Coating device, coating method

Abstract

To coat a fine three-dimensional structure and an uneven surface formed of protein or interstitial fiber protein constituting a pathological tissue made into a slide glass specimen with an electrically conductive carbon-based film.SOLUTION: A coating method comprises a slide glass electrode preparation step, an arrangement step and a voltage application step. In the slide glass electrode preparation step, a slide glass electrode is made which has a film of predetermined electric resistivity formed on a surface of a plate-like slide glass. In the arrangement step, a tissue segment is arranged directly on the slide glass electrode, which is arranged on a sample base as a negative electrode so as to feed electricity. In the voltage application step, a voltage is applied between a positive electrode and the negative electrode in a pressure-reduced gas atmosphere for plasma generation to generate plasma.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a coating device and a coating method for forming a thin film (preferably a conductive thin film) on the surface of a tissue section in order to enable observation of an insulator with a scanning electron microscope.

Background Art

[0002] As techniques for forming a thin film on the surface of a substrate to be processed, Patent Documents 1, 2, etc. are known. Patent Document 1 discloses a means for forming a thin film using plasma chemical vapor deposition (PCVD). In Patent Document 1, for forming a thin film, a device that applies a DC high voltage between an anode and a cathode to generate a glow discharge is used. 4 When using Os (osmium), it is disclosed that a conductive Os (osmium) metal thin film (Os thin film) can be efficiently formed with a substantially uniform thickness. Patent Document 2 discloses a prototype device that adopts a method using a conductive substrate to be processed as a plasma generation unit and the verification results using the same. In Patent Document 2, a device capable of forming a carbon-based thin film on the surface of a three-dimensional object metal is disclosed by using the method and the device. Further, Non-Patent Documents 1 and 2 introduce electron microscopes for observing tissue sections on which a thin film has been formed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, when applying the technology of Patent Document 1 to the formation of a thin film on the surface of a tissue section (biological sample), since it is a design using a DC power supply, in the space of the negative glow layer region between the electrodes, the formation of Os fine particles starts before reaching the tissue surface, and due to growing while overlapping on the tissue surface, there is a problem of overlapping fine structures. Furthermore, the Os thin film formed from Os 4 is an element of the sixth period, so the Os film overlapping the tissue surface easily blocks the backscattered electron (BSE) signal, and there is a problem that it becomes difficult to acquire signals derived from elements of the fourth period or lower in particular.

[0006] In addition, the possibility of forming a carbon-based film on the fine uneven surface of a tissue (biological sample) was examined by the ion implantation method using the self-discharge electrode method, which is the technology of Patent Document 2. The technology of Patent Document 2 is a method of implanting ions on the surface of a conductive substrate to be treated. However, since tissue sections cut thinly from a formalin-fixed and paraffin-embedded specimen and placed on a slide glass are insulator (non-conductive) samples, even if the technology of Patent Document 2 is directly applied to the production of a thin film on the surface of a tissue section, there is a problem that plasma cannot be uniformly generated on the surface of the substrate to be treated.

[0007] In order to solve the above-described problems, the present invention aims to enable a scanning electron microscope (SEM) to detect a secondary electron (SE) signal that characterizes a pathological tissue specimen by coating the surfaces of proteins and stromal fibrous proteins that constitute a cell membrane with fine irregularities in a tissue section cut thinly from a formalin-fixed and paraffin-embedded specimen and placed on a slide glass with a thin film (preferably, a conductive carbon-based film).

Means for Solving the Problems

[0008] The coating device of the present invention coats the surface of a tissue section with a film material. The coating device of the present invention includes a vacuum chamber, an exhaust section, an intake section, an anode, a cathode, a power supply section, and a slide glass electrode. The exhaust section evacuates the gas in the vacuum chamber. The intake section intakes gas into the vacuum chamber. The anode and the cathode are disposed in the vacuum chamber. The power supply section applies a voltage between the anode and the cathode. The slide glass electrode forms a film having a predetermined electrical resistivity on the surface of a plate-shaped glass. The cathode is a conductive sample stage. The tissue section is placed directly on the slide glass electrode. The slide glass electrode is disposed on the sample stage so as to be electrically connected to the sample stage.

