JP2001004506A - Apparatus and method for preparing sample for electron microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rapidly and effectively fix an organism tissue by irradiating a sample obtained by dipping the tissue in a fixative or the like contained in a container with a microwave, and applying a pressure to the sample. SOLUTION: The apparatus for manufacturing a sample of an electron microscope comprises a microwave irradiating means for irradiating a sample 4 obtained by dipping the tissue in a fixative or the like contained in a container 3 with a microwave 20, and a pressurizing means for applying a pressure to the tissue. That is, a waveguide 11 for guiding the microwave 20 generated from a magnetron 10 into a chamber 2 is opened so as to be opposed to the container 3. A pressurizing member 8 for pressurizing the sample 4 contained in the container 3 is provided vertically movably by a cylinder 7 above the chamber 2. Since this method rapidly permeates the tissue by the fixative and accelerates crosslinking of a protein for constituting the tissue, the sample can be rapidly and effectively prepared.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus and a method for quickly and securely fixing a sample for an electron microscope, and more particularly, to a method using microwave irradiation means and pressure means independently. Also, the present invention relates to an electron microscope sample preparation apparatus, an electron microscope sample preparation method, and the like that can quickly and reliably fix a biological tissue by using them in a freely combined manner.

[0002]

2. Description of the Related Art Conventionally, the results of observing biological tissues of animals and plants with an electron microscope largely depend on sample preparation techniques, and various studies have been made to fix collected biological tissues so as to maintain their state in a living body as much as possible. Is being done.

In this sample preparation technique, in recent years, attempts have been made to fix biological tissues by irradiating a microwave (microwave), which is one of electromagnetic waves, with some success.

Although there is no established knowledge as to why this microwave is effective for fixing biological tissues, it is generally understood as a heat fixing effect by microwaves and a rapid penetration effect of a fixing agent into biological tissues. Have been.

More specifically, the heat fixation effect of microwaves is such that water molecules, low-molecular sugars, amino acids, and the like in biological tissues efficiently absorb microwaves and cause molecular motion by microwave irradiation. It is thought that the heat is transmitted to the surroundings, and is promoted by promoting the cross-linking of the fixing agent penetrating into the biological tissue and the protein constituting the biological tissue.

[0006] The quick penetration effect of the fixative into biological tissues is due to the fact that water as a liquid usually exists as a pentamer due to hydrogen bonds between water molecules, but the hydrogen bond is broken by microwave irradiation, and the fixative is immobilized. Is likely to be caused by the ability to rapidly pass between water molecules or by the immobilization agent being guided by the microwave and momentarily carried into the biological tissue.

In any case, it has been clarified by various experiments that the sample preparation time can be greatly reduced by the microwave. Therefore, an apparatus and a method devised to effectively irradiate the sample with the microwave are disclosed. Various proposals have begun. For example, the inventor of the present application has developed an apparatus for preparing an electron microscope sample obtained by improving a microwave oven developed for commercial food cooking, and has achieved certain effects.

[0008] The structure of this device is shown in FIG.
Based on the above, a brief description will be given. Microwave oven 100 used
Can be appropriately selected. For example, Toshiba ER-4
35JF type (voltage 100 V, high frequency output 500 W, oscillation frequency 2450 MHz) can be used.

The microwaves 100 can be radiated stably by removing the wiring from the transformer (not shown) built in the microwave oven 100 into the magnetron (not shown) and adding a new heater transformer. To do.

On a turntable 103 provided in a microwave oven 102, a container 106 containing a sample 107 (a biological tissue immersed in a fixing solution) is placed. At this time, in order to prevent an excessive rise in the temperature of the sample 107, an ice water container 106 containing ice water 108 is installed, and the sample 107 (a biological tissue immersed in a fixing solution) is placed in the ice water of the ice water container 106. Can be put in the sample container 106 containing.

Then, when the power switch of the microwave oven 100 is turned on, the microwaves 1 diffused by the electromagnetic wave stirrer 101 disposed on the microwave oven 100 are turned on.
Irradiate the sample in the container 106 intermittently or continuously for a predetermined time.

The inventor of the present invention has confirmed the effect of the microwave 105 to quickly fix the sample 107 with the electron microscope sample preparation apparatus obtained by improving the commercially available microwave oven 100.

On the other hand, the present inventor has confirmed through experiments that applying a constant pressure to the sample further enhances the effect of fixing biological tissues. Therefore, a method of accommodating the sample in a commercially available syringe and pressurizing the sample is described. And so on.

Further, in the above-mentioned pressurizing method, there was a problem that the pressure could not be kept constant. For example, in order to keep the pressing force as constant as possible, a method of applying a constant pressure to the syringe via a vise was developed. In order to obtain the fixing effect by pressurization more objectively and universally, we have been studying hard.

[0015]

However, the above-described prior art has the following technical problems. In other words, in the microwave irradiation method using the improved apparatus of the cooking microwave oven, the microwaves are dispersed (diffused), so that irradiation of the sample cannot be performed intensively.
Fixation efficiency was inefficient.

Further, since the irradiation amount of the microwave to the sample is not constant, there remains a problem in reproducibility of the fixing condition of the sample. In a series of pressurizing methods using a syringe, it is difficult to maintain a constant pressurizing condition, and it is also difficult to control the pressure.

Furthermore, in any of the conventional electron microscope sample preparation techniques, there is a large variation in the fixed level of biological tissue during the sample preparation process, so that an electron microscope photograph can be obtained.
The failure was clear and the fundamental problem of universal technology needed to be solved.

Under these circumstances, the inventor of the present application may exhibit a synergistic sample fixing effect by using both the microwave irradiation method and the pressurizing method that exhibit the effect of promoting the fixing effect. Have a new idea that there is no
The effect of this new technical idea was confirmed at the experimental level.

In modern medicine, diagnosis accompanied by confirmation of pathological cells using an electron microscope is rapidly spreading. However, at present, it takes time and effort to prepare a sample. It takes about one week to about 10 days to provide an electron micrograph. For this reason, there has been an increasing demand from medical sites for rapid sample preparation in order to contribute to rapid diagnosis.

Therefore, the present invention makes it possible to use a microwave irradiating means and a pressurizing means freely in order to more quickly and surely prepare a sample for an electron microscope. It is an object of the present invention to provide an electron microscope sample preparation apparatus, an electron microscope sample preparation method, and the like, which can easily control pressure and pressure and can be widely applied to fixing various biological tissues.

[0021]

In order to achieve the above object, the following means are employed. In the electron microscope sample preparation apparatus according to claim 1, microwave irradiation means for irradiating a microwave on a sample in which biological tissue is immersed in a fixing solution or the like stored in a container, and applying pressure to the biological tissue. And a pressurizing unit to be added, so that the biological tissue can be subjected to microwave irradiation and / or pressurization. In this means, since both microwave irradiation and pressurization can be used in combination, the effect of fixing the biological tissue by the fixing solution can be enhanced. For example,
A relatively hard tissue such as a lens, a skin, a bone, a plant, or a microorganism, which has been difficult to fix conventionally, can be easily fixed. In addition, since either one of microwave irradiation and pressurization can be appropriately selected and used, optimal fixing conditions can be selected according to the type of biological tissue. Furthermore, since only pressurization can be performed, it is possible to easily and efficiently infiltrate the biological tissue with a resin (epoxy resin or the like), which is necessary for ultrathinning the biological tissue. Since sample preparation can be performed quickly (possibly within 2 to 3 days), it is useful for quick diagnosis.

