JP2567068B2 - Electron microscope sample preparation method and electron microscope sample preparation instrument - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biotechnology technique such as virology, immunology, and cell engineering, which utilizes an antigen-antibody reaction.

[Conventional technology and its problems]

In recent years, human health has been affected by new types of viral diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, non-A non-B viral hepatitis, etc., and there is a risk of new infection due to blood transfusion. Has been done. Early diagnostics for these infectious diseases are being developed worldwide. The conventional diagnosis of infectious diseases was mainly antibody testing. The reason is that the number of virus particles is very small immediately after infection, and it cannot be detected by the conventional test method. For example, a hemagglutination method or the like has been conventionally known as a method for directly detecting a virus. However, in this method, since the detection sensitivity is insufficient, it was necessary to culture the virus and grow it to 10 million or more. On the other hand, electron microscopy is the only means for morphological observation to confirm the presence of viruses. Morphological observation makes it possible to determine the virus group, and when a new virus is discovered, it is essential to finally confirm the virus under an electron microscope. However, the virus is 20
Since it is about 200 nm, it can be observed only at ultra-high magnification. Since an extremely narrow region on the order of μm will be observed, the observation will be extremely difficult unless the virus is present at a high concentration. For this reason, conventionally, electron microscope observation was virtually impossible unless there were 100 to 1 billion virus particles per ml.

Therefore, in the conventional method for directly detecting the virus, it was important to first establish a technique for culturing the virus.
However, in virus culture, different hosts are used for each virus, so it was necessary to find susceptible cells suitable for virus culture. Moreover, it is extremely difficult to search for the susceptible cells because it is not possible to culture the virus that infects humans using animal cells. Due to these circumstances, research on virology that infects humans is far behind that of animals. For example, since viral hepatitis is highly infectious, at present, there are many accidents in which medical personnel are infected by patients, which has become a social problem. As viral hepatitis, hepatitis A virus and hepatitis B virus are known, and vaccines are also manufactured. However, despite the fact that non-A and non-B viral hepatitis has been predicted for a long time, the existence of the virus has not been confirmed, so that there is no appropriate treatment method.

Cancer initially develops in one cell and develops over a long period of time. Most of the current diagnostic methods such as endoscopy, CT, and pathological examination are performed by the eyes of a doctor. Therefore, at the time when a cancer is diagnosed, cancer that cannot be visually diagnosed has metastasized to many people and often recurs even after surgery. If it can be diagnosed at the cellular level when there is no metastasis, recurrence will disappear, and it will be possible to cure cancer by immunotherapy alone without surgery. Currently, cancer immunodiagnostic methods such as tumor markers are being researched and developed, but few are in practical use.

The present inventors have conducted research on a laser magnetic immunoassay method or the like capable of detecting viral antigens and cells with extremely high sensitivity, and the results thereof were first described in Japanese Patent Application Nos. 61-224567, 61-252427, and 61-252427.
61-254164, 62-22063, 62-152791, 62
-152792, 62-184902, 62-264319, 62-
Patent applications have been filed as 267481 and 63-6050. These new immunoassays are characterized in that laser light is used to detect the presence or absence of an antigen-antibody reaction, and that magnetic fine particles are used as a labeling material, which enables ultratrace detection of picograms.

However, technology for efficiently observing specimens such as viruses, cancer cells, or lymphocytes with an electron microscope has no technological innovation in the method of concentrating and purifying specimens, and it is necessary to rely on the conventional centrifugal sedimentation method. Is the current situation. Therefore, a lot of labor is required for the sample adjustment, and the efficiency is extremely low. The present inventors have studied a method for capturing and separating viruses and cells by labeling magnetic fine particles with a specimen such as virus, cancer cells or lymphocytes, and the results of the study were previously published in Japanese Patent Application No. 63-102916, A patent application has been filed as No. 63-102919. These patents can efficiently collect and separate viruses and cells. Also, when screening hybridoma cells obtained by the cell fusion technology, a new screening method in which magnetic microparticles are labeled with hybridomas that produce the desired monoclonal antibody and this is selectively recovered by external magnetic force is also pending. is there. However, in order to efficiently observe the collected specimen with an electron microscope, it was necessary to improve the technique.