[0009] The coating method of the present invention coats the surface of a tissue section with a film material. The coating method of the present invention performs a slide glass electrode manufacturing step, an arranging step, and a voltage applying step. The slide glass electrode manufacturing step makes a slide glass electrode having a film with a predetermined electrical resistivity formed on the surface of a plate-shaped glass. The arranging step directly arranges a tissue section on the slide glass electrode and arranges the slide glass electrode so that it can be energized to a sample stage that is a cathode. The voltage applying step applies a voltage between the anode and the cathode in a plasma generating gas atmosphere under reduced pressure to generate plasma.

Advantages of the Invention

[0010] According to the coating apparatus and the coating method of the present invention, a tissue section that has been thinly sliced from a formalin-fixed and paraffin-embedded specimen and placed on a slide glass has a spongy surface due to its unevenness and the presence of minute vesicles inside, but plasma can be uniformly generated on the slide glass and the tissue section placed thereon. Therefore, a thin film (preferably a conductive carbon-based thin film) can be formed on the surface of the complex three-dimensional structure of the thinly sliced tissue section, so that secondary electrons from the tissue section can be efficiently emitted during observation with a scanning electron microscope. Therefore, a secondary electron signal can be detected with a scanning electron microscope.

Brief Description of the Drawings

[0011]

Figure 1

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Figure 9

[0012] Hereinafter, an embodiment of the present invention will be described in detail. Components having the same functions are given the same numbers, and duplicated explanations will be omitted. In the following description, scanning electron microscope observations using a low-vacuum scanning electron microscope (Non-Patent Document 1) were performed at an electron gun acceleration voltage of 5 to 15 kV. Scanning electron microscope observations using a field emission scanning electron microscope (Non-Patent Document 2) were performed at an electron gun acceleration voltage of 2.0 to 10 kV. EXAMPLES

[0013] FIG. 1 shows a flow chart of the coating method of the present invention and observation with a scanning electron microscope. FIG. 2 shows an example of the configuration of a coating device of the present invention. In the coating method of the present invention, the surface of a tissue slice is coated with a film material. The film that coats the surface of the tissue slice is preferably a carbon-based film or an osmium film, and more preferably a carbon-based film. The coating method (S100) includes a slide glass electrode preparation step (S110), a placement step (S120), and a voltage application step (S130). Then, observation with a scanning electron microscope (S200) is performed.

[0014] The slide glass electrode manufacturing step (S110) produces a slide glass electrode by forming a film with a predetermined electrical resistivity on the surface of a plate-shaped glass. The placement step (S120) directly places a tissue section on the slide glass electrode and arranges the slide glass electrode so that it can be energized to a sample stage that is a negative electrode. The voltage application step (S130) applies a voltage between the positive electrode and the negative electrode in a plasma generation gas atmosphere under reduced pressure to generate plasma.

[0015] The voltage application step (S130) may be executed using the coating device 200. The coating device 200 coats the surface of the tissue section with a film material. The coating device 200 includes a vacuum chamber 270, an exhaust section 210, an intake section 280, a positive electrode 290, a sample stage 230 that is a negative electrode, a power supply section 260, and a slide glass electrode 100. The slide glass electrode 100 is formed by forming a film with a predetermined electrical resistivity on the surface of a plate-shaped glass. The exhaust section 210 exhausts the gas 310 in the vacuum chamber 270.

[0016] The intake unit 280 sucks gas into the vacuum chamber 270. The intake unit 280 may be provided with flow rate adjustment units 281 and 282. With the structure shown in FIG. 2, two types of source gases 381 and 382 can be sucked in. The gas (source gas) to be sucked in is a gas for plasma generation. The gas for plasma generation can be appropriately selected according to the type of film for coating the tissue section. When forming a carbon-based film, as the gas for plasma generation, a hydrocarbon gas (e.g., acetylene), a nitrogen-containing compound (e.g., aniline), a mixed gas of a hydrocarbon gas (e.g., acetylene) and nitrogen gas, or a mixed gas of a hydrocarbon gas (e.g., acetylene) and a boron-containing compound may be used. When forming a highly conductive carbon-based film, as the gas for plasma generation, a nitrogen-containing compound (e.g., aniline), a mixed gas of a hydrocarbon gas (e.g., acetylene) and nitrogen gas, or a mixed gas of a hydrocarbon gas (e.g., acetylene) and a boron-containing compound may be used. When forming a low-conductive carbon-based film, as the gas for plasma generation, a hydrocarbon gas (e.g., acetylene) may be used. When forming an osmium film, as the gas for plasma generation, osmium tetroxide gas may be used. If two types of source gases 381 and 382 can be sucked in, it is easy to suck in a mixed gas.