In the electron microscope sample preparation apparatus according to the second aspect, the microwave irradiating means according to the first aspect has an opening formed so as to face a high-frequency oscillator for emitting a microwave and a container containing the sample. And a waveguide configured to be able to irradiate the microwave intensively toward the sample (container). By this means,
Since the microwave emitted from the high-frequency oscillator is irradiated to the sample accommodated in the container without diffusing it, microwave irradiation can be performed efficiently and the microwave irradiation conditions can be set. Reproducibility can be improved. In addition, since microwaves can be intensively irradiated, an effect of rapidly activating various enzyme reactions and antigen-antibody reactions is exhibited.

According to a third aspect of the present invention, there is provided a sample preparation apparatus for an electron microscope, wherein a heat conductive casing disposed in contact with an outer wall of the container according to the first or second aspect and a cold water circulation disposed inside the casing. And a cooling water circulating means provided with a passage, so that the temperature of the fixing liquid or the like inside the container can be maintained at a predetermined temperature or less. According to this means, since an excessive rise in the temperature of the sample solution which is heated to a high temperature by the microwave irradiation can be suppressed, the continuous irradiation of the microwave can be performed without worry. In addition, by employing a heat conductive casing, the effect of cooling the sample by cold water can be enhanced.

According to a fourth aspect of the present invention, there is provided an electron microscope sample preparation apparatus, wherein the casing according to the third aspect has a substantially U-shape when viewed from above so as to open toward the opening of the waveguide. The sample container is formed, and the sample container is housed in a deep position of the U-shaped opening of the casing, and is arranged so as to surround the container. In this means, the microwave emitted from the opening of the waveguide enters the U-shaped opening of the casing containing the cold water circulation path and is installed so as to be surrounded by the casing. The light passes through the side wall of the sample container and is intensively irradiated on the sample inside the container. Further, the microwave transmitted through the entire sample container is reflected by the inner wall of the casing that is in contact with the sample container, and is irradiated again on the sample solution. Therefore, according to this means, it is possible to efficiently irradiate the sample with a small number of microwaves and to reduce the loss of the microwaves, thereby facilitating the setting and reproduction of the experimental conditions.

According to a fifth aspect of the present invention, there is provided an electron microscope sample preparation apparatus provided with a stirring means for stirring a fixing solution or the like for fixing a biological tissue, which is accommodated in the container according to any one of the first to fourth aspects. In this means, a plurality of biological tissues that are immersed in the fixative and simultaneously fixed by stirring the fixative (and the buffer) contained in the container to make the solution temperature uniform, The level of fixation between them can be uniform.

According to a sixth aspect of the present invention, there is provided an electron microscope sample preparation apparatus, further comprising a temperature detecting means for detecting the temperature of the fixing solution or the like contained in the container according to any one of the first to fifth aspects. By this means, it is possible to detect when the sample solution (fixing solution and buffer solution) has reached a predetermined temperature by microwave irradiation, and to control the temperature of the sample solution not to be higher than necessary.

According to a seventh aspect of the present invention, there is provided an electron microscope sample preparation apparatus, wherein the pressurizing means according to any one of the first to fifth aspects is attached to a lid for closing an upper opening of a container containing the sample, and A pressure member having a pressure surface pressed from above, wherein the temperature detecting means according to claim 6 is held at the center of the pressure member, and a rod-shaped member protruding from a lower end of the pressure member. It is composed of a temperature sensor member. Then, the temperature sensor member is inserted into the inside of the container through a through hole formed in the center of the lid of the container, and is configured to detect the temperature of the sample that changes in temperature by irradiation of the microwave. With this means, the temperature of the sample solution (fixing solution and buffer solution) can be simultaneously detected and pressurized by simple means.

According to an eighth aspect of the present invention, an elastic member for preventing pressure escape is provided on the pressing surface according to the seventh aspect so as to surround the through-hole at the time of attachment. In this means, even if a configuration in which the temperature sensor member is inserted from the through hole formed in the lid of the sample container is adopted, the pressure for escaping from the slight gap between the inner wall of the through hole and the outer wall of the temperature sensor member is reduced. It can be effectively closed by an elastic member which is in close contact with the upper surface of the lid.

According to a ninth aspect of the present invention, the pressure member holding the temperature sensor member according to the seventh or eighth aspect is formed of a heat insulating member. In this means, the temperature of the sample solution transmitted to the temperature sensor member is not deprived by the member (pressing member) holding the temperature sensor member, so that more accurate temperature detection can be performed. I do.

According to a tenth aspect of the present invention, in the electron microscope sample preparation method, the pressing member according to any one of the seventh to ninth aspects is characterized in that:
It is configured to move up and down by a pressurizing cylinder arranged above. The outer diameter of the pressing surface formed at the lower end of the pressing member and in contact with the lid member is formed smaller than the inner diameter of the pressing cylinder. With this means, a larger pressure can be applied to the lid member with a small cylinder pressure. For example, if the area of the horizontal section and the area of the pressurizing surface of the pressurizing cylinder is 10: 1, then a cylinder pressure of 0.5 atm.
It is efficient because a pressure of (0.5 × 10) atmosphere can be applied.

[0031] In the method for preparing an electron microscope sample according to the eleventh aspect, by using the apparatus for preparing an electron microscope sample according to any one of the first to tenth aspects, the fixative can be quickly penetrated into the biological tissue, Since cross-linking with the protein constituting the cell tissue is promoted, a rapid and reliable sample preparation can be performed.

According to a twelfth aspect of the present invention, in the method for preparing an electron microscope sample according to the eleventh aspect, at least an aldehyde-based fixing agent is used as a fixing solution for biological tissue. For example, glutaraldehyde (1,5-pentanedial)
Has an aldehyde group (—CHO) having an affinity for the amino group of the protein constituting the cell tissue at both ends, and is therefore suitable as a protein cross-linking agent. Combined with the rapid infiltration into the inside, the quick fixation can be further promoted.

According to a thirteenth aspect, the biological tissue is stained by using the electron microscope sample preparation apparatus according to any one of the first to tenth aspects, whereby the stained liquid is added to the biological tissue as in the case of the fixative. Can be quickly penetrated.

In the method for activating an enzyme reaction or the method for activating an antigen-antibody reaction according to the fourteenth aspect, by using the apparatus for preparing an electron microscope sample according to any one of the first to tenth aspects, a target enzyme reaction or an antigen can be obtained. Since the antibody reaction can be quickly and reliably activated, the presence of an enzyme or the presence of an antigen-antibody reaction in a tissue or a cell can be visualized and easily known.

As described above, the present invention can rapidly and reliably and easily perform fixation, staining, enzymatic reaction activity, and antigen-antibody reaction activation of biological tissues, thereby contributing to rapid diagnosis in medical treatment. It is widely applied to fields such as enzyme histochemistry and immunology, and has the technical significance of contributing to the development of various industries.

