Specimen holder for water-containing preparations and method for using it; and high-pressure freezing device for the specimen holder

[0002] This invention claims priority of a German patent application DE 100 15 773.4, filed Mar. 30, 2000, which is incorporated by reference herein.

[0003] The invention concerns a specimen holder for water-containing preparations. The invention furthermore concerns a method for using the specimen holder for water-containing preparations. In addition, the invention also concerns a high-pressure freezing device that uses a specimen holder for water-containing preparations.

[0004] A method for rapid freezing of water-containing preparations under high pressure is known from DE Patent 1 806 741. The advantage of freezing water-containing preparations under high pressure can be explained as follows: If approximately 2000 bar of pressure is applied to the specimen during cooling, the cooling rate necessary for inducing vitrification (i.e., freezing with no formation of ice crystals) is reduced by a factor of 100. It is thus possible to vitrify specimens that are approximately 200 μm thick.

[0005] A first type of high-pressure freezing devices is represented by the Leica EM HPF and Balzers HPM 010 units. In devices of this type, liquid nitrogen is used as both the pressure transfer medium and the coolant. These high-pressure freezing devices have the disadvantage that they are comparatively large (approximately 0.8 m×1.6 m,×1.5 m) and weigh approximately 600 kg. The heavy construction means the price of such systems is high.

[0006] The freezing cycle in these systems proceeds as follows: In order to coordinate pressure buildup and cooling, the high-pressure chamber is first filled with ethanol. Cold liquid nitrogen is then fed into the pressure chamber by way of a high-pressure cylinder. The pressure chamber has an exhaust of substantially smaller dimensions than the dimensions of the supply line. Pressure in the chamber is built up by the restricted flow of pressurizing gas through the narrow exhaust. A disadvantageous effect in such systems is that a transition layer forms between the ethanol and the liquid nitrogen, which reduces the achievable cooling rate. The specimen, which is approximately 2 mm in diameter and 2000 μm thick, is located in two half-shells that are shaped in the pressure chamber.

[0007] The second type of high-pressure freezing device is represented by the Leica EM PACT unit. This high-pressure freezing device has separate circuits for pressure transfer and for cooling. This makes it possible to use relatively small equipment. A preparation holder that can withstand a pressure of 2000 bar is required, however. Pressure is built up in the preparation holder, and cooling is achieved by spraying a coolant, preferably liquid nitrogen, onto the exterior of the preparation holder. This requires a preparation holder with walls as thin as possible. One configuration of a possible preparation holder is known from EP 0 853 238 A1. The specimen is located in a tube with an inside diameter of approximately 0.3 mm. This small inside diameter has proven disadvantageous for many applications, since it can be particularly difficult to introduce the small and dimensionally unstable preparations into the tube. For many preparations (e.g. botanical preparations such as leaves or membranes) disk-shaped flakes are preferred, and as mentioned, these are difficult to introduce into the tube.

[0008] It is an object of the invention to create a specimen holder for water-containing specimens which is suitable for freezing planar specimen items under high pressure and which, in that context, makes possible easy handling of the specimen items. Attaining a high cooling rate is also intended.

[0009] This object is achieved by a specimen holder which is characterized in that a specimen retention element is arranged in the cutout of the specimen holder. The specimen retention element comprises at least a first part and a second part, a recess for specimen reception being shaped in the second part. The first and the second parts are joined to one another in such a way that the recess is sealed in pressure-tight fashion.

[0010] A further object of the invention is to create a method with which planar specimen items can be frozen under high pressure, thereby achieving efficient utilization of the coolant. The method is also intended to make possible easy handling of planar specimen items.

[0011] This is achieved, according to the present invention, by a method characterized by the following steps:

[0012] placing a specimen into the recess in the second part of the specimen retention element; and

[0013] pressing the second part of the specimen retention element against the first part of the specimen retention element by way of a screw that acts on the second part.

[0014] Lastly, it is an object of the invention to create a high-pressure freezing device which uses the specimen holder according to the present invention.

[0015] This is achieved, according to the present invention, by a high-pressure freezing device for using a specimen holder for water-containing specimens which is characterized in that

[0016] the specimen holder possesses a housing sheath in which a cutout is defined, and a specimen retention element is arranged in the cutout of the specimen holder;

[0017] the high-pressure freezing device defines a housing;

[0018] a first and a second nozzle are arranged with respect to the cutout in such a way that they lie directly opposite one another and thus direct coolant onto the specimen retention element from both sides; and that

[0019] a high-pressure coupling connects to the specimen holder independent of the first and the second nozzle.

