as according to FIG. 1 is shown cut open. The opening 36 is of a circular shape and is arranged in or near to the center of the middle plate 33 and the sample 1 is held between two enclosing discs including a first (bottom) disc 37 and a second (top) disc 38, e.g. circular enclosing elements 37, 38 in the form of discs made of sapphire, metal or another suitable material. The enclosing elements 37, 38 are fixed in the opening 36 and their distance is adjusted by fixing, gasketing and/or spacer means 39. The enclosing elements 37, 38 themselves may also comprise one or more recesses in which the sample 1 can be received when they are inserted into the opening 36, thereby reducing or eliminating the need for fixing, gasketing and/or spacer means 39. The enclosing elements 37, 38 also serve to protect the sample 1 since the rapid flow of refrigerant required for cooling would otherwise carry away the sample.

[0058] Further details and variants of a cartridge 30 and its middle plate 33 are described in the prior art mentioned. Again, it should be understood that the present disclosure is not limited to the specific configuration of the cartridge 30 and the middle plate 33. The entire cartridge 30 is dimensioned in such a way that the high pressures (e.g. above 2,000 bar) required for high pressure freezing can be built up and be maintained within a period of preferably 200 to 500 ms, whereby rapid freezing of sample 1 is achieved within this time interval.

[0059] FIG. 4 illustrates an incubation chamber for incubating a sample according to an embodiment of the present disclosure in a simplified sectional view.

[0060] The elements of the incubation chamber 100 shown in FIG. 4 are designed slightly differently from the incubation chamber 900 as shown in FIG. 1, but may likewise be designed in the same manner. The present disclosure is not characterized in or limited by specific parts of the incubation chamber 900 being present or not. Elements of the incubation chamber 100 shown in FIG. 4 are designated with identical reference numerals as for the incubation chamber 900 shown in FIG. 1.

[0061] Also here, for assembling the incubation chamber 100 shown in FIG. 4, an open silicone cylinder 110 is mounted to a glass plate forming a bottom 120 of the incubation chamber. The cylinder 110 is again closed by a glass lid 130. The whole assembly is, in the incubation chamber 100, covered by a metal cover 150 which fixes the glass lid 130 and no separate glass window 160 is provided. The gas to which the sample 1 is to be exposed may be provided and the gas atmosphere may be maintained in any manner conceivable.

[0062] A middle plate 33, e.g. as previously explained in connection with FIGS. 2 and 3, is attached to a lower surface of the bottom 120 of the incubation chamber 100 by using an adhesive not specifically shown for reasons of generality, and the middle plate 33 is detachable from the incubation chamber 100 by effecting a movement of the incubation chamber 100 as described hereinbefore in full detail for different embodiments. As shown the middle plate 33 is provided in an opened state, i.e. with only a lower enclosing element 37 being inserted, and the sample 1 is provided on the lower enclosing element. The bottom 120 of the incubation chamber 100 comprises an opening 180 for providing fluid access to the opening 36 of the middle plate 33.

[0063] FIGS. 5A and 5B illustrate steps of detaching a middle plate from an incubation chamber by tilting according to an embodiment of the present disclosure. The incubation chamber is embodied essentially as described for the embodiment shown in FIG. 4 and is therefore designated 100.

[0064] In general, as the middle plate 33 protrudes from the bottom 120 of the incubation chamber 100 when attached thereto, an engagement structure 11 may be provided as a recess with a shape complementary to at least a part of a shape of the middle plate which is adapted to receive at least a part of the middle plate 33 protruding from the bottom 120 of the incubation chamber 100. In the specific embodiment shown, in order to restrict a tilting movement, the engagement structure 11 is provided as a structure with an undercut into which a corresponding protrusion 13 provided at the middle plate 33 may be inserted, such that when tilting the incubation chamber 100 while maintaining the protrusion 13 of the middle plate 33 inserted into the undercut, as indicated with a white arrow in FIG. 5A, an overhang of the undercut “pulls off” the middle plate 33.

[0065] FIGS. 6A and 6B illustrate steps of detaching a middle plate from an incubation chamber by lifting according to an embodiment of the present disclosure. Also here, the incubation chamber is embodied essentially as described for the embodiment shown in FIG. 4 and is therefore designated 100.

[0066] Also here, in general, as the middle plate 33 protrudes from the bottom 120 of the incubation chamber 100 when attached thereto, an engagement structure 11 may be provided as a recess with a shape complementary to at least a part of a shape of the middle plate which is adapted to receive at least a part of the middle plate 33 protruding from the bottom 120 of the incubation chamber 100.

[0067] A lifting movement may specifically be restricted by providing a parallel set of guiding elements with undercuts as the engagement structure 11, e.g. in the form of a dovetail guide, which are adapted to slidingly receive complementary structures provided at the middle plate in the form of protrusions 13 in an insertion direction which is, in the illustration of FIGS. 6A and 6B, orthogonal to the paper plane. When lifting the incubation chamber 100 in a direction at an angle to the insertion direction, as indicated with a white arrow according to FIG. 6A, the middle plate 33 may be detached from the incubation chamber 100.

[0068] In FIGS. 5A to 6B, a device provided according to the present disclosure is shown in a partial view and is indicated 10. The device 10 can, as mentioned before, be a high-pressure freezing device adapted to perform all or at least a part of the further steps of high-pressure freezing as well, or the device 10 may be a dedicated device separate from a high-pressure freezing device, i.e. a separate constructional unit. The device 10 according to FIGS. 5A to 6B comprises the engagement structure 11. A surface of the device 10 is indicated 12.