[0036]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below with reference to examples and experimental examples. First, an external configuration of the apparatus 1 will be briefly described with reference to FIG. 1 which is an external perspective view of an electron microscope sample manufacturing apparatus (hereinafter, referred to as “apparatus”) 1 which is a preferred embodiment according to the present invention.

<Appearance Configuration of Apparatus 1> The apparatus 1 includes a casing 25 having a substantially rectangular parallelepiped shape.
Inside, a chamber 2 formed for installing a container 3 for accommodating a sample 4 and the like is provided.

The side walls 201, 201 and the upper and lower plates 202, 2
A chamber 2 having a substantially rectangular parallelepiped space surrounded by 02
A waveguide 11 for guiding a microwave 20 (see FIG. 2 and the like) emitted from a magnetron 10 (see FIG. 2), which is a high-frequency oscillator not shown in FIG. 3 (see opening 11a shown in FIG. 1 and the like).

The microwave 20 used in the apparatus 1
Is particularly preferable because a microwave having a frequency of 24.5 gigahertz (GHz) similar to that of a general cooking microwave oven easily vibrates water molecules.

A pressing member 8 which is a component of a pressing means for pressing the sample 4 (see FIG. 2) housed in the container 3 is not shown in FIG. 1 above the chamber 2. A cylinder 7 (see FIG. 2) is provided so as to be able to move up and down, and a rod-shaped temperature sensor 9 is protruded from the tip (lower end) of the pressure member 8 so as to hang down.

Around a vessel 3 installed at a predetermined location below the chamber 2, a chilled water circulating device 6 constituting chilled water circulating means for cooling the vessel 3 from the outside is provided. The irradiation plays a role of controlling the temperature of the sample in the container 3 not to rise excessively.

The chilled water circulation device 6 is configured to be detachable from the chamber 2. When the present apparatus 1 is used to activate an enzyme reaction or an antigen-antibody reaction other than the electron microscope sample preparation, for example, the cold water circulation device 6 is removed,
A dedicated sample container (container having various forms such as a rectangular shape or an elongated cylindrical shape) (not shown) can be installed in the chamber 2 and used. That is, various kinds of sample setting means inside the chamber 2 can be selected.

The device 1 is designed so that it can be used by setting various desired conditions. A keyboard 29 on which various operation keys used for setting conditions are arranged is provided on the right side of the front of the chamber 2. Note that a power switch 27 for driving the apparatus 1 is provided below the keyboard 29.

<Internal Configuration of Apparatus 1> The internal configuration of the apparatus 1 will be specifically described below mainly with reference to FIGS. FIG. 2 is a block diagram simply showing the internal configuration of the present apparatus 1, and shows the relationships among the respective members in an easy-to-understand manner.

(Sample Container and Others) First, a sample 4 in which a biological tissue (not shown) is immersed is placed in a substantially cylindrical container 3 installed below the chamber 2 and having an opening 31 above.
Is housed. The opening 31 is covered with a substantially disk-shaped lid 5 which is formed so as to be in close contact with the inner peripheral wall surface of the container 3 and has a through hole 51 formed at the center thereof, thereby closing the container 3. .

The material of the container 3 is preferably a synthetic resin having chemical resistance so that the sample 4 which can be composed of various chemicals can be safely contained. For example, an acetal resin (polyacetal resin) or the like is used. it can.

The container 3 is surrounded by the chilled water circulating device 6 configured to circulate chilled water via a cooling pump 14, and a sample 4 is placed below the container 3.
A magnet 12 for rotating a (magnetic) stirrer 121 to be introduced into the sample 4 is provided as a stirring member for stirring the sample.

(Microwave Irradiation Means) Next, a magnetron 10 connected to a microwave controller 13 controlled by a computer 17 is provided inside the chamber 2 on the back side. Microwave 20 is emitted from unit 10a.

Incidentally, the microwave oscillating device is a speed modulation tube,
A BK tube, a magnetron and the like can be appropriately selected, but a magnetron (also called a magnet tube) is particularly preferable in consideration of oscillation efficiency and the like.

The microwave 20 is supplied to the oscillation unit 10a
Is transmitted while being reflected on the inner wall surface of the rectangular waveguide 11 formed so as to include the inside of the waveguide 11 and enters the chamber 2 from the opening 11 a of the waveguide 11, and is circulated by the cold water circulation device 6.
The sample 4 is configured to be transmitted through the outer wall (side wall) 32 of the container 3 through the opening passage 67 (see FIG. 5).

(Temperature Detecting Means) The temperature sensor 9 of the pressurizing member 8 disposed above the container 3 is inserted through the through hole 51 of the lid 5 when the pressurizing member 8 descends. And the temperature of the sample 4 can be detected.

A signal relating to the sample temperature detected by the temperature sensor 9 is sent to a computer 17 and, when the temperature exceeds a predetermined temperature, a signal from the magnetron 10 is transmitted through the microwave controller 13. Is configured to control the oscillation of the microwave 20.

Incidentally, as the temperature detecting means of the present apparatus 1, a temperature detecting means using an infrared sensor or the like may be adopted. In this case, the lid 5 of the container 3 is formed of a transparent material (the through-hole 51 is unnecessary), and the temperature may be detected by irradiating infrared rays from above the lid 5, so that the temperature sensor 9 is inserted into the sample 4. There is an advantage that there is no need.

(Pressing Member, Pressure Sensor) Here, reference numeral 7 shown in FIG. 2 simply represents a pressing cylinder provided above the pressing member 8. Air is fed from a compressor 16 to a predetermined region inside the cylinder of the pressurizing cylinder 7 via an electromagnetic valve 15.

A pressure sensor 18 for detecting the pressure of the output air from the compressor 16 is provided between the compressor 16 and the solenoid valve 15 so as to sequentially send a pressure signal to a computer 17.

When the pressure value sent from the pressure sensor 18 reaches a predetermined pressure set in the computer 17 in advance, the microwave 20 is emitted from the magnetron 10 via the microwave controller 13. Have been.

(Control Unit) Reference numeral 28 in FIG. 2 indicates a display unit, and reference numeral 29 indicates a keyboard. The control unit is connected to the computer 17 to form a control unit indicated by reference numeral 19 together with the computer 17. I have. The keyboard 29 is provided for setting the output mode of the microwave 20 of the magnetron 10, setting the pressure, setting the temperature, setting the operation cycle, and the like. The display unit 28 displays these set values on a liquid crystal display. It is provided for display.

Next, the specific structure of each device constituting the pressurizing means, the cold water circulating means, and the stirring means constituting the present apparatus 1 will be described in more detail with reference to FIG.

<Pressing Means> First, the pressing means comprises a cylindrical pressing cylinder 7 having an internal cavity, and a vertical cross section T substantially arranged in the cylinder 7 internal space 71 so as to be capable of reciprocating in the vertical direction. A piston 72 having a U-shape and a pressurizing member 8 connected to the lower end of the piston 72 and exposed above the inside of the chamber 2 are provided. The pressurizing means is not limited to the one using a cylinder, but a cylinder type is preferable because the pressure can be easily controlled.