[0020] The advantage of the invention is that the specimen holder for water-containing specimens can be connected to a system for pressure generation in order to build up pressure within the specimen holder. Cooling is accomplished by spraying a coolant from outside onto the specimen holder, in particular in the region in which the water-containing specimen is introduced into a specimen retention element. It is particularly advantageous that the specimen retention element can be split, so as thereby to be able to receive preferably flat preparations. Shaped into a second part is a recess into which the specimen items can easily be placed. In addition, the first part and the second part are each configured in such a way that they have a contact surface which forms a thermal separation between the two parts and the housing sheath.

[0021] The subject matter of the invention is schematically depicted in the drawings and will be described below with reference to the Figures, in which:

[0022] FIG. 1 shows a schematic side view of the specimen holder;

[0023] FIG. 2 shows a three-dimensional view of the specimen holder;

[0024] FIG. 3 shows a schematic side view of the specimen holder with the high-pressure coupling attached; and

[0025] FIG. 4 schematically depicts the high-pressure freezing device with the specimen holder according to the present invention.

[0026] A side view of an embodiment of specimen holder 1 is depicted in FIG. 1. Specimen holder 1 must be configured in such a way that it exhibits pressure resistance greater than 2000 bar. Specimen holder 1 should have the lowest possible mass and the greatest possible thermal conductivity in order to allow high cooling rates.

[0027] In this embodiment, specimen holder 1 comprises a housing sheath 2 that is equipped with a cutout 4. A specimen retention element 6 is placed in cutout 4. Specimen retention element 6 comprises a first part 6aand a second part 6b.During cooling, the coolant, preferably liquid nitrogen, is sprayed onto specimen retention element 6 in cutout 4 from both sides, onto first and second parts 6aand 6b.

[0028] First part 6aof specimen retention element 6 possesses a constriction 8, i.e., a portion of first part 6awith a reduced cross section, at which first part 6alies against housing sheath 2 of specimen holder 1. Constriction 8 of first part 6afurthermore has, on the side facing toward housing sheath 2, a protrusion 9 at which first part 6alies directly against housing sheath 2. Second part 6bis provided opposite first part 6a.Configured in second part 6b,on the side located opposite first part 6a,is a recess 10 that is suitable for receiving the specimen items. Recess 10 is preferably disk-shaped, in order to be able to receive correspondingly shaped specimen items. Opposite recess 10, second part 6bpossesses a depression 12 that is preferably configured in the form of a half-sphere.

[0029] A screw 14 is also arranged in housing sheath 2 of specimen holder 1, in such a way that it acts on second part 6bof specimen retention element 6. Screw 14 possesses a conical tip 16 at the end located opposite depression 12 of second part 6b.As screw 14 is screwed in, conical tip 16 engages into depression 12 of second part 6b.Screw 14 presses second part 6bagainst first part 6a.The two parts 6aand 6bhave directly opposing surfaces which are configured such that the surfaces, fitted to and pressed against one another, seal off recess 10 from the outside in pressure-tight fashion. As already mentioned above, first part 6apossesses protrusion 9 which directly contacts housing sheath 2; and second part 6bpossesses depression 12 which is connected via the screw to housing sheath 2. It is clearly evident from FIG. 1 that the result of this is a small contact surface between housing sheath 2 and first part 6aand second part 6b,respectively. This arrangement minimizes thermal conduction between the specimen retention element 6 and the housing sheath 2, thereby resulting in a thermal separation, which yields a savings in coolant during cooling. In addition, rapid heating after cooling is prevented.

[0030] First part 6ais equipped with a bore 18 that aligns with a corresponding bore 19 in housing sheath 2. Bore 18 in the first part ends at the surface of first part 6awhich lies opposite recess 10 for specimen reception. A tube 20 is guided in bores 18 and 19 and, in the present embodiment, adhesively bonded in pressure-tight fashion to bore 18 in first part 6aand bore 19 in housing 2. Tube 20 possesses an end 22 that projects out of housing sheath 2. End 22 is shaped conically so that a high-pressure coupling (see FIG. 4) can be connected there.