The present invention has been made in view of the above circumstances, and its purpose is to observe various cells such as viruses, cancer cells, lymphocytes infected with AIDS and EB virus, and hybridomas producing monoclonal antibodies. Therefore, an object of the present invention is to provide an electron microscope sample adjusting method and an electron microscope sample adjusting instrument having high detection sensitivity and high observation efficiency.

[Means for solving the problem]

According to the first aspect of the present invention, a first step of magnetically labeling a specimen and a gradient magnetic field are applied to the specimen magnetically labeled in the first step to guide the specimen onto a mesh surface for electron microscope observation. An electron microscope sample preparation method is provided, which comprises at least a second step of fixing and a third step of negatively staining the magnetically labeled sample fixed in the second step in the gradient magnetic field. It

As a method for facilitating specimen inspection, if an antibody that specifically reacts with the specimen is added to the specimen suspension containing the specimen in advance, the specimen is aggregated, and the specimen is magnetically labeled, electron microscope observation is very easy. To be easier.

When the concentration / purification of the sample is particularly required, a gradient magnetic field is applied to the sample suspension containing the magnetically labeled sample after the first step of the first aspect of the invention so that only the magnetically labeled sample is locally applied on the water surface. It is preferable that after the concentration, a thin tube is inserted into the local concentration point and a sample concentration step of collecting the magnetically labeled sample is performed, and then the steps from the second step onward are performed.

Further, in the second step of the first invention, the electron microscope observation mesh is held on a detachable film, and a gradient magnetic field from the back surface of the film is applied so that the magnetic field of the electron microscope observation mesh portion is the highest. It is preferable to induce and fix the sample on the surface of the mesh for electron microscope observation by acting on.

Furthermore, according to the second invention, a mechanism for locally concentrating the magnetically labeled specimen on the water surface of the container, a mechanism for collecting the specimen, a mechanism for holding an electron microscope inspection mesh, and a mechanism for retaining the electron microscope observation mesh surface are provided. There is provided an electron microscope sample adjusting device comprising at least a sample guiding / fixing mechanism.

[Action]

According to the present invention, the sample is bound to the magnetic substance-labeled antibody by the antigen-antibody reaction and is magnetically labeled, so that it can be reliably guided and fixed on the electron microscope observation mesh by the external magnetic force. There are two types of methods for magnetically labeling a sample. One method is a method in which a magnetic substance-labeled antibody to which an antibody such as a monoclonal antibody that specifically reacts with a specimen is bound is directly reacted with the specimen and an antigen-antibody reaction, and the other method is that 1gG that specifically reacts with the specimen first. This is a method in which an antibody is added to the sample to agglutinate the sample in advance, and then a magnetic substance-labeled antibody bound to protein A that reacts with a 1 gG antibody is reacted with an antigen-antibody. The former method is a method of selectively capturing a target object such as a target virus or cell in a sample with a magnetic substance-labeled antibody by an antigen-antibody reaction. It is characterized by the high reliability of the inspection by probing the magnetically labeled antibody as a marker. On the other hand, in the latter method, the amount of protein A used for the magnetically labeled antibody is 1 g except for the target substance.
Since it also reacts with the G antibody, the selectivity is inferior to the former method. However, since the target object has been previously aggregated by the 1 gG antibody, if the target object is a microscopic body such as a virus, it is sufficient to search for the presence of the magnetically labeled aggregate, which is extremely useful when observing with an electron microscope. Can be efficient.

The magnetic fine particles used for the preparation of the magnetic substance-labeled antibody are preferably iron-based oxides such as magnetite, ferromagnetic substances made of transition metals or rare earth elements, and since these magnetic fine particles have a higher density than organisms, It looks black in the microscope field and its existence can be confirmed very easily.