The cylinder internal space 71 is partitioned by a piston 72 into an upper space 71a and a lower space 71b.
a is open, and an air inflow (discharge) port 21b is open in one lower space 72.

With this configuration, when the compressed air sent by the compressor 16 (see FIG. 2) not shown in FIG. 3 is sent from the air inlet (discharge) port 21a into the upper space 71a by the adjustment by the solenoid valve 15, The piston 72 descends and pushes down the pressurizing member 8. On the other hand, when the piston 72 is fed into the lower space 71 b from the air inlet (discharge) port 21 b, the piston 72 rises and pushes up the pressurizing member 8.

The electromagnetic valve 15 is configured to open and close a valve directly connected to the movable core in the electromagnetic coil by absorbing or separating the movable core in the electromagnetic coil by the exciting current. A command signal sent through the controller serves to adjust the respective air flow rates to the air inlet (discharge) ports 21a and 21b by an on / off operation. Thereby, a desired pressure can be obtained.

The pressure member 8 is formed of a heat insulating synthetic resin selected as appropriate, and is designed so as not to deprive the temperature of the temperature sensor 9 held at the center.
It enables more accurate temperature detection.

Here, the structure of the pressing means of the present apparatus will be described more specifically with reference to FIG. 4 showing a state where the pressing member 8 is actually pressing. At the lower end of the pressing member 8, a cylindrical portion 8 having a smaller diameter than the upper diameter is provided.
The lower end surface 82 of the cylindrical portion 81 comes into contact with the upper surface 52 of the lid 5 closing the container 3 and presses the lid 5 downward. At this time, the lower end face 8
The tip of the rod-shaped temperature sensor 9 projecting downward from 2 is inserted into the container 3 through the through hole 51 of the lid 5,
It is immersed in sample 4.

Here, reference numeral 22 (22a, 2a, 2a) shown in FIG.
2b, 22c) represent O-ring members, respectively. First, the O-ring 22 a is provided around the outer peripheral wall of the lid 5 and is pressed against the inner peripheral wall of the container 3. The O-ring 22a serves to prevent pressure applied to the sample 4 from leaking from the gap 23a between the outer peripheral wall of the lid 5 and the inner peripheral wall of the container 3 when the lid 5 is pressed. I have.

Next, the O-ring 22b is attached to the lower end surface 82a of the pressing member 8 in a ring shape as viewed from below.
At the time of pressurization, it is pressed against the upper surface of the lid 5. The gap 2 formed between the inner peripheral wall surface of the through hole 51 of the lid 5 and the outer cylindrical wall surface of the temperature sensor 9 by the O-ring 22b.
The pressure is prevented from escaping from 3a.

Further, the O-ring 22c is connected to the stopper 91 of the temperature sensor 9 which contacts the bottom surface 83 of the inner cylinder of the pressure member 8.
Is attached in a ring shape when viewed from below, and is pressed against the bottom surface 83 of the inner cylindrical portion. This O-ring 22
The pressure c prevents the pressure from escaping upward from the gap 23d between the outer cylinder portion of the temperature sensor 9 and the pressure member 8. Thus, the O-ring 22 (22a, 22b, 22
According to c), the pressure can be applied from the pressing member 8 to the lid 5 without loss of pressure.

Here, assuming that the diameter of the inner cylinder portion of the cylinder 7 is R, the diameter of the pressing surface 82 is r ₁ (R> r ₁ ), and the cylinder pressure is P, the pressing force p ₁ transmitted from the pressing surface 82
Depends on the area _{ratio, p 1 = P × (πR} 2 / πr 1 2)
Becomes

Further, if the pressure applied to the sample 4 is p ₂ , it depends on the horizontal cross-sectional area of the inner cylinder of the container 3. Therefore, if the inner cylinder of the container 3 is r ₂ (R> r ₂ ), p 2 ₂ = P x
The ^{_{(πR 2 / πr 2 2)}} . Therefore, in the pressurizing means of the present apparatus 1, a larger pressure p ₂ can be obtained with a smaller cylinder pressure P.
Is added to the sample 4.

<Cold Water Circulation Means> Next, the cold water circulation means will be described. The cold water circulating means is provided in order to suppress an excessive rise in temperature of the sample 4 due to the irradiation of the microwave 20 and to enable continuous irradiation of the microwave 20 without worry.

The structure will be described. An aluminum casing 68 formed in a substantially U-shape in plan view and a cold water circulation path 61 disposed inside the casing 68 in a substantially U-shape are provided. Have.

The cooling water circulation path 61 includes a cooling pump 14
Chilled water 64 of 3 ° C. or less is supplied through a chilled water supply pipe 62 from a supply port 66 a opened on the lower surface of the chilled water circulation path 61, and from a discharge port 66 b opened in an upper region of the chilled water circulation path 61. Discharge pipe 63
, And the low-temperature cold water 64 is always circulated.

The opening passage 67 formed inside the substantially U-shaped casing 68 is arranged so as to face the opening 11 a of the waveguide 11. The microwave 20 that has entered the chamber 2 from the waveguide 11 passes through the opening passage 67 and enters the outer cylinder of the container 4 installed at the innermost portion of the internal passage 67, and The sample 4 is irradiated through the member.

Then, the microwave 20 passing through the container 4
Is reflected on the inner wall surface 69 of the internal passage 67 in contact with the container 3 (see the arrow W), and is again irradiated on the sample 4. With the above configuration, the microwaves 20 are intensively and efficiently applied to the sample 4, which is preferable.

The means for supplying cold water can be selected as appropriate, and a method using ice water or a cooling element can be used. Reference numeral 65 shown in FIG. 3 is a chilled water discharge valve provided in advance to discharge chilled water in the chilled water circulation path 61 in order to prevent spillage of chilled water inside when the casing 68 is removed.

<Stirring Means> Next, the structure of the stirring means will be described. The stirring means includes a magnet stirrer 121 which is charged into the sample 4 and deposited on the bottom of the container 3.
And a magnet 12 attached to the bottom plate 202 of the chamber 2 directly below the container 4.

According to this configuration, when a predetermined switch member is turned on, a magnetic force is generated in the magnet 12, and the magnet stirrer 121 rotates at a desired speed to form a vortex in the fixing solution (and the buffer solution) of the sample 4. Therefore, the fixative is uniformly stirred, and the sample temperature is made uniform. Therefore, the effect of fixing the biological tissue (not shown) immersed in the sample 4 can be made uniform.

<Sample Case> The configuration of a dedicated sample case developed for accommodating a biological tissue will be described below with reference to FIGS. The biological tissue formed into a thin shape is accommodated in a predetermined sample case 24 so as not to be dispersed by micro irradiation or stirring. A plurality of the sample cases 24 are accommodated in the container 3 so as to be stacked (see FIG. 4), and immersed in the fixing solution accommodated in the container 4, thereby fixing the biological tissue.

The interior of the sample case 24 is largely divided into four regions for the purpose of facilitating the identification of the type of biological tissue, and each of the partitions 243 has a plurality of cutouts. The tissue fixing places 244 are arranged side by side in the circumferential direction.

