Refrigeration – Processes – Treating an article
Reexamination Certificate
2001-03-30
2003-02-11
Capossela, Ronald (Department: 3744)
Refrigeration
Processes
Treating an article
C062S373000
Reexamination Certificate
active
06516620
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German patent application DE 100 15 773.4, filed Mar. 30, 2000, which is incorporated by reference herein.
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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 &mgr;m thick.
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.
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 &mgr;m thick, is located in two half-shells that are shaped in the pressure chamber.
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.
SUMMARY OF THE INVENTION
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.
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.
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.
This is achieved, according to the present invention, by a method characterized by the following steps:
placing a specimen into the recess in the second part of the specimen retention element; and
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.
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.
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
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;
the high-pressure freezing device defines a housing;
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
a high-pressure coupling connects to the specimen holder independent of the first and the second nozzle.
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.
REFERENCES:
patent: 4107937 (1978-08-01), Chmiel
patent: 1 806 741 (1969-06-01), None
patent: 0 853 238 (1998-07-01), None
Leica EM HPF High-Pressure Freezer, pp. 1-4, 1994.
Capossela Ronald
Leica Mikrosysteme GmbH
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