[0017] The positive electrode 290 and the negative electrode (sample stage 230) are arranged inside the vacuum chamber 270. The positive electrode 290 may be arranged to cover the inside of the vacuum chamber 270, or the vacuum chamber 270 itself may serve as the positive electrode 290. The "positive electrode arranged in the vacuum chamber" includes the case where the positive electrode 290 is a separate body from the vacuum chamber 270, the case where the inner surface of the vacuum chamber 270 is the positive electrode 290, and the case where the vacuum chamber 270 itself is the positive electrode 290.

[0018] The sample stage 230 is conductive and is the cathode. The sample stage 230 is insulated from the outside by the insulating stage 220. The slide glass electrode 100 is arranged on the sample stage 230 so as to be electrically conductive with the sample stage 230. The sample stage 230 is connected to the power supply unit 260 via the conducting wire 250. The slide glass electrode 100 has a structure that is electrically integrated with the sample stage 230 (cathode). The current introduction terminal 240 connects the conducting wire 250 outside and inside the vacuum container 270. The anode 290 may be grounded. With such a structure, the electric field strength near the cathode 230 becomes stronger, and it becomes easier to generate plasma near the cathode 230.

[0019] The tissue section is placed directly on the slide glass electrode 100. The tissue section is, for example, a tissue section thinly sliced from a formalin-fixed and paraffin-embedded specimen. Also, the film with a predetermined electrical resistivity that coats the slide glass electrode 100 produced in the slide glass electrode production step (S110) may be any film that does not cause charge-up when coating the tissue section. Therefore, a conductive film is preferably used, but it does not need to be a general conductive material, and any material of a film having a predetermined electrical resistivity may be used. For example, for the "predetermined electrical resistivity", the electrical resistivity may be 100 Ωm or less. The film thickness may be any thickness that is likely to become a plasma generation site and a film formation reaction site. Note that a carbon-based film is preferably used as the film that coats the slide glass electrode 100. Also, the electrical resistivity and the film thickness are preferably an electrical resistivity of 0.25 Ωm or more and 57 Ωm or less, and a film thickness of 3 nm or more and 100 nm or less.

[0020] The power supply unit 260 applies a voltage between the anode 290 and the cathode (sample stage 230). Specifically, a negative voltage pulse (high voltage millisecond pulse) of 2 kV or more at 1 Hz or more and 100 kHz or less may be applied to the sample stage 230, which is the cathode, with respect to the ground potential. The power supply unit 260 may be able to apply, for example, a maximum of -25 kV. When the sample has few irregularities, the anode 290 may be grounded and a negative DC voltage of 1 kV or more may be applied to the cathode (sample stage 230).

[0021] The surface of the slide glass electrode 100 of the coating device 200 has a structure that is energized with a film having a predetermined electrical resistivity (preferably, a carbon-based film) between the conductive sample stage 230. Therefore, a current of up to 10 A can be passed from the power supply unit 260 through the conducting wire 250. In a reduced-pressure state, the raw material gases 381 and 382 are inhaled by the flow rate adjustment units 281 and 282 to create a plasma generation gas atmosphere. Then, when a high-voltage pulse is applied to the sample stage 230, plasma is generated near the composition section.

[0022] As the tissue section, for example, a tissue section thinly sliced from a formalin-fixed and paraffin-embedded specimen can be used. The thickness of the tissue section can be appropriately adjusted according to the observation target, but is, for example, about 0.1 to 10 μm. The tissue section may have a thickness of less than 10 μm by the method disclosed in Patent Document 2 and Reference Non-Patent Document 1 (K. Baba and R. Hatada, Deposition of diamond-like carbon films by plasma source ion implantation with superposed pulse. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, 2003.). Further, after removing paraffin as necessary, various stainings (for example, staining with a fluorescent dye observable with a fluorescence microscope, staining with a dye observable with an optical microscope) may be performed and then dehydrated and dried. In the coating with the conductive carbon-based film, even in the case of a tissue section subjected to Mayer's hematoxylin staining containing Al, detection and imaging of the SE signal characterizing the tissue morphology become possible. In order to increase BSE, the tissue section may be doped with a metal such as gold (Au). In particular, since the carbon-based film is less likely to block BSE, by coating the tissue section doped with Au or the like with the carbon-based film, the BSE signal derived from Au or the like can be favorably acquired. Further, by doping with Au and coating with the carbon-based film, good detection and visualization of the BSE signal can be achieved without using the heavy metal U that requires management. Although Au is introduced into the tissue section by Grocott staining, it is possible to obtain a good SEM image by coating the Grocott-stained tissue section with a conductive carbon-based film and imaging the BSE signal and the SE signal with a scanning electron microscope. For example, it is also possible to confirm fungi such as Aspergillus and mucus vacuoles in the SEM image. Then, it may be placed on the sample stage 230 of the coating device 200 in a state of being placed on the slide glass electrode.