Since the sample case 24 is immersed in a fixing solution or the like, it is made of a chemically resistant synthetic resin.
The lid member 241 and the container member 242 formed in a substantially dish shape are
It is configured to be detachably attached.

<How to Use the Present Apparatus 1> Hereinafter, the operation procedure 3
A method of using the present apparatus 1 will be described with reference to FIGS. The container 3 containing the sample 4 is placed in the chamber 2
Once installed in the predetermined position, follow the operation procedure below.

(Procedure 31) First, in a start procedure 31, various data setting operations are performed. Specifically, the mode setting, the pressure setting, the temperature setting, the operation cycle setting, and the on-time / off-time setting of the magnetron 10 are performed.

The "mode setting of the magnetron 10"
The oscillation amount of the microwave 20 is changed to “High” level and “Lo”.
This is an operation of selecting any one of the “w” levels and inputting and setting it to the computer 17 by a predetermined key operation.

Incidentally, the "High" level can provide an output at 100 watts, and the "Low" level can provide an output at 50 watts. If it is desired to raise the temperature over time, the latter will be selected.

“Setting the pressure” means that the desired pressure member 8
Is input to the computer 17 by operating a predetermined key (“Press” key). The pressure is a pressure level for the sample 4 and can be set at 0 to 10 atm. "Temperature setting" means microwave 2
The upper limit temperature of the sample 4 whose temperature is increased by the irradiation of 0 is set to the computer 1 by operating a predetermined key (“Temp” key).
This is the work of inputting and setting the number 7. The temperature can be set in the range of 0 to 112 ° C.

The "setting of the operation cycle" means that the irradiation cycle of the microwave 20, for example, a predetermined key (“Cycle” key) determines how many times the continuous irradiation for a certain time or the intermittent irradiation at a certain interval is repeated. This is an operation of inputting and setting to the computer 17 by operation.

The computer 17 of the apparatus 1 is configured so that past operation cycles can be stored (stored) in the computer 17 by operating a predetermined key (“Store” key). When using the experimental conditions as is, it is only necessary to select the stored data by operating a predetermined key (“Recall” key).

"Setting the on-time / off-time"
The microwave irradiation time (referred to as “on time”) and the microwave non-irradiation time (referred to as “off time”) are appropriately determined by a predetermined key (“OnTime” key or “OffTime”).
e) key) is an operation of input setting to the computer 17 by operation. If the off time is set to 0, continuous irradiation becomes one cycle, and if the off time is set to a value other than 0,
Intermittent irradiation (irradiation → non-irradiation) is one cycle. Note that all the setting values described above are displayed on the display unit 28 of the liquid crystal.

(Procedure 32) After performing the above-mentioned various settings in procedure 31, the start switch 27 (see FIG. 1) is turned on.

(Step 33) The compressor (pump) 16 is operated until the set pressure is reached.

(Step 34) When the set pressure value is reached, the magnetron 10 is operated to start the oscillation of the microwave 20. The pressure is maintained at a set pressure.

(Procedure 35) Check whether the temperature and the pressure do not exceed the set values. If it exceeds, irradiation of microwave 20 and pressurization are stopped.

(Step 36) The time set in the on-time is checked, and if the set time is reached, the magnetron 1
0 is stopped, and the irradiation of the microwave 20 is stopped.

(Step 37) The time set as the off time is checked, and when the set time is reached, the magnetron 10 is operated again.

(Step 38) The number of cycles is checked, and when the number of cycles reaches a preset number, "memory 1"
(A memory that is always called when the setting cycle is completed) is called and operated under the setting conditions stored in the memory 1. By this operating means, for example, after ending the irradiation cycle of the microwave 20 under constant pressurization, only the microwave irradiation can be ended, and the “pressing only operation” stored in the memory 1 can be continued. it can. If nothing is stored in the memory 1, the process ends with the set cycle operation.

(Step 39) When the operation set (stored) in the memory-1 is completed, the magnetron 10 is stopped (stopped), the pressurizing member 8 is raised, and all the series of operations are completed.

<Sample Preparation Method> An electron microscope sample preparation method using the present apparatus 1 will be described below. The sample preparation method can be roughly divided into “selection of fixative”, “selection of buffer”, “slice form of tissue piece”, “microwave irradiation”,
It is necessary to consider five points of “pressurization”.

(Fixing solution) There is "fixing" as a first step operation for observing a biological tissue sample with an electron microscope.
The term "fixation" refers to adding an artificial operation to produce a biological tissue sample so as to keep the original form and structural contents as much as possible so as not to change, and is used in this fixing operation. The reagent solution is called a fixative.

As the fixative, 2 (-2.5%) glutaraldehyde, which is particularly preferable among aldehyde-based fixatives, may be used alone, but 2% glutaraldehyde (hereinafter referred to as "GA"). And 4% paraformaldehyde (hereinafter referred to as "PFA") in a mixture of sucrose and CaCl
₂ , an electrolyte such as MgCl ₂ may be added.

Sucrose keeps the environment inside and outside the cell constant,
Addition of an electrolyte such as CaCl ₂ has an effect of suppressing elution of cellular components.

The fixative must penetrate into the biological tissue as quickly as possible. The reason is that if the permeation takes a long time, the pH in the biological tissue rises and GA becomes multimerized, making it difficult to permeate. However, when the fixative is rapidly permeated, the fixative (GA) becomes This is because it is rapidly penetrated into the biological tissue in the form of a monomer, so that the irradiation of the microwave facilitates the formation of a crosslink between the fixing agent and the protein of the biological tissue.

Table 1 shows that 2% GA and 4% PFA
Please refer to the prescription of the fixative of the above.

[0103]

[Table 1] (Buffer) Phosphate buffer is used as the buffer used in the fixing method of performing microwave irradiation. Generally, cacodylate buffer (see Table 1 above) or a buffer obtained by adding sucrose to this buffer is used. Liquids can also be used. In addition, a biochemical buffer (Good's buffer) can be used.

[0104] Also by microwave irradiation means and the pressure means (sliced form of samples), since it becomes difficult to secure the large biological tissue piece uniformly and quickly, if the size of within 3 mm ^3, the thickness Good results can be obtained by thinning and slicing into thin cushions.

(Microwave Irradiation) There are two types of microwave irradiation methods, continuous irradiation and intermittent irradiation. Intermittent irradiation can keep the temperature of the fixing liquid in the sample container 3 more constant than continuous irradiation. Since it is easy, the damage of the sample due to heat can be suppressed to a relatively small amount, so that it is generally preferable.

In continuous irradiation, thermal damage to the sample due to a rapid temperature rise poses a problem. Therefore, it is important to shorten the irradiation time (on-time) as short as possible to suppress the temperature rise.

The intermittent irradiation is performed by continuously performing microwave irradiation until the set temperature is reached, and thereafter repeating the on-time and the off-time, and the on-time and the off-time for an arbitrarily set number of seconds from the start of the microwave irradiation. It can be divided into a method that repeats time and a method that repeats time. The setting of the irradiation cycle may be appropriately selected depending on the type of biological tissue, the size of the tissue piece, and the like.