By the way, it is usual that a large number of foreign bodies other than the target object are contained in the sample much more than the target object. Since these foreign bodies interfere with the inspection of the electron microscope,
One of the purposes of specimen adjustment of an electron microscope is to purify a target object. Since organisms do not respond to external magnetic forces,
The method of the present invention in which the analyte is magnetically labeled and only the target object is separated is extremely convenient for purification of the target object. Also,
In the method of the present invention, as an external magnetic force, a gradient magnetic field designed to be locally concentrated on the water surface is applied to the sample suspension containing the magnetically labeled sample, whereby the target object is concentrated at the local concentration point. It will be. Then, by inserting a thin tube into the local concentration point and collecting the target object, purification and concentration of the sample can be achieved at the same time. The reason for concentrating on the surface of the water is to reduce contamination of foreign matter other than the target object as much as possible. For example, if the bottom of the container is chosen as the local concentration point, contamination of foreign matter and precipitated cells is unavoidable.

The concentrated / purified sample is usually placed on a copper mesh with a diameter of about 3 mm for electron microscope observation, and stained for easy observation. In the conventional process, after the sample is dropped and attached on a copper mesh on which a supporting film such as formvar is attached, the excess sample is absorbed by filter paper, and a stain solution such as phosphotungstic acid is dropped to stain it. After that, the dyeing solution was sucked again with a filter paper, naturally dried, and then subjected to electron microscope observation. Since the step of removing such a sample and the staining solution with a filter paper was taken, most of the dropped sample was not retained on the copper mesh but was absorbed by the filter paper. As mentioned above, one of the reasons that the practical virus concentration required for observing virus particles is 100 million or more per ml is that only a small amount of the sample remains on the mesh in the above process. This is because. According to the method of the present invention, in the step of dropping the sample on the mesh and dyeing, the gradient magnetic field such that the magnetic field at the center of the mesh is the highest is applied from the back surface of the mesh holding film in which the mesh can be attached and detached. The sample is guided and fixed on the central surface of the electron microscope observation mesh by the action of. Therefore, when the excess sample and the staining solution are sucked with the filter paper or the like, the magnetically labeled sample is magnetically sucked onto the mesh, so that it is not sucked with the filter paper or the like. For this reason, for example, in the case of observing virus particles, it is possible to easily observe an electron microscope even with a diluted sample of about several tens per ml.

By using the electron microscope sample adjusting device of the present invention, anyone can easily carry out the electron microscope sample adjusting method of the present invention described above.

Hereinafter, the present invention will be described in more detail with reference to the drawings, but the following is merely an example of the present invention and does not limit the scope of the present invention.

[Example 1] Fig. 1 is a process chart showing an example of an electron microscope sample preparation method according to the present invention. The first step is to magnetically label a sample, and the magnetically labeled sample is a mesh for electron microscope observation. It consists of a second step of inducing and fixing in a gradient magnetic field, and a third step of negative staining in a gradient magnetic field. The samples used in this example were influenza A viruses (A / Ishikawa / 7/8
It is 2 (H3N2), and it is confirmed from the hemagglutination reaction and the hemacytometer that 1 million viruses are present per ml. Influenza virus at this concentration was
The virus was serially diluted in two-fold and the electron microscopic observation limit of the virus was examined. First, when the specimen was observed by an electron microscope by a conventional method without magnetic labeling, the presence of the virus particles could not be confirmed.

Next, the first step of this example will be described. The antibody used for magnetic labeling was a 1 gG antibody obtained by purifying rabbit hyperimmune serum against the virus, which was covalently bound to magnetic fine particles composed of magnetite with an average particle size of 10 nm coated with dextran to give magnetic properties. A body labeled antibody was obtained. The magnetic label of the sample is 35 ml of 1 ml of the sample and 10 μl of the magnetic substance-labeled antibody.
It was carried out under the conditions of incubation at 2.5 ° C. for 2.5 hours.

