Electron staining device

Abstract

The present invention addresses the problem of providing an electron staining device capable of obtaining a good contrast and achieving an electron staining process with high working efficiency when observing a sample such as a polymer material or a biological material with an electron microscope.　An electron staining device (1) comprises a sample chamber (10) for installing a sample to be electron stained, an electron staining agent introduction means (11) for introducing an electron staining agent vapor into the sample chamber (10), and an evacuation means (12) for evacuating the inside of the sample chamber (10). The electron staining device (1) has a volume changing means (13) for changing the volume of the sample chamber (10), and by reducing the volume of the sample chamber in a state in which the electron staining agent is introduced into the sample chamber, the pressure in the sample chamber is increased, and the abovementioned problem can be solved.

Description

electronic staining equipment

[0001] The present invention relates to an electron staining device used when applying electron staining to a sample to be observed with an electron microscope.

[0002] Electron microscopes are used to observe polymeric and biological materials. Samples observed with electron microscopes are often composed of light elements such as hydrogen, carbon, oxygen, and nitrogen, which result in weak scattering energy in response to incident electrons. For this reason, a technique is used to obtain high contrast by adding heavy metal elements to the sample to be observed in advance to increase the scattering energy. This technique is generally called electron staining, and the reagents containing heavy metal elements used to enhance the contrast of samples during electron staining are called electron stains.

[0003] Many electronic stains contain heavy metal elements and are therefore harmful to the human body. Therefore, safety precautions are necessary when performing electronic staining, and electronic staining is often performed in an exhaust facility such as a draft chamber. Various technologies have been proposed to perform electronic staining safely and efficiently.

[0004] For example, Patent Document 1 discloses an electronic stain in which ruthenium tetroxide, osmium tetroxide, or the like is adsorbed and aggregated in the pores of a porous material such as silica gel. The electronic stain of Patent Document 1 is stored in a frozen state, and when used, is left at room temperature to generate vapor of ruthenium tetroxide, etc., allowing electronic staining work to be performed. With the electronic stain of Patent Document 1, the ruthenium tetroxide held on the surface and inside of the silica gel when heated is liquefied and sublimated, and the ruthenium tetroxide, etc. is re-adsorbed into the pores of the porous material when re-cooled, so that the ruthenium tetroxide, etc. does not stick to the bottom of the container, and the material can be maintained in an easy-to-handle form.

[0005] Patent Document 2 discloses a sample staining device that detoxifies staining agents such as osmium tetroxide and ruthenium tetroxide remaining in the staining device after a staining process by converting them into low-oxidized osmium compounds and ruthenium compounds using a reducing agent.

[0006] Patent Literature 3 discloses a method and an apparatus for use in the method, which includes a step of acquiring an image of a surface layer of a sample and a step of removing the surface layer of the sample, thereby bringing the next thin section to the surface, and a step of exposing the sample to a staining agent after at least one of the removals of the surface layer. In the method of Patent Literature 3, the step of exposing the sample to a staining agent is performed in a vacuum chamber or a low-pressure chamber.

[0007] Patent Document 4 discloses an electron staining apparatus including a vacuum vessel connected to an exhaust system and provided with a mounting table on which a polymeric material to be electron stained is placed, an exhaust opening / closing means for connecting and disconnecting the vacuum vessel to the exhaust system, an electron staining gas introducing means for sublimating and gasifying a solid electron stain, a gas opening / closing means for connecting and disconnecting gas from the electron staining gas introducing means to the vacuum vessel, and a pressure detecting means for detecting the vacuum pressure and gas pressure within the vacuum vessel. In the apparatus of Patent Document 4, the electron staining process is carried out by introducing an electron staining gas into the vacuum vessel after the desired vacuum pressure has been achieved within the vacuum vessel in which the sample (polymeric material) is placed.

