JP6281844B2 - Preparation method of electron microscope observation sample - Google Patents

Description

  The present invention relates to a method for preparing an electron microscope observation sample capable of observing the original fine structure in a cell without destroying the cell structure, and a preparation kit for an electron microscope observation sample for use in such a preparation method.

  Observation with a high-resolution electron microscope is indispensable for observing the intracellular microstructure. In order to observe with an electron microscope, since an electron beam is used, it is necessary to observe in a vacuum. However, since 80% or more of the cell components are water, it is difficult to observe the cells alive in a vacuum with an electron microscope.

  In view of this, conventionally, a method has been generally used in which intracellular structures are crosslinked and fixed by a chemical fixing method using a crosslinking agent such as glutaraldehyde, and dehydrated with alcohol for observation. However, the cross-linking agent has a problem that the penetration into the cell is slow, the structure changes before fixing, and the original fine structure in the cell is not maintained.

  In order to overcome the problem of the chemical fixation method, a method of observing the intracellular structure of a living cell by instantly rapidly freezing the cell has been proposed. For example, in order to instantly freeze cells rapidly, a biological sample is pressed against a metal block cooled to liquid helium temperature for a short time to prepare a sample for observing the biological sample using a transmission electron microscope. A method of rapidly freezing and fixing (see Patent Document 1) or a mixed coolant in which ethane is mixed with propane at a ratio of 10 to 90% is cooled by a coolant to a temperature lower than the melting point of the propane, and this cooling state A sample rapid freezing method (see Patent Document 2) in which a sample is immersed in a mixed coolant and rapidly frozen, or a cooled metal surface cooled with liquid helium, liquid nitrogen, or the like, or dropped into the coolant A method of rapidly freezing (see Patent Document 3) has been proposed. Such rapid freezing has an advantage that the structure in a living cell can be maintained as it is, unlike chemical fixation.

  However, the quick freezing equipment required to perform such a method is very expensive due to its complicated structure and requires advanced techniques, and the cells are pressed against a metal block or dropped into a coolant. There was a problem that the original fine structure of the cells could not be maintained due to physical impact due to. Furthermore, there is a problem that the water in the cells becomes ice crystals during the freezing process, and the ice crystals grow to destroy the structure in the cells.

  It is considered that by increasing the freezing rate, the intracellular water can be made into “amorphous ice” and the destruction of the cell structure can be prevented. However, in general, cells are attached to a cover glass, and when rapid freezing is performed with the cells attached to the cover glass, there is a certain amount of culture solution around and above the cells. Since the thickness of the cells is about 5 to 10 μm, it is necessary to remove the culture medium layer as much as possible in order to freeze it to become amorphous ice. However, if the layer of the culture solution is removed as much as possible, the culture solution is easy to dry immediately. As a result, the cells are deteriorated or die, so that advanced techniques and devices are required. Moreover, since the cover glass is made of glass, its thermal conductivity is low. Therefore, the freezing speed from the cover glass side is slow, and the freezing proceeds from the side of the cell not close to the cover glass. As a result, the part close to the cover glass takes time to freeze, and does not turn into amorphous ice well. Therefore, there has been a demand for a method for preparing an electron microscope observation sample in which cells are rapidly frozen so as to be amorphous ice to prevent destruction of the cell structure.

JP-A-10-142123 JP-A-5-126698 JP-A-5-18874

  An object of the present invention is to provide a method for preparing an electron microscope observation sample that is capable of observing the original fine structure in a cell without destroying the cell structure.

  The inventors of the present invention conducted a test using an aluminum foil having high thermal conductivity instead of a commonly used cover glass when rapidly freezing cells. By freezing the cells by attaching them to an aluminum foil coated with a carbon thin film, the cells can be rapidly frozen from the side in contact with the aluminum foil without removing the culture medium around or above the cells. As a result, it was found that ice crystals were not seen in the cells and an electron microscope observation sample maintaining the original fine structure in the cells could be prepared, and the present invention was completed.

That is, the present invention is as follows.
(1) A method for preparing an electron microscope observation sample, comprising the following steps (a) to (d):
(A) fixing a thermally conductive film whose surface is coated with a thin film of carbon or noble metal with a fixing device;
(B) a step of adhering cells to the heat conductive film fixed in step (a);
(C) a step of rapidly freezing the cells by immersing the heat conductive film to which the cells are adhered in the step (b) in a coolant cooled to −150 ° C. or lower;
(D) a step of preparing an electron microscope observation sample from the cells rapidly frozen in the step (c);
(2) The method for preparing an electron microscope observation sample according to the above (1), wherein the carbon or noble metal thin film is subjected to a hydrophilic treatment.
(3) The method for preparing an electron microscope observation sample according to the above (1) or (2), wherein the coolant is liquid propane or liquid isopentane.
(4) The method for preparing an electron microscope observation sample according to any one of (1) to (3) above, wherein the fixing device is an O-ring.
(5) In the step (d), the cells rapidly frozen in the step (c) are embedded in a resin, the heat conductive film is peeled off from the resin in which the cells are embedded, and an ultrathin section is produced. The method for preparing an electron microscope observation sample according to any one of (1) to (4) above.
(6) A kit for preparing an electron microscope observation sample, comprising: a heat conductive film whose surface is coated with a carbon or noble metal thin film; and a fixing device for fixing the heat conductive film.

