JPS6135945Y2 - - Google Patents

Description

[Detailed Description of the Invention] This invention relates to an apparatus for rapidly freezing samples such as biological tissues when observing biological tissues using an electron microscope.

When observing biological tissue with an electron microscope, the tissue must be fixed by some means.
Conventionally, a so-called chemical fixation method, in which tissue is chemically fixed, has generally been adopted as this fixing means. However, when the purpose is to study biological phenomena, it is necessary to investigate the distribution of protein particles that move inside and outside the cell membrane, but the conventional chemical fixation methods described above only utilize the coagulation effect of proteins. As a result, floating protein particles aggregate and solidify due to this coagulation effect, resulting in the production of giant protein particles that are different from the living state, or the tendency for protein particles to become unevenly distributed in specific locations. It has a fatal flaw, as mentioned above.

Therefore, in order to immobilize specimens in a living state, such as biological tissues, which maintain life only in the presence of water, so-called freezing techniques have been developed, in which tissues are frozen and immobilized without chemical fixation. Fixed methods have been proposed. This freeze-fixation method physically fixes the tissue, so it is the most effective means of preventing the movement and aggregation of migrating protein particles. The movement of people must be taken into consideration.

The growth of ice crystals is highly dependent on the freezing rate of the sample, so to prevent the growth of ice crystals, it is necessary to cool the sample from a state above the freezing temperature of water as quickly as possible. Research reports have shown that fresh samples without any agents require a cooling rate of 1000°C or more per second. Therefore, in order to rapidly freeze the sample, the sample is sufficiently cooled with cryogenic liquefied gas such as liquid helium, and the sample is pressed onto the surface of a metal block that has a sufficiently large heat capacity compared to the sample. The method of absorbing it into this metal block is adopted. However, in this case, the sample must be kept above the freezing temperature of water and instantly cooled by pressing it onto a metal block. Otherwise, ice crystals may grow and the surface of the sample may deteriorate before the sample is rapidly frozen. Freezing occurs, destroying the sample structure and impeding heat absorption.

This idea was developed by focusing on these problems when freezing a sample, and it involves keeping the sample above the freezing temperature until just before pressing it onto the metal block, and then instantly pressing it onto the metal block from this state. It is designed to freeze quickly, so that a good frozen state can be obtained from the surface of the sample to the deep part of the sample.

The configuration of this invention will be explained in detail below based on an illustrated embodiment.

As shown in FIG. 1, a liquefied gas 1 such as liquid helium, liquid hydrogen, or liquid nitrogen is stored in a container 2 that is insulated from the outside. Container 2 shown
consists of multiple vacuum containers 3 and 4 with double inner and outer walls, and a cooling body 5 such as liquid nitrogen is housed between these multiple containers 3 and 4 for pre-cooling.

A holder 6 made of copper or silver having good heat conductivity is housed in the container 2, and the holder 6 is fixed to the lid 8 of the container 2 by a support 7. This holder 6 houses a metal block 9 made of a metal with high thermal conductivity such as high-purity copper, and this metal block 9 and the holder 6 make the sample s larger overall than the sample s to be frozen. Heat capacity is ensured.

The liquefied gas 1 stored in the container 2 is supplied from a container (not shown) through the transfer tube 10, and its supply is stopped by closing the valve 11 of the transfer tube 10. On the other hand, the gas evaporated in the container 2 is discharged through the exhaust tube 12.
through to the atmosphere or to a recovery tank (not shown), either naturally or forced by vacuum pump 13.

A sample introduction pipe 14 is provided on the lid 8 of the container 2 from the outside toward the upper surface of the metal block 9. This sample introduction pipe 14 is made of a material with relatively high electrical resistance, such as stainless steel, and a current is passed through it through lead wires 15, 15, and the heat generated thereby causes the pipe 14 to The inside is kept warm. As shown in FIG. 2, the outer periphery of this sample introduction pipe 14 is covered with a heat insulating member 16, and this heat insulating member 16 includes, for example, a fluororesin 17 that covers the outer periphery of the sample introduction pipe 14, and a foamed resin 17 that covers the outer periphery of the sample introduction pipe 14. Resin 18
It consists of such things as

A shutter 19 is provided at the lower end of the sample introduction pipe 14 facing the top surface of the metal block 9, and this shutter 19 is used to protect the introduction pipe 1 from the introduction pipe 1 during normal times.
Plunger 20 for sample insertion.
Developed when the system is introduced.

To cool the sample s with this device, the container 2
The liquefied gas 1 is stored inside the holder 6, and the metal block 9 is sufficiently cooled through the holder 6.
A current is passed through the sample introduction pipe 14 to keep the inside of the pipe warm so that it does not fall below the freezing temperature of water, that is, 0°C. Generally, it is desirable to maintain the temperature near the tip of the sample introduction pipe 14 at about 4 to 5°C.

Next, the sample s is attached to the sample holder 21 at the tip of the plunger 20, and the plunger 20 is introduced into the sample introduction pipe 14 and allowed to fall freely from an appropriate height. Then, the plunger 20 falls while pushing open the shutter 19 with its tip, and the sample holder 21
hits the top surface of the metal block 9, so the sample s is pressed against the metal block 9. Therefore, when the sample s is inside the sample introduction pipe 14, the temperature is 0°C.
Since it is kept at a higher temperature, it does not freeze, but the moment it is pressed against the metal block 9, the heat it retains is absorbed by the metal block 9, and it is quickly cooled and frozen.

As explained above, according to this invention, the sample s is introduced through the sample introduction pipe 14 kept at a temperature higher than the freezing temperature of water, so the sample s is frozen until just before it is pressed onto the metal block 9. It freezes rapidly from the moment it is crimped. Therefore, the growth of ice crystals and freezing of the sample surface can be prevented, and a good frozen state can be obtained.

In addition, since a current is passed through the sample introduction pipe 14 and it generates heat itself, it is possible to keep the temperature uniform without causing unevenness, for example when a heater is wrapped around the pipe, and the temperature can be easily controlled. Become.

[Brief explanation of the drawing]

FIG. 1 is a longitudinal sectional side view showing an embodiment of this invention, and FIG. 2 is a half sectional perspective view showing an embodiment of the sample introduction pipe. 1...Liquefied gas, 9...Metal block, 14
...Sample introduction pipe, 19...Shutter, 20...
...plunger, s...sample.

Claims (1)

[Claims for Utility Model Registration] 1. By striking the tip of a plunger against the surface of a metal block that has good heat conduction and is cooled by liquefied gas such as liquid helium, a sample attached to the tip of the plunger can be attached to the metal block. In the sample quick freezing device, which is crimped onto the surface of a block and allows the metal block to absorb the heat held by the sample and quickly freeze the sample, a sample introduction pipe is provided that introduces a plunger from the outside, and the sample is A sample rapid freezing device in which the introduction pipe is made of a metal material with relatively high electrical resistance, and the inside of the pipe is kept at a temperature above the freezing temperature of water by passing an electric current through the pipe and generating heat. 2. The freezing device according to claim 1, in which the sample introduction pipe is made of stainless steel. 3. The freezing device according to claim 1 or 2 of the utility model registration, wherein a shutter is provided at the tip of the sample introduction pipe facing the surface of the metal block and is opened by introducing a plunger.