(Pressurization) The pressurization condition must be appropriately selected depending on the type of biological tissue, the size of the tissue piece, and the like.
In general, good results can be obtained by pressurizing under a pressure of 2 to 3 atm for a few hours. After pressurizing simultaneously with microwave irradiation, only microwave irradiation is terminated, and only pressurization is performed for the remaining time.

Hereinafter, an experimental example will be described with reference to electron micrographs (FIGS. 8 to 14) obtained by the experiment.

<Experimental Example 1: Sample preparation method using the present apparatus 1> An electron micrograph obtained in Experiment 1 is shown in FIG.
Shown in (Biological tissue) This is a rat bone marrow tissue. The sample preparation conditions are as follows. (Prefix) Fixative: 2% GA and 0.1 M cacodylate buffer (cac
A fixative solution of Odylate buffer was used.
The pH of the buffer is 7.4. Microwave irradiation level:
The mode setting of the magnetron 10 is at the “High” level. Irradiation cycle: A cycle of 10 seconds on-time and 20 seconds off-time, with a total irradiation time of 40 seconds (4 cycles). Pressurizing condition: Continuous pressurization at 3 atm for 2 hours. This pre-fixing operation is performed to fix the protein inside the cell tissue (the same applies hereinafter). (Washing) Washing was performed with a 0.1 M cacodylate buffer overnight. This washing operation is an operation to discharge the remaining GA from the tissue in order to prevent a redox reaction between the GA and osmium tetroxide used for post-fixation (the same applies hereinafter). (Fixed after) 2% osmium tetroxide (OsO4) and 0.1
A fixative of M cacodylate buffer was used. Buffer pH
Is 7.4. Pressurizing condition: 1.5 hours at 3 atm. The post-fixation operation is performed to fix lipids such as phospholipids present in cell tissues, particularly in a biological membrane system (the same applies hereinafter). The (dehydration) was performed in a series of increasing ethanol concentration. Specifically, 60%, 70%, 80%, 90%, 95%, 98%
Was dehydrated with ethanol of each concentration for 20 minutes, and finally, a total of three times of dehydration was performed with ethanol of 100% concentration for 30 minutes. The dehydration operation is performed for the purpose of reducing the volume change of the cell tissue and facilitating the subsequent operation of infiltrating the hydrophobic resin. Substitution was performed three times for 30 minutes each using (substituted) propylene oxide (hereinafter referred to as “PO”).
This replacement operation is performed using PO having affinity for both the dehydrating agent (ethanol) and the resin in order to facilitate the next resin penetration. (Resin penetration) 2: 1 mixture of PO and epoxy resin
Time, then 2: 1 with a 1: 1 mixture of PO and epoxy resin
Time, 2 hours with a 1: 2 mixture of PO and epoxy resin,
The infiltration work was performed sequentially (the resin was manufactured by Luft method
812 mixture. A: B = 3: 7, DMP-30 is 1.5
% Addition). The mixture was pressurized overnight using the present apparatus 1. The pressure is 3 atm. In this resin infiltration work, by applying pressure, a resin having a high viscosity can easily penetrate into the cell tissue. Note that this resin infiltration operation is an operation necessary for performing the next cell tissue thinning operation. (Resin embedding and polymerization) New resin (epoxy resin) 1
After immersion for 12 hours at 37 ° C, 12 hours at 45 ° C, 6 hours
The polymerization was carried out at 0 ° C. for 12 hours and at 65 ° C. for 24 hours. (Preparation of Thin Section) A 0.5 μm-thick toluidine blue-stained section was observed with an optical microscope, a necessary portion was trimmed, and a section of about 80 nm was prepared using an ultramicrotome. (Electron staining) After staining with 3.5% uranyl acetate staining solution for 30 minutes, washing with distilled water, and staining with lead citrate staining solution for 5 minutes.
Stained for minutes. (Electron microscope observation and photography) JEOL JEM-1200E
Observation was performed at an acceleration voltage of 80 kV at X, and photographing was performed. Experimental result. Since bone tissue is an extremely hard tissue, the permeability of the drug solution is insufficient, and the bone was previously permeated by a reduced pressure treatment. But,
When simultaneously observing a hard bone tissue and a soft bone marrow tissue as in the present sample, even if a sample block having high machinability is prepared by increasing the permeability by decompression, the morphology of the bone marrow cells is likely to be deformed in the tissue image. However, due to the combination of microwave irradiation and pressurization, there was no morphological change of bone marrow cells or detachment of osteoblasts
Bone tissue was also observed favorably (see a drawing substitute photograph in FIG. 8).

<Experimental example 2: Fixing method by decompression treatment without using the present apparatus 1> An electron micrograph obtained in Experiment 1 is shown in FIG. (Biological Tissue) This is a rat bone marrow tissue similar to that of Experimental Example 1. The sample preparation conditions are as follows. (Pre-fixed) A reduced pressure processing method using a conventional vacuum pump was used. Immediately after the tissue excision, the tissue was immersed in a pre-fixation solution, subjected to a pressure reduction treatment with an oil rotary pump for 1 hour before fixation, and then returned to normal pressure for 2 days for additional fixation. Fixative: A fixative of 2% GA and 0.1 M cacodylate buffer was used. The pH of the buffer is 7.4. (Washing) The membrane was washed overnight with a 0.1 M cacodylate buffer. (Fixed after) 2% osmium tetroxide (OsO4) and 0.1
This was performed for 2 hours using a fixing solution of M cacodylate buffer.
pH is 7.4. (Dehydration) (Substitution) Experiment 1 was performed in the same manner. (Resin penetration) Half-day with a 2: 1 mixture of PO and epoxy resin, then half-day with a 1: 1 mixture of PO and epoxy resin, half-day with a 1: 2 mixture of PO and epoxy resin Work was performed sequentially. Then, the resin mixture was subjected to a pressure reduction treatment with a vacuum pump overnight (the resin was manufactured by the Luft method Quet).
olq812 mixed solution. A: B = 3: 7, 1.5% DMP-30 added).

(Embedding and polymerization of resin)-(Electron microscopy observation
Photographing) The same as in Experimental Example 1. Experimental result. Even if a sample block with good machinability was prepared by increasing the permeability by decompression, the morphology of the bone marrow cells was deformed in the histological image. The morphological change of the bone marrow cells and the detachment of the osteoblasts also occurred (see the substitute photograph in FIG. 9 for comparison with FIG. 8).