[0023] In the coating device 200, the slide glass electrode 100 coated with a film having a predetermined electrical resistivity (preferably, a carbon-based film) is configured to be energized via the sample stage 230. As a result, a high-voltage millisecond pulse generated by the power supply unit 260 is applied, so that the surface of the slide glass electrode 100 and the thin tissue section on the slide glass electrode 100 become a plasma generation site and a reaction site for film (preferably, carbon-based film) formation. The coating device 200 forms a thin film (preferably, a conductive thin film) on the surface of the tissue section based on such a principle (coats with a film constituent material (preferably, a conductive substance)), so the slide glass electrode 100 may have a structure electrically integrated with the sample stage 230 as long as it can apply a high-voltage millisecond pulse.

[0024] In the coating device 200, within a voltage range that does not cause arc discharge in a plasma generation gas atmosphere under reduced pressure, a negative high-voltage pulse can be repeatedly applied with respect to the ground potential. Therefore, the tissue section on the slide glass electrode 100 on the sample stage 230 can be made into a plasma generation site and a reaction site for film formation by substantially using it as a self-discharge electrode. Thus, in the coating device 200, ions are generated inside and on the surface of the sponge-like tissue section and are attracted around the structural proteins inside the tissue section, so that film (preferably, carbon-based film) formation proceeds on the surface of the structural proteins.

[0025] In the observation with a scanning electron microscope, in order to achieve good secondary electron emission, the film covering the surface of the tissue section is preferably conductive. The electrical resistivity of the film formed on the surface of the tissue section is, for example, 585 Ωm or less. The inventors of the present invention have confirmed that by covering the surface of the tissue section with a carbon-based film having an electrical resistivity of 27.2 Ωm to 585 Ωm by the method of the present invention and observing with a scanning electron microscope, secondary electrons are efficiently emitted from the tissue section, enabling good visualization through the acquisition of secondary electron signals. When only a hydrocarbon gas (e.g., acetylene) is used as the source gas, a carbon-based film with such low conductivity that the electrical resistivity cannot be measured even with a resistance measuring instrument with a measurement limit of 2 GΩ at a film thickness of 35 nm can also be formed. If the film thickness of the film covering the surface of the tissue section is too thick, it will cover the fine structure of the sample surface and make it difficult to observe with an optical microscope. Therefore, it is better to be as thin as possible. However, if it is too thin, the film will not be continuous and will cause charging. By setting the film thickness of the carbon-based film to, for example, 3 nm to 130 nm (preferably 35 nm to 130 nm), good observation with an optical microscope and detection / imaging of SE signals with high spatial resolution become possible. The film thickness of the osmium film is, for example, about 1 nm to 10 nm (e.g., 2.5 to 5 nm).

[0026] After the coating method (S100), observation with a scanning electron microscope (S200) is performed. In the observation, the tissue section whose surface is coated with a film (preferably a conductive carbon-based film) by the coating method (S100) may be observed with a scanning electron microscope such as Non-Patent Documents 1 and 2.

[0027] FIG. 3 is a diagram showing an example in which a carbon-based film is formed on the surface of silicon having a trench structure. FIG. 3(A) is a diagram showing the entire trench structure, and FIG. 3(B) is an enlarged view of the solid-line portion in FIG. 3(A). In this example, a silicon trench structure having an opening width of 10 μm in depth is placed on the sample stage 230, and acetylene is introduced as a source gas with its concentration adjusted by the flow rate adjuster 281, and a carbon-based film is formed at a pulse voltage of -18 kV and a pulse frequency of 1 kHz. After the treatment, the silicon wafer is cut, and when the cross section is observed by SEM for the formation state of the carbon-based film, it is possible to confirm the carbon-based film formed also on the side surface of the trench of the silicon substrate (FIG. 3).