  The electron microscope observation sample prepared by the method for preparing the electron microscope observation sample of the present invention does not destroy the cell structure, and can observe the original fine structure in the cell. In addition, the method for preparing an electron microscope observation sample according to the present invention does not require a complicated apparatus and does not require a high level of skill, so that an electron microscope observation sample can be easily produced at low cost.

It is a figure which shows the side surface, upper surface, and lower surface of the aluminum foil fixed with the stainless steel O-ring. It is a figure which shows the state which made the cell adhere | attach on aluminum foil. It is a figure which shows a mode that the aluminum foil which adhere | attached the cell in liquid propane cooled with liquid nitrogen is put and immersed. (A) A state in which cells are adhered to an aluminum foil coated with a carbon thin film, (b) a state in which cells are frozen, (c) a state in which cells are embedded with resin, and (d) a state in which the aluminum foil is peeled off It is a figure which shows a concept. It is a figure which shows the result of having observed the ultra-thin section produced by making cells adhere to a cover glass and quick-frozen with a transmission electron microscope. It is a figure which shows the result of having observed the ultra-thin section produced by making cells adhere to aluminum foil and rapidly freezing with a transmission electron microscope. It is a one part enlarged view of FIG.

As a preparation method of the electron microscope observation sample of the present invention,
(A) fixing a thermally conductive film whose surface is coated with a thin film of carbon or noble metal with a fixing device;
(B) a step of adhering cells to the heat conductive film fixed in step (a);
(C) a step of rapidly freezing the cells by immersing the heat conductive film to which the cells are adhered in the step (b) in a coolant cooled to −150 ° C. or lower;
(D) a step of preparing an electron microscope observation sample from the cells rapidly frozen in the step (c);
If it is a preparation method of the electron microscope observation sample provided with these process (a)-(d), it will not restrict | limit in particular, Gold, platinum, etc. can be mentioned as a noble metal.

  The electron microscope observation sample preparation kit of the present invention includes a heat conductive film whose surface is coated with a thin film of carbon or a noble metal, and a fixing device for fixing the heat conductive film. The kit is not particularly limited as long as it is a kit used for the preparation of an observation sample. By using such a kit, an electron microscope observation sample capable of observing the original fine structure in a cell can be easily produced. It becomes possible.

  The material of the heat conductive film is not particularly limited as long as it has heat conductivity, and examples thereof include aluminum, gold, silver, copper, and diamond. From the viewpoint, aluminum or gold is preferable, and aluminum is more preferable.

  As thickness of the said heat conductive film, 5-20 micrometers, Preferably it is 7-15 micrometers, More preferably, 10-12 micrometers can be mentioned.

  The fixing device is not particularly limited as long as it is a device that can be fixed so that the thermally conductive film can maintain a stretched state, and examples thereof include an O-ring or a square ring that can be fitted with the thermally conductive film interposed therebetween. Can do. Fixing the heat conductive film with a fixing device prevents deformation of the heat conductive film, facilitating handling such as freezing and resin embedding, and adhesion between cells attached to the heat conductive film Can be prevented.

  Although it does not restrict | limit especially as a material of a fixing device, Stainless steel, iron, aluminum, copper etc. can be mentioned.

  The thermally conductive film may be polished and smoothed, and the polishing method may be a physical polishing method or a chemical polishing method. When the rapidly frozen cells are embedded with a resin, the thermal conductive film is polished and smoothed to facilitate peeling of the thermal conductive film from the embedded resin. In addition, by placing the cells on a heat conductive film (substrate) that has been smoothed, the heat conductive film is peeled off from the embedded resin, and then an ultrathin section is cut out from the resin surface with a microtome. Alternatively, a clean ultrathin section can be taken from the second sheet, and it becomes possible to observe the site of cells attached to the heat conductive film.

  Examples of the thickness of the thin film include 10 to 200 nm, preferably 20 to 100 nm. Examples of the coating method include a vacuum deposition method and a sputtering method. By coating the thin film, it becomes easy to peel the heat conductive film from the resin in which the cells are embedded.