<Experimental Example 3: Use of the present apparatus 1> (Biological tissue) Tissue from human surgery (normal skin tissue for skin transplantation). The sample preparation conditions are as follows. (Pre-fixation) After excision of the tissue, the tissue was immersed in a pre-fixation solution and fixed by microwave irradiation and pressurization. Fixative: A fixative of 2% GA and 0.1 M cacodylate buffer was used. pH is 7.4. Microwave irradiation level: The mode setting of the magnetron 10 is “Low” level. Irradiation cycle: A cycle of 10 seconds on-time and 20 seconds off-time, with a total irradiation time of 40 seconds (4 cycles). Pressurizing condition: Continuous pressurization at 3 atm for 2 hours. (Washing) The membrane was washed overnight with a 0.1 M cacodylate buffer. (Fixed after) 2% osmium tetroxide (OsO4) and 0.1
A fixative of M cacodylate buffer was used. pH 7.4
It is. The fixed time is one hour. Incidentally, no pressure was applied during the subsequent fixing. The (dehydration) was performed in a series of increasing ethanol concentration. Specifically, 60%, 70%, 80%, 90%, 95%, 98%
Was dehydrated with ethanol of each concentration for 10 minutes, and finally, a total of three times of dehydration was performed with ethanol of 100% concentration for 20 minutes. Using (substituted) propylene oxide (hereinafter referred to as “PO”), substitution was performed twice for 30 minutes each. (Resin penetration) 1: 1 with a 2: 1 mixture of PO and epoxy resin
Time, then 1: 1 mixture of PO and epoxy resin
Time, 1 hour with a 1: 2 mixture of PO and epoxy resin,
The infiltration work was performed sequentially (the resin was manufactured by Luft method
812 mixture. A: B = 3: 7, DMP-30 is 1.5
% Addition). The mixture was pressurized overnight using the present apparatus 1. The pressure is 3 atm. (Embedding and Polymerization of Resin) The same conditions as in Experiment 1 were used. (Preparation of Thin Section) (Staining) (Shooting) A 0.5 μm-thick toluidine blue-stained section was observed with an optical microscope and photographed. Experimental result. The drawing substitute photograph shown in FIG. 10 shows the necessary parts before electron microscopic observation.
It is an optical microscope photograph taken for confirming (observation area is limited in electron microscope observation). As this picture shows,
In the sample preparation by microwave irradiation and pressurization using the present apparatus 1, the collagen fibers in the dermis have excellent staining properties (that is, the fixation state is good), and the resin penetration is sufficient, so that the stratum corneum and No exfoliation from the resin was observed at all, indicating that a good sample was obtained.

<Experimental Example 4: Ordinary sample preparation method without using the present apparatus 1> (Biological tissue) Tissue removed from human operation (normal skin tissue for skin transplantation)
(Same as Experiment 3). The sample preparation conditions are as follows. (Pre-fixed) A reduced pressure processing method using a conventional vacuum pump was used. Immediately after the tissue excision, the tissue was immersed in a pre-fixation solution, subjected to a pressure reduction treatment with an oil rotary pump for 1 hour before fixation, and then returned to normal pressure for 2 days for additional fixation. Fixative: A fixative of 2% GA and 0.1 M cacodylate buffer was used. pH is 7.4. (Washing) (Post-fixation) (Dehydration) (Replacement) (Resin penetration) (Resin embedding and polymerization) The same as in Experiment 2 above. (Preparation of thin section) (staining) (photographing) The same as in Experiment 3 above. Experimental result. The normal sample preparation method is unsuitable because a piece with poor resin penetration is broken at the boundary between the stratum corneum (outermost layer) and the resin, and is not suitable.

<Experiment 5: Sample preparation method using the present apparatus 1> (Biological tissue) Normal human skin cells. The sample preparation conditions are as follows. (Pre-fixation) to (Resin embedding and polymerization) The same as in Experiment 3. (Preparation of Thin Section) to (Electron Microscope Observation / Photographing) The same as in Experiment 1. Experimental result. Even in the epidermal cells of normal skin, the stratum corneum is hard and lacks resin permeability, so that the boundary between the stratum corneum and the resin usually peels off under electron microscopic observation. However, good observation is possible with penetration by pressure. In addition, the basal cells and the basement membrane in the epidermis are well preserved, and the intercellular connection of spiny cells is also well preserved, indicating that the fixation situation is good.

<Experiment 6: Ordinary resin permeation method> Skin tissue of human palmoplantar keratosis. The sample preparation conditions are as follows. (Prefix) to (replacement) Same as Experiment 5. Experimental result. The stratum corneum was thickened ten times as much as the normal stratum corneum, indicating that the sample was extremely poor in resin penetration by the usual method.

<Experiment 7: Resin infiltration method by low-temperature substitution method> (Biological tissue) The stratum corneum of human palmar pad keratosis skin tissue.
The sample preparation conditions are as follows. (Prefix) to (replacement) Same as Experiment 5. (Resin penetration) 2: 1 mixture of PO and epoxy resin
Time, then 2: 1 with a 1: 1 mixture of PO and epoxy resin
Time, 2 hours with a 1: 2 mixture of PO and epoxy resin,
The infiltration work was performed sequentially (the resin was manufactured by Luft method
q812 mixed solution. A: B = 3: 7, DMP-30 is 1.
5% added). Then, the above resin mixture solution (all without an accelerator added) was subjected to pressure infiltration under the conditions of 3 atm and 4 ° C for 2 days. Subsequently, the PO was washed twice for 10 minutes to remove the accelerator-free resin. Further, a 2: 1 mixture of PO and epoxy resin for 2 hours, a 2: 1 mixture of PO and epoxy resin for 2 hours, a 2: 1 mixture of PO and epoxy resin for 2 hours, The infiltration operation was performed sequentially (the resin was a mixed solution of Luft method Quetolq 812. A:
B = 3: 7, DMP-30 1.5% added). And
The above resin mixture (both with an accelerator added) was subjected to pressure infiltration at 3 atm and 4 ° C for 2 days, and then replaced with a new resin mixture (with an accelerator added) at 3 atm and room temperature. Was performed under pressure for 1 day (resin was manufactured by Luft method Que).
tolq812 mixture. A: B = 3: 7, DMP-30
Is 1.5% added). (Resin embedding and polymerization) The same as in Experiment 2 above. (Preparation of thin section) (staining) (photographing) The same as in Experiment 3 above. Experimental result. When the pressurization and low-temperature substitution methods are used together, the resin penetration is good,
It was observed over almost all layers without peeling.

The electron microscope sample preparation apparatus 1 and the electron microscope sample preparation method using the apparatus 1 according to the present invention described above include, in the electron microscope sample preparation step, pre-fixing only by microwave irradiation, and microwave irradiation. In addition to speeding up fixation in pre-fixation using pressure and post-fixation using only pressure and promoting resin penetration by pressurization, improving the reactivity of immunostained specimens in optical microscope specimen preparation work, activating during immunostaining It can be used in a wide range of fields such as chemical treatment (antigen-antibody reaction activation) and application to enzyme histochemistry by enzyme reaction activation.

