JPH0717051Y2 - Cryomicroscope sample cooling device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION << Industrial Application Field >> In recent years, it relates to a freezing technique for preserving living tissues including human bodies such as blood components, bone marrow cells, spermatozoa, fertilized eggs, etc. while maintaining the activity of the tissues and cells. Research is actively carried out,
This has been useful in the advancement of biotechnology and medical fields, or in securing and preserving excellent genetic resources.

In such research, observing the freezing process of various samples as described above using a cryomicroscope is
It is an extremely important method for finding the optimum freezing process and freezing technology.

The present invention mainly relates to a sample cooling device for a cryomicroscope used for such a purpose.

<< Prior Art >> As a cooling device of this type, as shown in FIG.
A simple container-shaped housing d having a space a inside and observation windows b and c in the center of both surfaces is used, and a refrigerant supply pipe e and an exhaust pipe f are internally provided in the space a. The space a of the housing d in which the sample g is stored is cooled by spraying the refrigerant onto the space.

As another sample cooling device, a device having a cooling chamber in a gas seal case (housing) is disclosed in Japanese Patent Laid-Open No. Sho 59-59.
-218417.

The cooling device of this known example is generally the same as that shown in FIG. 7 in which the entire device is covered with a housing and the inside of the housing is filled with a dry gas.

<Problems to be solved by the invention> However, such freezing of a biological sample is performed under strict temperature control in order to maintain its activity at a high level, and even to a very low temperature of, for example, −150 ° C. or lower. Therefore, it is necessary to cool the liquid, and as the cold source, liquefied nitrogen suitable for cryogenic cooling, and sometimes cryogenic liquefied gas refrigerant such as liquefied helium is often used, and the temperature control means is often by program control.

However, according to the conventional device of FIG. 7, the refrigerant is sprayed into the space a of the housing d in which the sample g is immediately stored to cool the space. Therefore, the space a is only the wall d ′ of the housing d. Since it is in contact with the outside through the outside, there is a large amount of heat invasion (release of cold) from the outside, and it directly affects the internal atmosphere temperature, making it difficult to maintain it at an extremely low temperature. Shows a temperature gradient as shown in FIG. 8, and from this point as well, the temperature tends to be unstable, and inconvenience is likely to occur in terms of temperature control.

Therefore, in order to avoid this, it is conceivable to form the housing d with a heat insulating material such as urethane foam.
In a cryo-microscope, as shown in FIG. 7, the space for the cooling device of the sample table i is limited due to the working distance of the objective lens h, so that the thickness of the heat insulating material cannot be made large, for example. It is not possible to improve the insulation performance to the extent that it can maintain −150 ℃ for several minutes.

Further, since the refrigerant is directly sprayed into the space a of the housing d accommodating the sample g, an air flow is generated inside, which causes the sample g to sway (vibrate), which causes a problem in observation. Have

Further, the known cooling device can solve the above-mentioned fog problem because the inside of the housing is filled with the dry gas, but the sample is rapidly cooled in a stable frozen atmosphere in a heat insulation state with the outside world. It does not satisfy the requirement of freezing and does not solve the instability of the sample due to the fluidity of the refrigerant.

The present invention has been made in view of the above problems of the conventional device, in which the central portion surrounded by the intermediate portion is
A cooling chamber formed of a sample mounting plate made of a translucent plate-shaped body is provided with a structure arranged in a substantially central portion of a container-shaped housing internal space that is sealed from the outside. The cooling chamber can be cooled by spraying the cooling medium into the hollow portion of the cooling chamber, and the cooling chamber is surrounded by the housing through the internal space, and the supply pipe and the exhaust pipe or the hot air supply pipe are provided. Only with the cooling chamber communicating with the outside, the cooling chamber can be rapidly cooled and it is easy to maintain stable cryogenic temperature, and strict temperature control can be realized. The sample is surrounded by the hollow part of the cooling chamber, and the sample mounting plate is cooled by the thermal contact with the cooling chamber, following the cooling chamber temperature. In addition, avoid the inconvenience that the sample is shaken by being placed outside the cooling chamber where the refrigerant is sprayed, and the internal space of the housing is separately purged with dry nitrogen gas or the like. The purpose is to prevent the window for microscope observation, the window for the light source, the micro slide glass, and the like from being fogged by frosting or the like.

<< Means for Solving the Problem >> The sample cooling device for a cryomicroscope according to the present invention is characterized by the following means in order to achieve the intended purpose. That is, in a sample cooling device for observing a process in which a sample is cooled and frozen through cryogenic liquefied gas with a cryogenic microscope of an optical system, a cooling chamber, a housing, a cryogenic liquefied gas supply pipe, and an exhaust pipe are provided. And a cooling chamber having a sample mounting plate formed of a translucent plate and a hollow portion surrounding the sample mounting plate, and a container shape The housing has an internal space sealed to the outside, and a purge gas supply system and a discharge system for keeping the internal space in a dry state, and face each other across the internal space. Of the two housing walls that are present, one side has a microscope observation window and the other side has a light source window, and these windows face each other.
And the cooling chamber is arranged between the microscope observation window and the light source window in the inner space of the housing, and a gap is provided between the cooling chamber and each housing wall, and The low-temperature liquefied gas supply pipe and the exhaust pipe penetrate the housing wall and communicate with the hollow portion of the cooling chamber.