  In order to improve the adhesion of cells, it is preferable to hydrophilically treat the coated thin film. Examples of the hydrophilic treatment include plasma treatment and corona discharge treatment. Alternatively, the upper surface of the thin film may be coated with an adhesion factor such as collagen, gelatin, fibronectin, or elastin, or polylysine, polyethyleneimine, spermidine, spermine, or other polycation.

  As a method of adhering cells to the fixed heat conductive film, a method of adding a culture solution containing cells on the fixed heat conductive film and allowing to stand, or adding a culture solution and cells on the heat conductive film And a method of culturing.

  The cell may be a single cell, a tissue cell, or a plurality of cultured cells. Examples of cell types include animal cells, plant cells, bacteria, and protists. .

  As a method of rapidly freezing cells by immersing the heat conductive film to which the cells are adhered in a cooling agent cooled to −150 ° C. or less, the heat conductivity in which the cells are adhered in a state of being fixed by a fixing device. A method of immersing the film in a coolant cooled to −150 ° C. or lower, preferably −160 ° C. or lower can be mentioned.

  As a cooling agent, liquid propane, liquid isopentane, and liquid ethane can be mentioned, and liquid propane or liquid isopentane can be mentioned suitably.

  In the present invention, the method for preparing an electron microscope observation sample from the cells rapidly frozen in step (c) is not particularly limited, but (1) the cells rapidly frozen in step (c) are embedded in a resin, A method of peeling a thermally conductive film from an embedded resin to prepare an ultrathin section, (2) a method of preparing an ultrathin section directly from frozen cells, and (3) a replica by cutting a frozen sample. The method etc. can be mentioned.

  In the method of preparing the ultrathin section by embedding the cells rapidly frozen in the step (c) (1) with a resin, peeling the heat conductive film from the resin in which the cells are embedded, Examples of the method of embedding with a resin include a general method of embedding from a resin such as an epoxy resin, a methacrylic acid resin, or a polyester resin. The rapidly frozen cells may be further fixed with acetone containing a reducing agent such as glutaraldehyde or formaldehyde or an oxidizing agent such as osmic acid, and then embedded in a resin.

  Moreover, as a method of peeling a heat conductive film from the resin which embedded the said cell, the method of peeling a heat conductive film physically using instruments, such as tweezers, or a finger can be mentioned. It is preferable to remove the heat conductive film from the fixture before peeling off the heat conductive film.

  Furthermore, the method for preparing the ultrathin section is not particularly limited, and a general method used in the preparation of an electron microscope observation sample can be used. The method of producing using a microtome according to the method as described in "Technology" Kyoritsu Shuppan publication 20-31 (2008)) can be mentioned.

  Examples of the method (2) for preparing an ultrathin section directly from frozen cells include a method for preparing ultrathin sections from frozen cells using a cryomicrotome. The prepared ultrathin sections can be observed directly with a cryo-electron microscope, or can be observed with an immunoelectron microscope after reacting with an antibody or the like.

  (3) As a method for cleaving frozen cells to produce a replica, freeze fracture replica method in which a frozen cell is cleaved and carbon or platinum is deposited on a fractured surface to produce a replica, or a frozen cell An example is a freeze fracture replica method in which ice is sublimated (etched) in a vacuum after cleaving, and then a replica is prepared by vapor-depositing carbon or platinum on the surface thereof. The replica produced should be observed with an electron microscope in accordance with the method described in the literature (Juro Usukikura, “Bioelectronic Microscopy Techniques Understandable”, Kyoritsu Shuppan Publishing, pages 75-85 (2008)). Can do.

  The preparation kit for the electron microscope observation sample of the present invention may include an instruction describing the preparation method of the electron microscope observation sample of the present invention, an instrument such as tweezers for holding the fixing instrument, a coolant, and the like.

  An aluminum foil having a thickness of 12 μm was sandwiched between a stainless steel O-ring having an inner diameter of 12 mm, a width of 1 mm, and a thickness of 0.3 mm and a stainless steel O-ring having an inner diameter of 10 mm, a width of 1 mm, and a thickness of 0.3 mm. The aluminum foil was smoothed by being immersed in a polishing solution (85% phosphoric acid, 12% nitric acid) at 85 ° C. for 2 to 4 seconds. Furthermore, using a vacuum evaporation system JEOL JEE-400 (manufactured by JEOL Ltd.), carbon was deposited on the upper surface of the aluminum foil for 10 seconds to coat a carbon thin film (100 mm), and PIB-10 (manufactured by Vacuum Device Inc.) A plasma treatment was performed for 10 seconds to perform a hydrophilic treatment. The side, top and bottom surfaces of the aluminum foil fixed with a stainless steel ring are shown in FIG.