Next, the second step will be described. Fig. 2 shows that a magnetically labeled sample is guided in a gradient magnetic field onto an electron microscope observation mesh.
FIG. 1 is a sectional view of a fixture to be fixed, in which reference numeral 1 is an electromagnet
1a is an electromagnet core, 2 is a magnetic pole piece (A), 3 is a mesh mount, 4 is an adhesive film, 5 is a mesh holder, 6 is a mesh, 7 is a thin tube, and 8 is a cap. A conical pole piece (A) 2 made of pure iron is placed on the electromagnet iron core 1a, and the periphery thereof is covered by a non-magnetic mesh mount 3 made of aluminum. The tip of the pole piece (A) 2 is cut so that the magnetic field is not excessively concentrated, and the diameter of the end face is
It is 2 mm. In this embodiment, the electromagnet 1 was excited so that the magnetic field at the end face of the magnetic pole piece was about 3 KG. The mesh mounting table 3
A through hole having a diameter of 4 mm is provided at the center of the mesh, the end face of the magnetic pole piece (A) 2 is located at the center of the through hole, and is 0.5 mm lower than the surface of the mesh mounting table 3. The through hole is not always necessary, but the reason for providing the through hole is that the mesh 6 and the pole piece (A) 2 can be easily brought close to each other to obtain a strong magnetic field, and the center of the mesh 6 and the pole piece (A) 2 can be easily obtained. It is easy to match. A copper mesh 6 having a diameter of 3 mm on which the formvar support film is attached is lightly removably attached to an adhesive parafilm (adhesive film) 4 and held by a mesh holder 5. That is, as shown in FIG. 3, the mesh holder 5 is an acrylic resin having an outer diameter of 20 mm, an inner diameter of 8 mm, and a thickness of 1 mm, and the adhesive film 4 described above is attached to the bottom surface of the mesh holder 5.
Is lightly adhered to the adhesive film 4 at the center of the mesh holder 5. Since the mesh 6 is small and thin, it has conventionally been necessary to handle it with care. However, if the mesh 6 as in the present embodiment is handled, the sample adjustment is easy and the mesh 6 after the sample adjustment is transported, Easy to store. When the mesh 6 is taken out, if the adhesive film 4 on the back surface is lightly pushed out with a finger or the like, the mesh 6 can be easily peeled off from the adhesive film 4 and the mesh 6 can be taken out with tweezers.

Now, when the mesh holder 5 is placed on the center of the mesh table 3 and the electromagnet 1 is excited, the magnetically labeled sample in the capillary tube 7 is compressed by the cap 8 and a few μl is dropped on the mesh 6. A gradient magnetic field is generated by the magnetic pole piece (A) 2 so that the central portion of the mesh becomes the highest, so that the magnetically labeled sample is reliably guided and magnetically attracted to the central portion of the mesh 6. Approximately 1 minute after the dropping of the sample, the excess sample is sucked with a filter paper while the electromagnet 1 is excited. By the above processing, only the magnetically labeled sample is fixed on the mesh 6.

Next, in the third step, with the electromagnet 1 still energized, a negative staining solution consisting of 1% phosphotungstic acid having a pH of 7 is dropped onto a few μl mesh 6, and after 30 seconds, the excess staining solution is absorbed with a filter paper. Allow to air dry.

As a result of applying the above-mentioned electron microscope sample preparation method to the above-mentioned influenza virus and examining the detection limit, it was possible to very easily search for viruses with a magnification of 50,000 times even at a virus concentration of about 10,000 cells / ml. did it. Photo 1 is an electron micrograph of influenza virus that was magnetically labeled. The diameter of the virus particle is 100 nm, and as is clear from Photo 1, it can be seen that the magnetic substance-labeled antibody appearing black is bound around the virus.