<Operation> On the lower surface of the sample mounting plate of the cooling chamber in the sample cooling device configured as described above, the sample stored in the micro slide glass or the like is attached to aluminum tape, mylar tape, or the like, and the cooling chamber is attached. The housing lid of the housing and the housing body are joined with a flange or the like, and the O-ring is used for sealing.

Further, nitrogen gas or the like is introduced from the dry gas container such as nitrogen gas into the housing through the purge gas supply pipe, and the inside of the housing is purged while exhausting from the purge gas exhaust pipe,
In a dry state, a refrigerant at a desired temperature is supplied from a cryogenic refrigerant container such as liquefied nitrogen to the cooling chamber and exhausted from an exhaust pipe.

At this time, since the cooling chamber is arranged in the inner space of the housing with a gap around it, and only the supply pipe and the exhaust pipe are in thermal communication with the outside, heat intrusion from the outside is extremely high. Therefore, the cooling chamber can be cooled rapidly, and it becomes easy to maintain a cryogenic temperature, and strict temperature control can be performed.

The sample is also surrounded by the cooling chamber, and the temperature is almost the same as that of the cooling chamber due to solid heat conduction through the sample mounting plate.

Since the space in which the sample is stored is inside the housing and there is no gas flow due to the refrigerant, there is no inconvenience that the sample vibrates during cooling or observation.

<< Embodiment >> An embodiment of the present invention will be described below with reference to the drawings.

As shown in FIGS. 1 to 3, the cooling chamber 1 is formed in a hollow donut shape, and the hollow portion 2 surrounds the central hole portion 3.

The cooling chamber 1 is preferably formed of a metal material having good heat conductivity such as copper and aluminum, and is formed into a seal structure by means of welding or brazing the constituent members.

The upper bottom portion of the cooling chamber 1 in the drawing is preferably formed by a sample mounting plate 4 made of a light-transmissive plate such as sapphire, hard glass, or quartz glass having good thermal conductivity, and a sample mounted in contact therewith. 5 can be cooled by solid heat conduction, the micro slide glass 6 accommodating the sample 5 can be easily attached, and a normal slide glass 6'can be used as shown in FIG.

In the cooling chamber 1, a supply pipe 7 for supplying a cryogenic liquefied gas refrigerant such as liquefied nitrogen into the hollow portion 2 of the cooling chamber 1 at an appropriate position, for example, the upper surface 1a as shown in the drawing, The exhaust pipe 8 is connected by a sealable means such as welding or brazing. At this time, the supply pipe 7 and the exhaust pipe 8 are preferably provided in a symmetrical arrangement as shown in the drawing. .

Further, if the spray nozzle 9 is provided at the tip of the supply pipe 7, the refrigerant is easily atomized, which is advantageous in that it is cooled more uniformly.

The sample mounting plate 4 is arranged on the lower surface of the cooling chamber 1 in the inverted microscope and on the upper surface of the cooling chamber 1 in the upright microscope, and is arranged in the working distance range of the objective lens. As is known, in the inverted microscope, the objective lens is upward, and in the upright microscope, the objective lens is downward.Because the illustrated example shows the case of the inverted microscope, The lens 10 is arranged upward as shown in FIG.

It should be noted that the various cryogenic microscopes exemplified above are of optical systems, as is obvious. Housing as shown in Fig. 1
Reference numeral 11 includes a container-shaped housing body 11a, and a housing lid 11b which is obtained by previously suspending the cooling chamber 1 by a supply pipe 7 and an exhaust pipe 8 and combining them.

At the center of the housing body 11a and the housing lid 11b, a transparent plate-shaped objective lens 10, that is, a microscope observation window 12 and a light source window 13 are provided.

The housing body 11a and the housing lid 11b are detachably fastened, for example, by screwing or by fastening the respective flanges 14 and 15 with bolts, and the housing 11 is sealed from the outside by an O-ring 16 or the like.

In this way, the cooling chamber 1 is held in the central portion of the internal space 17 of the housing 11 with a gap from the surroundings and is suspended in this state only by the supply pipe 7 and the exhaust pipe 8. Therefore, no other person has thermal communication with the outside.

Further, a purging nitrogen gas inlet pipe 18 and a purging nitrogen gas exhaust pipe 19 are respectively connected to the housing body 11a, and the temperature sensor 20 is in contact with or not in contact with the micro slide glass 6. It is arranged.

When the cooling chamber 1 is cooled, a hot air pipe (not shown) is separately arranged, and hot air is also supplied to the cryogenic liquefied gas supplied from the supply pipe 7 to control the temperature of the cooling chamber 1. You may do it.

Further, the cooling chamber 1 is not limited to the circular donut shape, but may be a quadrangle or an arbitrary polygonal shape in addition to the circular donut shape.

Further, it can be applied to an upright low-temperature microscope by reversing the above-mentioned configuration. Further, the entire cooling chamber 1 is formed of a metal material or the like, and then the sample mounting plate 4 is placed on the lower surface.
It is also possible to arrange.