[0119]

According to the electron microscope sample manufacturing apparatus and the electron microscope sample manufacturing method of the present invention, the following effects are mainly obtained. (1) Since both microwave irradiation and pressurization can be used for a sample in which biological tissue is immersed in a fixing solution or the like stored in a container, the effect of fixing the biological tissue by the fixing solution can be enhanced. it can. (2) Since any one of microwave irradiation and pressurization can be appropriately selected and used, optimal fixing conditions can be selected according to the type of biological tissue. (3) It is very convenient because resin penetration into biological tissue can be promoted only by pressurization. (4) The fixation of hard tissues such as skin tissue and bone tissue, which has been difficult in the past, can be facilitated, so that the range of biological tissue observation using an electron microscope can be greatly expanded. (5) The microwave emitted from the magnetron is not scattered but is radiated intensively to the sample contained in the container, which contributes to improving the reproducibility of the condition setting and various enzyme reactions. This is convenient because it can quickly activate the antigen-antibody reaction. (6) By providing the cold water circulation means and the temperature detection means, it is possible to control and prevent an excessive temperature rise of the sample solution which is heated by the microwave irradiation, so that the continuous irradiation of the microwave can be safely performed. It can be carried out. (7) Since the sample stirring means is employed, the temperature of the fixing solution contained in the container can be made uniform, and the level of fixation among a plurality of biological tissues immersed in the fixing solution can be made uniform. . (8) In the method for preparing an electron microscope sample according to the present invention, a fixative solution is promptly penetrated into a biological tissue, and cross-linking with a protein constituting a cell tissue is promoted. And contribute to quick diagnosis.

[Brief description of the drawings]

FIG. 1 is an external perspective view of an electron microscope sample preparation apparatus according to a preferred embodiment of the present invention.

FIG. 2 is a block diagram showing a configuration of the apparatus.

FIG. 3 is a diagram showing a structure around a sample setting chamber of the apparatus.

FIG. 4 is an enlarged view showing a state in which a pressing member is in contact with and pressurizes a sample container installed in the chamber.

FIG. 5 is a plan view showing a configuration of a chilled water circulating means disposed around a sample container of the apparatus.

FIG. 6A is a plan view of the biological tissue housing case housed in the sample container as viewed from above. FIG. 6B is a side view of the case viewed from the side.

FIG. 7 is a flowchart of the operation of the apparatus of the apparatus.

FIG. 8 is a drawing-substituted electron micrograph of rat bone tissue when microwave irradiation and fixation by pressure and resin infiltration by pressure are performed.

FIG. 9 is an electron micrograph instead of a drawing of the same structure obtained by fixation by decompression treatment and preparation of a resin-penetrated sample.

FIG. 10 is an electron micrograph as a substitute for a drawing of human surgically excised skin tissue when fixation by microwave irradiation and pressurization and resin permeation by pressurization are performed.

FIG. 11 is a drawing-substituted electron micrograph of the same tissue after normal fixation and resin infiltration.

FIG. 12 is a scanning electron micrograph of human normal skin epidermal cell tissues when fixed by microwave irradiation and pressurization, and subjected to resin penetration by pressurization.

FIG. 13 is an electron micrograph of a skin epidermal cell tissue of palmoplantar keratosis when fixation by microwave irradiation and pressurization and resin permeation by pressurization were performed.

FIG. 14 is a transmission electron microscope photograph showing a transmission electron microscope image of the stratum corneum portion of the drawing substitution photograph.

FIG. 15 is a diagram showing a usage mode of a conventional microwave improved microwave irradiation technique.

[Explanation of symbols]

REFERENCE SIGNS LIST 1 electron microscope sample preparation device 3 (sample) container 4 sample 5 lid (of container 3) 6 cold water circulation device 7 pressurizing cylinder 8 pressurizing member 9 temperature sensor (member) 10 magnetron 11 waveguide 11a (waveguide 11) Opening 12) Magnet 20 Microwave 22 (22a, 22b, 22c) (It is an elastic member)
O-ring 31 Opening 32 (of container 3) Outer wall 51 (of container 3) 51 Through-hole 61 (of lid 5) 61 Cold water circulation path 67 Open passage 68 Casing (of cold water circulation device 6) 82 Pressing surface 121 Make up) magnetic stirrer

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 1/28 F (71) Applicant 599088519 NMG Co., Ltd. 2-2, Kotobukicho, Atsugi-shi, Kanagawa No. 18 (71) Applicant 599088520 Yoshinori Matsuda 1-14-4, Tateoka Fueda, Murayama City, Yamagata Prefecture (72) Inventor Shichiro Miyazawa 1-33-16 Tobi, Atsugi City, Kanagawa Prefecture (72) Inventor Noriko Nemoto Machida, Tokyo Morino 3-18-18-203 (72) Inventor Yoshinori Matsuda 1-14-4 Fueda Tateoka, Murayama City, Yamagata Prefecture

Claims (14)

[Claims] 1. A microwave irradiation means for irradiating a microwave on a sample in which a biological tissue is immersed in a fixative or the like stored in a container, and a pressurizing means for applying pressure to the biological tissue. An electron microscope sample preparation apparatus comprising: performing microwave irradiation and / or pressurization on the biological tissue. 2. The microwave irradiating means includes a high-frequency oscillator for emitting microwaves, and a waveguide having an opening facing the container, and directing the microwaves toward the sample. 2. The electron microscope sample preparation apparatus according to claim 1, wherein the apparatus is configured to be capable of intensive irradiation. 3. Fixing the inside of the container by a cold water circulating means having a heat conductive casing disposed in contact with the outer wall of the container and a cold water circulating passage disposed inside the casing. 3. The electron microscope sample preparation apparatus according to claim 1, wherein the temperature of the liquid or the like is maintained at a predetermined temperature or lower. 4. The casing is formed in a substantially U-shape when viewed from above so as to open toward the opening side of the waveguide, and is arranged so as to surround the container. The electron microscope sample preparation apparatus according to claim 3, wherein 5. The electron microscope sample preparation apparatus according to claim 1, further comprising a stirring means for stirring the fixing solution and the like contained in the container. 6. The electron microscope sample preparation apparatus according to claim 1, further comprising a temperature detecting means for detecting a temperature of the fixing solution or the like contained in the container. 7. The temperature detecting means includes a pressing member having a pressing surface which comes into contact with a lid for closing an upper opening of the container accommodating the sample and presses from above. Is held at the center of the pressure member,
The pressure sensor comprises a rod-shaped temperature sensor member protruding from a lower end of the pressure member. The temperature sensor member is inserted into the inside of the container through a through hole formed in the center of the lid to irradiate the microwave. The electron microscope sample preparation apparatus according to claim 6, wherein the apparatus is configured to detect the temperature of the sample that changes in temperature due to the temperature. 8. An electron microscope sample preparation apparatus according to claim 7, wherein an elastic member for preventing pressure escape is provided on said pressing surface so as to surround said through hole at the time of contact. . 9. The electron microscope sample preparation apparatus according to claim 7, wherein the pressure member holding the temperature sensor member is formed of a heat insulating member. 10. The pressurizing member is configured to move up and down by a pressurizing cylinder disposed above, wherein an outer diameter of the pressurizing surface is formed smaller than an inner diameter of the pressurizing cylinder. The electron microscope sample preparation apparatus according to any one of claims 7 to 9, wherein: 11. A method for preparing an electron microscope sample, comprising using the electron microscope sample preparation apparatus according to claim 1. 12. The method for preparing an electron microscope sample according to claim 11, wherein at least an aldehyde-based fixing agent (having an aldehyde group) is used as a fixing solution for biological tissue. 13. A biological tissue staining method using the electron microscope sample preparation apparatus according to any one of claims 1 to 10. 14. A method for activating an enzyme reaction or an antigen-antibody reaction, comprising using the electron microscope sample preparation apparatus according to any one of claims 1 to 10.