[Example 2] Before the first step of magnetically labeling the sample described in Example 1, a step of aggregating the virus was performed. That is, 20 μl of the 1 gG antibody also used in Example 1 was added to 1 ml of the sample used in Example 1, and the viruses were aggregated by incubation at 35 ° C. for 2.5 hours and then at 4 ° C. overnight. After this step, sample preparation was performed in the same step as in Example 1. The difference from Example 1 is only the magnetic substance-labeled antibody used for magnetically labeling the sample. That is,
In this example, protein A was bound to dextran-coated magnetic particles. Since protein A binds only to the IgG antibody, the virus aggregated by the IgG antibody is magnetically labeled. In this way, pre-treatment for agglutinating the viruses with each other makes it easier to observe with an electron microscope.
I was able to easily search even about l. Photo 2 is an electron micrograph of the aggregated influenza virus magnetically labeled. It can be seen at a glance that dozens of virus particles are aggregated and the magnetic substance-labeled antibody that looks black is bound around the virus.

The method of aggregating a sample in advance to facilitate observation with an electron microscope as in this example is conventionally known as immunoelectron microscopy, but the present invention is the first method of magnetically labeling a sample. The detection sensitivity was improved by 5 orders of magnitude or more as compared with the conventional immunoelectron microscopy.

[Example 3] After the virus aggregation step and the first step of magnetically labeling the sample described in Example 2, the sample concentration / purification step is performed, and then the second step and the third step of Example 2 are performed. Sample preparation was performed. Next, the sample concentration / purification process will be described in detail.

FIG. 4 is a diagram for explaining the sample concentration / purification process,
(A) shows the concentration / purification process, and (b) shows the recovery process. In the figure, reference numeral 10 is a container, 11 is a magnetically labeled specimen,
11-1 is the collected specimen, and 12 is the magnetic pole piece (B). Since the magnetic circuit is formed so that the magnetic flux generated from the electromagnet 1 collects on the magnetic pole piece (B) 12, a gradient magnetic field is generated so that the magnetic field on the water surface directly below the magnetic pole piece (B) 12 is the highest.
In this example, the maximum magnetic field was 8KG. The pole piece (B) 12 has a hollow center so that the capillary tube 7 can be inserted therein. The container 10 contains 1 ml of a sample suspension containing the magnetically labeled sample 11 after the first step.

In the concentration / purification step (a), when the container 10 is inserted between the electromagnet 1 and the magnetic pole piece (B) 12 and the electromagnet 1 is excited, the magnetically labeled specimen 11 is directly below the magnetic pole piece (B) 12. It is induced and concentrated on the water surface of. At this time, since various contaminants in the sample suspension do not react to the magnetic force, only the magnetically labeled sample 11 is present at the concentration point.
Will be simultaneously concentrated and purified.

In the sample collecting step (b), when the electromagnet 1 is kept excited, the capillary tube 7 is inserted into the through hole of the magnetic pole piece (B) 12 and brought into contact with the water surface at the concentration point. Phenomenon, and magnetically labeled analytes are collected in the capillaries by magnetic attraction. In this example, a capillary tube having an outer diameter of 1.1 mm and an inner diameter of 0.5 mm was used as the capillary tube 7.
About 5 μl of the solution was naturally sucked into the capillary tube 7 due to the capillary phenomenon. The cap 8 is used in the next step of dropping the collected sample on the mesh.

Conventionally, in the electron microscope sample preparation, if the sample was not sufficiently purified, the electron microscope observation was significantly hindered.However, the purification effect of the present example makes the electron microscope observation very easy, and the concentration effect causes detection sensitivity. It became possible to search in a short time even when the virus concentration was several hundreds / ml or less.

The sample adjusting method described above can be easily and efficiently performed by anyone by using the sample adjusting device described below.

FIGS. 5 and 6 are views showing the structure of the electron microscope specimen adjusting device, in which reference numeral 13 is a yoke (A) holding the pole piece (B) 12, and 14 is a yoke (B). is there. The yoke (A) 13 is connected to the yoke (B) 14 by a yoke clamp screw 15 and can be fixed in any direction as shown in FIG. Electromagnet 1
After passing through the container 10, the generated magnetic flux is focused by the magnetic pole piece (B) 12 and a magnetic circuit is formed so as to return to the electromagnet 1 through the yoke (A) 13 and the yoke (B) 14. It When the distance between the electromagnet 1 and the pole piece (B) 12 is 11 mm, and a current of 1 A is applied to the electromagnet 1, the magnetic field at a position 0.5 mm directly below the pole piece (B) 12 is 8 mm.
It was KG. The container 10 is set on a container guide surface 17 provided on the container support 16 and attached to a height adjusting stage 19 by a container support clamp screw 18. The container support 16 can be adjusted to any height by the height adjustment stage 19, and can be fixed in any direction as shown in FIG.

