Refrigeration – Storage of solidified or liquified gas – Including cryostat
Reexamination Certificate
2000-06-07
2001-08-07
Doerrler, William (Department: 3744)
Refrigeration
Storage of solidified or liquified gas
Including cryostat
C062S078000
Reexamination Certificate
active
06269649
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of the Swiss Patent Application CH1260/99.
FIELD OF THE INVENTION
The invention relates to a high-pressure freezing system in accordance with the characterizing clause of claim
1
. In the sense of the invention, a high-pressure freezing system is understood to be a freezing device for the rapid freezing (vitrification) of aqueous samples under high pressure.
BACKGROUND OF THE INVENTION
Information on the physics of such a freezing process can be found in the German Patent DE-PS-1806741 and the European Patent Application EP-A-0 853238.
In professional circles, for example, such a known system of the applicant is employed, which is successfully marketed under the name “Leica EM HPF”™ and described in the publication “LEICA EM HPF, High Pressure Freezer,
1
.K.-LEICA EM HPF-E-6/94, Jun. 1994”.
The “Leica EM HPF”™ allows the vitrification of conventional samples under a pressure of ca. 2000 bar at a cooling rate of 10
3
-10
5
K/s. For the said appliance, the crucial cooling phase from ambient temperature to −100° C. takes ca. 10 ms (cooling rate 10
4
K/s) at the surface of the sample. Consequently, all samples with a thickness of ca. 200&mgr;m are cooled to -100° C. in ca. 50 ms.
The known cooling technology and the physical processes show clearly: Applying an infinitely high cooling rate to the surface of a biological sample only partially determines the rate of cooling in its centre. A sample of thickness 200 &mgr;m, for example, exhibits a cooling rate in its centre of ca. 6000 K/s, regardless of whether an infinite cooling rate is employed at the surface or a cooling rate of ca. 10,000 K/s. The cooling rate is not determined by the pressure. By increasing the pressure, a lower cooling rate is sufficient for vitrification. Therefore, the cooling rate for the vitrification of biological samples is under standard pressure ca. 10
5 -
10
6
K/s, tinder 2000 bar, however, the cooling rate is only 10
3-
10
4
K/s, i.e., it is still possible to vitrify biological samples under high pressure using cooling rates a hundredfold lower.
The inventor referred in the article “A NEW CONCEPT AND MACHINE FOR HIGH PRESSURE FREEZING” from 15.3.1999, published in the internet under the address “www-mem.unibe.ch/~ danis/abstract HPF”, to these and further aspects of high-pressure freezing. He proposed increasing the pressure from 1 bar to 2000 bar within 10 ms by employing a compressed-air cylinder in place of the well-known very bulky and heavy equipment.
Pneumatic cylinders are already employed in a wide variety of applications. Using them to quickly build up high pressures, however, necessitates the use of large and heavy pumps.
SUMMARY OF THE INVENTION
Consequently, the first problem to be solved in the invention is the construction of a high-pressure freezing system, which is lighter than the hitherto known systems but which nevertheless allows a rapid build-up of pressure.
The above problem is solved by the features of independent claim
1
and claim
11
which is a reduction in the constructional size and yet an accelerated pressure build-up.
The dependent claims
2
to
6
and
12
to
18
refer to improved solutions with extensive integration and extensive advantages over the state of the art.
However, the invention is based on a further problem: The aim is a simple pressure adjustment in the range of ca. 1500-2000 bar. The reason for this being that it is known from technical literature that biological samples exposed to pressures exceeding 1600 bar can exhibit irreversible damages. In accordance with the invention, this is possible by merely varying the pneumatic pressure at the prestressed pneumatic cylinder; i.e., altering the input pneumatic pressure at the pneumatic cylinder during the prestressing phase can result in a different driving power at the output end leading to a different or adjustable pressure build-up in the high-pressure cylinder.
In addition, as, in accordance with the invention, pressure build-up and cooling are separated, it is possible to cool the sample using liquid nitrogen to 196° C. In boundary ranges of 200&mgr;m, in particular, the cell structure remains undamaged using the procedure of the present invention, which can be allowed for with no difficulty in the selection of the sample dimensions.
During vitrification, very low cooling temperatures, as acquired using liquid and undercooled nitrogen, for example, are of course advantageous, provided that the nitrogen itself —as indicated sporadically in the state of the art —is not exposed to these high pressures. If the undercooled nitrogen were to be used as pressure carrier, this would constitute a potential source of danger at the high pressures and it would not be possible to attain a temperature of -196° C. as the nitrogen is already solid at -196° C. under pressures exceeding 1500 bar.
The dependent claims
7
to
10
and
19
to
24
relate to improvements which can be applied independently to advantage other known high-pressure freezing systems or those still to be designed. This concerns in particular a novel and improved holding device for a sample container in accordance with the European Patent Application EP-A-0 853 238 of the inventor. A new holding device should facilitate the handling of the known sample container and accelerate the changing of sample containers or samples. In particular, this should accelerate the throughput of samples through a vitrification plant and thus achieve an improvement in economic efficiency.
REFERENCES:
patent: 4559298 (1985-12-01), Fahy
patent: 4688387 (1987-08-01), Conaway
patent: 4745764 (1988-05-01), Sitte et al.
patent: 5493865 (1996-02-01), Wohlwend
patent: 1 806 741 (1969-06-01), None
patent: 87 13 605 (1988-01-01), None
patent: 0 853 238 (1998-07-01), None
LEICA EM HPF; High Pressure Freezer; 1.K.-LEICA EM HPF-E; Jun. 1994; 2 Sheets.
Doerrler William
Foley & Lardner
Leica Mikrosysteme AG
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