[0028] FIG. 4 is a diagram showing a scanning electron microscope image (SEM image) for showing the difference in the backscattered electron (BSE) signals when a carbon-based film is formed and when an Os film is formed. FIG. 4(A) is an example of a carbon-based film with a thickness of 35 nm, FIG. 4(B) is an example of a carbon-based film with a thickness of 130 nm, FIG. 4(C) is an example of an Os film with a thickness of 2.5 nm, and FIG. 4(D) is an example of an Os film with a thickness of 5 nm. In the coating apparatus 200, various compositions capable of forming a conductive carbon-based film are used as source gases in the evacuated vacuum chamber 270. For example, vaporized acetylene is introduced as the source gas 381 in a predetermined amount by the flow rate adjuster 281, and at the same time, nitrogen gas is introduced as the source gas 382 in a predetermined amount by the flow rate adjuster 282. Then, the vacuum chamber 270 is maintained at 3 Pa, and a high-voltage millisecond pulse of 1 kHz at -8 kV is continuously applied to the slide glass electrode 100 on the sample stage 230 from the power supply unit 260 via the lead wire 250 for 26 minutes, whereby a conductive carbon-based film with a thickness of 35 nm is formed. When the application is continued for 98 minutes, a conductive carbon-based film with a thickness of 130 nm is formed (FIGS. 4(A) and (B)). In the case of the Os film, only the thickness increases from 2.5 nm to 5 nm, and Os is superimposed on the tissue surface, and when it becomes larger than this, the BSE signal cannot be obtained (FIGS. 4(C) and (D)), but in the case of the carbon-based film, the BSE signal can be obtained without any problem even when the thickness is 130 nm.

[0029] FIG. 5 shows scanning electron microscope images (SEM images) showing the difference in secondary electron (SE) signals between a carbon-based film and an Os film. FIG. 5(A) shows an example of a highly conductive carbon-based film, FIG. 5(B) shows a low-conductivity carbon-based film, and FIG. 5(C) shows an example of an Os film. FIG. 5(A) shows an example of a conductive carbon-based film with an electrical resistivity of 471 Ωm formed by applying a pulse voltage of −8 kV and 1 kHz for 20 minutes to a film thickness of 35 nm while introducing a predetermined amount of vaporized aniline as a raw material gas 381 by a flow rate adjustment unit 281 using a coating device 200. In the case of FIG. 5(A), the fine uneven structure around the mucus vesicles and the delicate villus structure of the cell surface can be confirmed in the SEM image detecting the secondary electron (SE) signal. FIG. 5(B) shows an example of forming a carbon-based film with a thickness of 35 nm and low conductivity, whose electrical resistivity is not measurable by an insulation resistance meter (manufactured by Sanwa Electric Measurement Co., Ltd., DM1528S) with a measurement limit of 2000 kΩ (2 GΩ) by applying a pulse voltage of −8 kV and 1 kHz for 20 minutes while introducing a predetermined amount of acetylene as a raw material gas 381 into a vacuum chamber using a coating device 200 by a flow rate adjustment unit 281. In the case of FIG. 5(B), it is difficult to observe the fine uneven structure around the mucus vesicles and the villi on the cell surface. FIG. 5(C) shows an example of forming an Os film with a thickness of 2.7 nm. In the case of FIG. 5(C), it is difficult to observe the fine uneven structure around the mucus vesicles and the villi on the cell surface, as in the case of FIG. 5(B). For these reasons, the carbon-based film, which has a higher electrical conductivity than a coating with a low-conductivity carbon-based film or an Os film, enables detection and imaging of SE signals with a high spatial resolution.

[0030] FIG. 6 shows an example of a scanning electron microscope image (SEM image) in which a carbon-based film with an electrical resistivity of 471 Ωm is formed and secondary electron (SE) signals are imaged. All of FIG. 6(A) to (D) are SEM images in which mouse lung tissue is stained with Mayer's hematoxylin, and then a predetermined amount of aniline vaporized using the coating device 200 is introduced into the vacuum chamber as the raw material gas 381 by the flow rate control unit 281, while a pulse voltage of -8 kV and 1 kHz is applied for 20 minutes so that the film thickness becomes 35 nm, forming a carbon-based film with an electrical resistivity of 471 Ωm. When the carbon-based film with an electrical resistivity of 471 Ωm is coated, even with Mayer's hematoxylin staining containing Al, the SE signal that characterizes the tissue morphology can be detected and imaged.