Now, the sample purification / concentration step of the present embodiment described above is performed by assembling the present adjusting device as shown in FIG. First, with the capillary tube 12 removed from the magnetic pole piece (B) 12, the container 10 containing the sample is placed on the container guide surface 17, and the magnetic pole piece (B) is placed.
The height adjustment stage 19 adjusts the distance between the water surface of the container 12 and the water surface of the container 10 to be 5 mm. Next, the electromagnet is energized at 1 A to induce and concentrate the magnetically labeled specimen on the water surface directly below the magnetic pole piece (B) 12. After about 1 minute, set the capillary tube 7 to the magnetic pole piece (B).
Insert into the 12 through holes and use the height adjustment stage to
When 10 is lifted up and the capillary 7 is brought into contact with the water surface of the container 10, the magnetically labeled sample is instantaneously sucked into the capillary 7 together with the sample suspension liquid by magnetic attraction. In order to ensure the recovery of the sample, it is preferable to repeat the operation of bringing the capillary tube 7 into contact with the water surface. This is because the sample is concentrated on the water surface, so that the moment the capillary tube 7 comes into contact with the water surface is most easily collected, and if the capillary tube 7 is put too much into the water surface, it becomes difficult to collect the sample. If the capillary tube 7 is taken out from the magnetic pole piece (B) 12 after the energization of the electromagnet 1 is stopped, the sample is collected in the capillary tube 7.

Next, a method for carrying out the second step and the third step using the present adjusting instrument will be described. In the second step and the third step, the present adjusting device is assembled as shown in FIG. That is, the pole piece (B) 2 and the container support 16 which were on the electromagnet 1 are loosened by rotating the yoke clamp screw 15 and the container support clamp screw 18, respectively, and retracted. On the iron core 1a of the electromagnet 1, as shown in FIG. 2, the magnetic pole piece (A) 2 and further the magnetic pole piece (A).
The mesh mounting table 3 is placed on top of 2. The mesh holder 5 shown in FIG. 3 is placed on the center of the platform 3. Next, a current of 0.5 A is applied to the electromagnet 1 to excite the electromagnet 1, and the collected specimen is dropped into the capillary tube 7. Since the gradient magnetic field at which the magnetic field at the center of the magnetic pole piece (A) 2 is highest acts from the back surface of the mesh holder 5, the magnetically labeled sample is magnetically attracted onto the central surface of the mesh 6 and fixed to the mesh 6. . At this time, if the magnetic field is too strong, the Holmvar support film of the mesh 6 may break, so
It is preferable to weaken the magnetic field as compared with the step of concentrating and purifying the sample as described above. In this sample adjustment device, the electromagnet 1
When 0.5 A was applied, the magnetic field on the mesh 6 was about 2.5 KG.

〔The invention's effect〕

As described in detail above, according to the electron microscope specimen preparation method according to the present invention, the specimen is magnetically labeled by binding with the magnetic substance-labeled antibody in the antigen-antibody reaction, so that it can be reliably applied to the electron microscope observation mesh by the external magnetic force. Can be guided and fixed to
The detection sensitivity of the sample is dramatically improved. Moreover, a series of sample adjusting steps can be efficiently and easily carried out by using the sample adjusting device of the present invention. In the above-mentioned examples, the case of influenza virus was explained as a sample, but the sample preparation method of the present invention is not limited to viruses, and can be of course applied to electron microscope observation of various cells such as cancer cells and lymphocytes. .