The sample mounting plate 4 of the cooling chamber 1 is provided with the central hole portion 3 and the bottom portion is a translucent plate-like body as in the above-mentioned embodiment, but the present invention is not limited to this, and is shown in FIG. As illustrated, by closing the central hole portions 3'3 'provided on the upper surface 1a and the lower surface 1b of the cooling chamber 1 with a light-transmissive plate member, the sample mounting plate can be placed on the upper and lower surfaces 1a, 1b. 4, 4 may be provided so as to abut the hollow portion 2, and as shown in FIG. 5, the central hole portion 3 is penetrated through the cooling chamber 1, and the central hole portion 3
The sample mounting plate 4 can be surrounded by the hollow portion 2 by disposing the translucent plate-shaped body at a high place in the center of the inside.

However, when the sample mounting plate 4 is provided inside the central hole 3, the problem of the distance between the sample 5 and the objective lens 10 housed in the micro slide glass 6 or the like attached to the sample mounting plate 4 Since the distance becomes longer, the microscope observation window 12 on the side of the objective lens 10 in the housing 11 is dropped inward by a required dimension as illustrated in FIG. 5 to avoid the above problem. Good

In FIG. 1, 21 is a dry gas container, 22 is a cryogenic refrigerant container, 23 and 24 are observation window fixing rings, 25 is a refrigerant solenoid valve, 26 is a refrigerant on-off valve, 27 is a purge gas solenoid valve, 28 is an on-off valve for purge gas, 29 is a solenoid valve for releasing purge gas, 30
Is a heater, and 31 is a controller for controlling the above 25, 27 and 30 by an input signal from the temperature sensor 20.

<< Effects of the Invention >> The sample cooling device for the cryomicroscope according to the present invention has the following effects.

One of them is that the sample held by the sample mounting plate is rapidly cooled and frozen in a stable frozen atmosphere in a heat insulation state with the outside world.

In other words, the cryogenic chamber, the light source, etc. that cause the heat fluctuation are not included in the housing containing the cooling chamber, and the sample mounting plate is in the hollow part (refrigerant supply part) of the cooling chamber in the housing. Since it is surrounded by, the temperature distribution in the housing is good with a cryogenic region near the sample mounting plate, and therefore
The sample follows the cooling chamber temperature due to the close thermal contact between the hollow portion and the sample mounting plate, and is frozen rapidly and stably.

The other is that only the cooling chamber and its related members are arranged in the housing, so that the temperature near the sample mounting plate can be strictly controlled mainly by the cooling chamber.

Furthermore, the other is that the fluidity of the refrigerant does not spread to the sample at all, so that the state of holding the sample by the sample mounting plate is stable, and therefore, there is no hindrance during microscope observation.

One other than the above is that the interior space of the housing is filled with the drying gas (purging gas), so that the window for microscope observation, the window for the light source, and others do not fog due to frost formation. In this respect, there is no problem in microscopic observation.

[Brief description of drawings]

FIG. 1 is a vertical sectional front view of an embodiment showing a sample cooling device for a cryomicroscope according to the present invention, and FIGS. 2 (a) and (b) are plan views and vertical sectional front views of a cooling chamber in the cooling device, respectively. FIG. 4 is a vertical sectional front view of a cooling chamber showing a state where a sample is placed on a sample mounting plate in the cooling device using a normal slide glass, and FIG. 4 is a vertical sectional front view showing another embodiment of the cooling chamber in the cooling device. Fig. 5 is a vertical sectional front view showing a different embodiment of the cooling device, Fig. 6 is a temperature gradient diagram in the cooling device, Fig. 7 is a vertical sectional front view showing a conventional cooling device, and Fig. 8 is conventional. 3 is a temperature gradient diagram in the cooling device of FIG. 1 ... Cooling chamber 2 ... Hollow part 4 ... Sample mounting plate 7 ... Supply pipe 8 ... Exhaust pipe 11 ... Housing 12 ... Microscope observation window 13 ... Light source window 17 ... Internal space 18 ...... Purge gas inlet pipe 19 ...... Purge gas exhaust pipe

Claims (1)

[Scope of utility model registration request] 1. A sample cooling device for observing a process in which a sample is cooled and frozen through a cryogenic liquefied gas by a cryogenic microscope of an optical system, comprising: a cooling chamber, a housing, a cryogenic liquefied gas supply pipe, An exhaust pipe, and the cooling chamber has a sample mounting plate formed of a translucent plate and a hollow portion surrounding the sample mounting plate, and The container-shaped housing has an internal space sealed to the outside and a purge gas supply system and a discharge system for keeping the internal space in a dry state. Of the two housing walls facing each other, a microscope observation window is formed on one of them, and a light source window is formed on the other side, and these windows are opposed to each other.
And the cooling chamber is arranged between the microscope observation window and the light source window in the inner space of the housing, and a gap is provided between the cooling chamber and each housing wall, and A sample cooling device for a low-temperature microscope, wherein a low-temperature liquefied gas supply pipe and an exhaust pipe penetrate a housing wall and communicate with a hollow portion of a cooling chamber.