[0031] FIG. 7 is a diagram verifying the necessity of Grocott staining of Aspergillus. FIG. 7(A)-(C) are examples of Grocott staining of Aspergillus. FIG. 7(D)-(F) are examples of not Grocott staining of Aspergillus. FIG. 7(A) and FIG. 7(D) are images taken by an optical microscope. FIG. 7(B) and FIG. 7(E) are SEM images of BSE signals. FIG. 7(C) and FIG. 7(F) are SEM images of SE signals. In the example of FIG. 7, Aspergillus, which is a deep fungal infection, is visualized by Grocott staining, and a predetermined amount of aniline vaporized in the coating device 200 is introduced into the vacuum chamber as the raw material gas 381 by the flow rate adjustment unit 281, while applying a pulse voltage of -8 kV and 1 kHz for 20 minutes so that the film thickness is 35 nm, and a carbon-based film with an electrical resistivity of 471 Ωm is formed. When SEM images of the BSE and SE signals are obtained, if the sample is coated with a carbon-based film with an electrical resistivity of 471 Ωm, Aspergillus can be confirmed in the SEM images of the BSE and SE signals without the need for Grocott staining.

[0032] FIG. 8 is a diagram verifying that the characteristics of mucus cells in mucus epithelial metaplasia occurring in the bronchial epithelium can be emphasized by Grocott staining. Next, for the mucus epithelial metaplasia occurring in the bronchial epithelium, mucus vesicles were stained by Grocott staining, and then a predetermined amount of aniline vaporized using the coating device 200 was introduced into the vacuum container 270 from the flow rate adjustment unit 281 as the raw material gas 381, while applying a pulse voltage of -8 kV and 1 kHz for 20 minutes so that the film thickness was 35 nm, forming a carbon-based film with an electrical resistivity of 471 Ωm. FIG. 8(A) is an example of the BSE signal obtained and imaged by a scanning electron microscope, in which the mucus vacuoles stained by Grocott staining are emphasized. FIG. 8(B) is an example of the SE signal obtained and imaged by a scanning electron microscope of the mucus vacuoles characterized by Grocott staining. Figures 8(C) and 8(D) show the distribution of gold introduced by Grocott staining, detected by energy dispersive X-ray spectroscopy (EDX).

[0033] FIG. 9 shows the results of observing the Grocott-stained bronchial epithelium with an optical microscope and a scanning electron microscope. In FIG. 9(A) to FIG. 9(D), after staining the bronchial epithelium with Grocott staining, a predetermined amount of aniline vaporized using the coating device 200 was introduced into the vacuum container 270 from the flow rate control unit 281 as the raw material gas 381, while applying a pulse voltage of -8 kV and 1 kHz for 20 minutes so as to obtain a film thickness of 35 nm or 130 nm, thereby forming a carbon-based film with an electrical resistivity of 471 Ωm. FIG. 9(A) and FIG. 9(B) are examples of optical microscope images. FIG. 9(C) and FIG. 9(D) are examples of images obtained by acquiring SE signals with a scanning electron microscope. In FIG. 9(A) and FIG. 9(C), the film thickness is 35 nm, and in FIG. 9(B) and FIG. 9(D), the film thickness is 130 nm. When the tissue was coated with a conductive carbon-based film having a thickness of 35 nm and 130 nm, good optical microscope images and scanning electron microscope images were obtained simultaneously. Fig. 9(E) shows an example in which the bronchial epithelium was stained with Grocott staining, and then, as in Fig. 4(A), acetylene and nitrogen gas were introduced into the vacuum chamber 270 as source gases using the coating device 200, a pulse voltage was applied, the surface of the tissue slice was coated with a conductive carbon-based film, and an SE signal was obtained with a scanning electron microscope to produce an image.

[0034] The coating device 200 forms a carbon-based film on a tissue slice cut from a formalin-fixed, paraffin-embedded specimen to prepare a sample for SEM. Although the incident energy of electrons irradiated from an electron gun built into the scanning electron microscope is not so high, the electrons pass through the carbon-based film formed on the specimen, and secondary electrons or transmitted electrons can be detected. It has been found that this makes it possible to observe tissue slices using a scanning electron microscope or the like, and that the charge accumulated on the surface of the specimen is released through the carbon-based film, so that no charge-up problem occurs. According to the present invention, even a tissue slice cut from a formalin-fixed, paraffin-embedded specimen used in pathological diagnosis can be observed at high magnification without deforming it or damaging the state of the specimen itself.