[0031] The method for using specimen holder 1 comprises the following steps. Before the specimen is put in place, the specimen must be correctly cut to a specific shape. The specimen piece (or cut piece) is placed into recess 10 of second part 6b.Second part 6bis then placed against first part 6a.Screw 14 is screwed in, and depression 12 and conical end 16 of screw 14 thereby contact, causing second part 6bto be pressed against first part 6a.The housing sheath 2, together with the specimen located between the first and second parts 6aand 6b,is then inserted into the high-pressure freezing device. In this context, end 22 of tube 20 projecting out of housing sheath 2 is correspondingly connected to a high-pressure coupling.

[0032] The coolant (liquid nitrogen) is then sprayed in the region of cutout 4 of housing sheath 2. The small contact surfaces between second part 6band screw 14, and between first part 6aand housing sheath 2, act thermally separate the specimen retention element 6 from the housing sheath 2 and screw 14. During cooling, as already mentioned above, the two parts 6aand 6bare entirely bathed in coolant, so that they cool down substantially more quickly than housing sheath 2. A considerable amount of liquid nitrogen for cooling is thereby saved, since a smaller mass needs to be cooled. Once cooling under pressure is complete, the low temperature is maintained and specimen holder 1 is opened with screw 14, and the preparation is removed along with part 6b.For subsequent specimen preparation, for example cryosubstitution, it is advisable to leave the specimen in second part 6bacting as a support, in order to simplify handling.

[0033] A perspective view of specimen holder 1 is depicted in FIG. 2. Specimen holder 1 is of substantially cylindrical configuration. Cutout 4 in housing sheath affords a view of first part 6aand second part 6bof specimen retention element 6. Cutout 4 is arranged in such a way that first and second parts 6aand 6bof specimen retention element 6 are freely accessible from both sides of specimen holder 1. Also evident is conical tip 16 of screw 14 which presses second part 6bagainst first part 6a.

[0034] FIG. 3 shows a cross section through specimen holder 1 with a high-pressure coupling 30 attached. High-pressure coupling 30 has shaped onto it a connector piece 32 that fits conformingly with end 22 of tube 20. The connector piece 32 transfers the pressure provided by the high-pressure coupling 30 via tube 20 to recess 10 of second part 6bof the specimen retention element 6. Screw 14, screwed into housing 2, secures second part 6bagainst the high pressure and, because it is screwed in, ensures that recess 10 is sealed off in pressure-tight fashion from the outside.

[0035] A schematic overall view of a high-pressure freezing device 40 that uses specimen holder 1 according to the present invention is depicted in FIG. 4. High-pressure freezing device 40 comprises a housing 42. An electronics unit 44, which processes user inputs and converts them into corresponding control signals, is provided in housing 42. Electronics unit 44 is connected to high-pressure generator 43 to ensure that the desired pressure acts on the specimen in recess 10. High-pressure coupling 30 couples the pressure from high-pressure generator 43 into specimen holder 1. Electronics unit 44 is also connected to a coolant reservoir 46 in order to maintain or interrupt the delivery of coolant into the region of specimen retention element 6. An interruption in coolant delivery is necessary when specimen retention element 6 has been cooled to the desired temperature. Liquid nitrogen is used as the coolant. The coolant is introduced into the region of specimen retention element 6 by way of a first and a second nozzle 47aand 47b.In the present embodiment, the coolant is sprayed out from the first and second nozzles 47aand 47bonto specimen retention element 6. The path traveled by the coolant from the first and second nozzles 47aand 47bonto specimen retention element 6 is depicted by arrows 48aand 48b. Also provided, on the exterior of the housing, is an input unit 49 with which a user can easily start the cooling operation or modify parameters.

[0036] The invention has been described with reference to one embodiment, but it is apparent that one skilled in the art can make modifications without thereby leaving the range of protection of the claims recited hereinafter.

[0037] 1 Specimen holder

[0038] 2 Housing sheath

[0039] 4 Cutout

[0040] 6 Specimen retention element

[0041] 6aFirst part

[0042] 6bSecond part

[0043] 8 Constriction

[0044] 9 Protrusion

[0045] 10 Recess

[0046] 12 Depression

[0047] 14 Screw

[0048] 16 Conical tip

[0049] 18 Bore

[0050] 19 Bore

[0051] 20 Tube

[0052] 22 End

[0053] 30 High-pressure coupling

[0054] 32 Connector piece

[0055] 40 High-pressure freezing device

[0056] 42 Housing

[0057] 43 High-pressure generator

[0058] 44 Electronics unit

[0059] 46 Coolant reservoir

[0060] 47aFirst nozzle

[0061] 47bSecond nozzle

[0062] 48aArrow

[0063] 48bArrow

[0064] 49 Input unit