  On the aluminum foil coated with a carbon thin film, 0.05 ml of a culture solution containing cells of cellular slime molds was added and allowed to stand for 30 minutes to adhere the cells on the aluminum foil. FIG. 2 shows a state where cells are adhered on the aluminum foil.

  The O-ring to which the aluminum foil to which the cells are adhered is fixed is held with tweezers, and immediately immersed in a liquid portion of liquid propane or liquid isopentane cooled with liquid nitrogen at −196 ° C. as shown in FIG. The cells were snap frozen. In addition, as shown in the said FIG. 2, since the cell is adhere | attached with the aluminum foil with high heat conductivity, freezing of a cell progresses rapidly from the site | part adhere | attached with the aluminum foil. Therefore, it is not necessary to remove the culture solution in order to rapidly freeze the cells, and there is no problem that the cells are dried. In addition, since the cells are immersed and immersed in liquid propane or liquid isopentane while being adhered to the aluminum foil or the culture solution, physical impact on the cells can be minimized.

  The frozen cells were embedded with resin by the following method. First, it was immersed in a solution containing osmium and acetone at −80 ° C. for 2 hours to replace the water in the cells with acetone, and then immersed at −20 ° C. for 1 hour, further at 4 ° C. for 1 hour, and at room temperature for 1 hour. . Next, it was immersed in acetone for 1 hour, acetone and propylene oxide (1: 1) for 1 hour, and propylene oxide for 1 hour. Furthermore, after being immersed in Suprr resin (manufactured by Polyscience) and propylene oxide (1: 1) for 2 hours and in Suprr resin for 1 day, it was cured by treatment at 70 ° C. for 8 hours. (A) A state in which cells are adhered to an aluminum foil coated with a carbon thin film, (b) a state in which cells are frozen, (c) a state in which cells are embedded with resin, and (d) a state in which the aluminum foil is peeled off The concept is shown in FIG.

  Further, after removing the O-ring and peeling off the aluminum foil from the resin in which the cells were embedded, the cell part was cut out from the resin using a thread saw, and the literature (Kyotsuji Usukura, “Bioelectronic Microscopy Technology”, published by Kyoritsu Shuppan 20 To 31 (2008)), ultrathin sections were prepared using a microtome.

  As a control, the cells were adhered to a cover glass (without smoothing treatment), and quick-frozen, resin-embedded, and ultrathin sections were prepared as described above.

  The obtained ultrathin sections were observed with a transmission electron microscope (CM-120: manufactured by Philips or Quanta3D FEG: manufactured by FEI) after electron staining with uranium acetate and lead acetate in a conventional manner, and the results are shown in FIGS. . FIG. 5 shows a case where an ultrathin section is prepared by attaching cells to a cover glass and quick freezing, and FIG. 6 shows a case where an ultrathin section is prepared by attaching cells to an aluminum foil and quick freezing. FIG. 7 is an enlarged view of a part of FIG. 6. As shown in FIG. 5, when an ultrathin section is prepared by adhering a cell to a cover glass and rapidly freezing, ice crystals are formed in the cell, and the fine structure in the cell is destroyed, resulting in a mesh. It was in the shape. On the other hand, as shown in FIGS. 6 and 7, when an ultrathin section is prepared by adhering cells to aluminum foil and rapidly freezing, ice crystals are not observed in the cells, and the fine structure is maintained. It was.

  The electron microscope observation sample prepared by the method of the present invention is capable of observing the original fine structure inside the cell without destroying the cell structure, and observing the fine structure such as cells, foods and biological materials. It is available to do.

Claims (5)

An electron microscope observation sample preparation method comprising the following steps (a) to (d):
(A) A step of polishing and smoothing the surface, and further fixing a thermally conductive film coated with a carbon or noble metal thin film with a fixing device;
(B) a step of adhering cells to the heat conductive film fixed in step (a);
(C) a step of rapidly freezing the cells by immersing the heat conductive film to which the cells are adhered in the step (b) in a coolant cooled to −150 ° C. or lower;
(D) a step of preparing an electron microscope observation sample from the cells rapidly frozen in the step (c); 2. The method for preparing an electron microscope observation sample according to claim 1, wherein the carbon or noble metal thin film is subjected to a hydrophilic treatment. The method for preparing an electron microscope observation sample according to claim 1 or 2, wherein the coolant is liquid propane or liquid isopentane. The method for preparing an electron microscope observation sample according to any one of claims 1 to 3, wherein the fixing device is an O-ring. In the step (d), the cells rapidly frozen in the step (c) are embedded with a resin, the heat conductive film is peeled off from the resin in which the cells are embedded, and an ultrathin section is produced. The preparation method of the electron microscope observation sample in any one of 1-4.