[0035] According to the coating device and coating method of the present invention, plasma can be generated uniformly even in a formalin-fixed, paraffin-embedded specimen having an uneven surface, or in a tissue section cut thin from a formalin-fixed, paraffin-embedded specimen having a sponge-like structure due to the presence of minute vesicles inside the section. Therefore, a thin film (preferably a conductive carbon-based thin film) can be formed on the surface of the tissue section, so that secondary electrons can be efficiently emitted from the tissue section when observed with a scanning electron microscope. As a result, a secondary electron signal can be detected by the scanning electron microscope.

[0036] It should be noted that the embodiments disclosed herein are illustrative and not restrictive in all respects. The scope of the present invention is not limited to the embodiments disclosed herein, and is intended to include all modifications within the scope indicated by the claims or within the scope equivalent to the claims. [Explanation of symbols]

[0037] 100 slide glass electrode 200 coating device 210 Exhaust section 220 Insulating stand 230 Sample stage 240 Current input terminal 250 Conductor 260 Power supply section 270 Vacuum container 280 Intake section 281, 282 Flow rate adjustment section 290 Anode 310 Gas 381, 382 Source gas

Claims

1. A coating device for coating the surface of a tissue section with a film material, comprising: a vacuum chamber; an exhaust unit for exhausting the gas in the vacuum chamber; an intake unit for taking in gas into the vacuum chamber; a positive electrode and a negative electrode disposed in the vacuum chamber; a power supply unit for applying a voltage between the positive electrode and the negative electrode; a slide glass electrode having a film with a predetermined electrical resistivity formed on the surface of a plate-shaped glass; and the negative electrode is a conductive sample stage, the tissue section is directly disposed on the slide glass electrode, and the slide glass electrode is disposed on the sample stage so as to be electrically connected to the sample stage. A coating device characterized by the above.

2. The coating device according to claim 1, wherein the tissue section is a tissue section thinly sliced from a formalin-fixed and paraffin-embedded specimen, and the film of the slide glass electrode has an electrical resistivity of 100 Ωm or less. A coating device characterized by the above.

3. The coating device according to claim 1 or 2, wherein the material of the film of the slide glass electrode is a conductive carbon-based film material, and the gas taken in from the intake unit is a nitrogen-containing compound, a mixed gas of a hydrocarbon gas and a nitrogen gas, or a mixed gas of a hydrocarbon gas and a boron-containing compound. A coating device characterized by the above.

4. The coating device according to any one of claims 1 to 3, wherein the positive electrode is grounded, and the power supply unit repeatedly applies a negative voltage pulse of 2 kV or more at 1 Hz or more and 100 kHz or less to the negative electrode. A coating device characterized by the above.

5. The coating device according to any one of claims 1 to 3, wherein the positive electrode is grounded, and the power supply unit applies a negative DC voltage of 1 kV or more to the negative electrode. A coating device characterized by the above.

6. A coating method for coating the surface of a tissue section with a film material, comprising: a slide glass electrode manufacturing step of manufacturing a slide glass electrode having a film with a predetermined electrical resistivity formed on the surface of a plate-shaped glass; an arrangement step of directly disposing the tissue section on the slide glass electrode and disposing the slide glass electrode so as to be electrically connected to a sample stage which is a negative electrode; a voltage application step of applying a voltage between a positive electrode and the negative electrode in a plasma generation gas atmosphere under reduced pressure to generate plasma; and executing the above steps.

7. The coating method according to claim 6, The tissue section is a tissue section thinly sliced from a formalin-fixed and paraffin-embedded specimen, and the film of the slide glass electrode has an electrical resistivity of 100 Ωm or less. A coating method characterized by this.

8. The coating method according to claim 6 or 7, wherein the material of the film of the slide glass electrode is a material of a conductive carbon-based film, and the gas for plasma generation is a nitrogen-containing compound, a mixed gas of a hydrocarbon gas and a nitrogen gas, or a mixed gas of a hydrocarbon gas and a boron-containing compound. A coating method characterized by this.

9. The coating method according to any one of claims 6 to 8, wherein the positive electrode is grounded, and the voltage applied to the negative electrode is a negative voltage pulse of 2 kV or more at 1 Hz or more and 100 kHz or less. A coating method characterized by this.

10. The coating method according to any one of claims 6 to 8, wherein the positive electrode is grounded, and a negative DC voltage of 1 kV or more is applied to the negative electrode. A coating method characterized by this.

Images