Further, not only the method of negatively staining the sample on the mesh, but also the method of embedding the sample in resin and observing the thin section with a transmission electron microscope can be applied. That is, in the case of preparing a thin slice, a method of advancing the process of fixing, dehydrating, and embedding the sample was repeated while repeating the operation of centrifuging the sample, but if the sample preparation method of the present invention is applied, Instead of centrifugation and sedimentation, the sample can be locally guided and held in a gradient magnetic field, so that the sample can be easily fixed and dehydrated with alcohol, and the sample can be concentrated locally even when it is embedded, so it can be cut with a microtome. Easy to do.

According to the present invention, an extremely small amount of unknown virus that cannot be cultured can be directly detected, which greatly contributes to virology. For example, it can contribute to the discovery of non-A, non-B hepatitis viruses. It is also effective for early diagnosis of the cell level of cancer cells and the like, and it is possible to observe a very small amount of cancer cells which are released from the cancer cells and are transferred to body fluids such as blood.
As described above, the effects of the present invention in the fields of medicine / medical science, science such as molecular biology, and fields of biotechnology such as cell engineering and genetic engineering are immeasurable.

[Brief description of drawings]

FIG. 1 is a process diagram showing an embodiment of an electron microscope specimen preparation method according to the present invention, and FIG. 2 is a sectional view of an instrument for inducing and fixing a magnetically labeled specimen on an electron microscope observing mesh in a gradient magnetic field. FIG. 3 is a schematic view of a mesh holder, FIG. 4 is a diagram for explaining a sample concentration / purification step, (a) is a concentration / purification step, (b) is a recovery step, and FIGS. The figure is a diagram showing a configuration of an electron microscope sample adjusting device. 1 ... electromagnet, 1a ... electromagnet iron core, 2 ... pole piece (A), 3 ... mesh mount, 4 ... adhesive film, 5
…… Mesh holder, 6 …… Mesh, 7 …… Small tube,
8 ... Cap, 10 ... Container, 11 ... Magnetically labeled specimen, 11-1 ... Recovered specimen, 12 ... Magnetic pole piece (B),
13 ... Yoke (A), 14 ... Yoke (B), 15 ... Yoke clamp screw, 16 ... Container support, 17 ... Container guide surface, 18 ...
… Container support clamp screws, 19… Height adjustment stage,
20 cars.

Claims (5)

(57) [Claims] 1. A first step of magnetically labeling a sample, and a gradient magnetic field is applied to the magnetically labeled sample in the first step to guide and fix the magnetically labeled sample on the surface of a mesh for electron microscope observation. An electron microscope specimen preparation method comprising at least a second step and a third step of negatively staining the magnetically labeled specimen fixed in the second step in the gradient magnetic field. 2. The electron microscope specimen preparation method according to claim 1, wherein an antibody that specifically reacts with the specimen is added to the specimen suspension containing the specimen in advance before the first step to aggregate the specimen. A method for preparing an electron microscope specimen, which comprises performing steps, and then performing the steps after the first step. 3. The electron microscope sample preparation method according to claim 1, wherein a gradient magnetic field is applied to the sample suspension containing the magnetically labeled sample after the first step to locally concentrate only the magnetically labeled sample on the water surface. After that, a thin tube is inserted at the local concentration point, and a sample concentration / purification step of collecting the magnetically labeled sample is performed, and then the steps from the second step onward are performed. . 4. The electron microscope specimen adjusting method according to claim 1, wherein the electron microscope observing mesh used in the second step is held on a removable film, and the electron microscope observing mesh is held from the back surface of the film. A method for preparing an electron microscope specimen, which comprises inducing and fixing the specimen on the surface of the mesh for electron microscope observation by causing a gradient magnetic field having the highest magnetic field of the part to act on the magnetically labeled specimen. 5. A mechanism for locally concentrating a magnetically labeled specimen on the water surface of a container, a mechanism for collecting the specimen, a mechanism for holding an electron microscope observation mesh, and a mechanism for guiding the specimen onto the surface of the electron microscope observation mesh. An electron microscope specimen adjusting device comprising at least a fixing mechanism.