[0008] The use of these conventional techniques has the advantage of making it easier to ensure worker safety compared to performing electronic staining in a draft chamber, etc. However, even with these conventional techniques, it takes a long time to obtain sufficient contrast for the specimen, and there are still issues in terms of work efficiency and productivity. For this reason, there is a strong demand for the establishment of a technology that can efficiently perform high-quality electronic staining work.

[0009] JP 2014-137293 A JP 2015-030945 A JP 2008-046127 A JP 2005-308505 A

[0010] The present invention has been made in view of the above-mentioned background art, and an object of the present invention is to provide an electron staining apparatus that can obtain good contrast and achieve an electron staining process with high work efficiency when observing samples such as polymeric materials and biological materials with an electron microscope.

[0011] The present inventors have conducted extensive research to solve the above problems and have discovered the following facts, which have led to the completion of the present invention.

[0012] In other words, the electronic staining agent used in electronic staining does not have a high vapor pressure, and when an apparatus for performing electronic staining under vacuum or low pressure, as described in Patent Documents 3 and 4, is used, safety for the operator can be ensured, but there are drawbacks such as difficulty in allowing the electronic staining agent to sufficiently penetrate the sample, resulting in insufficient staining or requiring a long time for staining.

[0013] If the electron staining process could be carried out under pressure, it would be possible to stain the sample sufficiently in a short time, thereby overcoming the above drawbacks.

[0014] Specifically, in a sealed electron staining device, the volume of the sample chamber for placing the sample can be varied, and by reducing the volume of the sample chamber while the electron stain is being introduced into the sample chamber, the pressure inside the sample chamber can be increased and a space with a higher molecular density of the sublimation gas (electron stain vapor) can be created. This makes it possible to sufficiently stain the sample to be electron stained in a short period of time.

[0015] The present invention thus completed is as follows:

[0016] An electron staining apparatus having a sample chamber for placing a sample to be electron stained, an electron stain introduction means for introducing electron stain vapor into the sample chamber, and an exhaust means for exhausting the inside of the sample chamber, characterized in that the electron staining apparatus also has a pressure change means for changing the pressure inside the sample chamber when electron staining is performed.

[0017] The electron staining apparatus of the present invention can perform electron staining with the specimen chamber pressurized, thereby providing an electron staining apparatus that can obtain good contrast and achieve highly efficient electron staining processes when observing specimens such as polymeric materials and biological materials with an electron microscope.

[0018] The electron staining apparatus of the present invention can easily change the volume of the sample chamber while the electron stain is being introduced into the sample chamber. Therefore, by reducing the volume of the sample chamber, the pressure inside the sample chamber can be easily increased, achieving good contrast and high work efficiency.

[0019] On the other hand, depending on the type of sample, if the electron stain penetrates too deeply into the sample, it may actually cause difficulties during observation using an electron microscope. In the present invention, the volume of the sample chamber can be easily changed, so the pressure within the sample chamber can be easily adjusted according to the characteristics of the sample, allowing for flexible adaptation to electron staining of various samples.

[0020] FIG. 1 is a schematic diagram of an electronic staining apparatus of the present invention. FIG. 1 is a schematic diagram showing a state in which a sample to be electronically stained is placed in a sample chamber in an electronic staining apparatus of the present invention. FIG. 2 is a schematic diagram showing a state in which an electronic staining agent vapor is filled in a sample chamber in an electronic staining apparatus of the present invention. FIG. 3 is a schematic diagram showing a state in which electronic staining is being performed in an electronic staining apparatus of the present invention. FIG. 4 is a schematic diagram showing a state in which electronic staining agent vapor remaining in the sample chamber is being removed after electronic staining is completed in an electronic staining apparatus of the present invention. FIG. 5 is a schematic diagram of an electronic staining apparatus of the present invention (when a replacement gas introduction means is provided). FIG. 6 is a schematic diagram showing a state in which electronic staining is being performed in an electronic staining apparatus of the present invention having a replacement gas introduction means. FIG. 7 is a schematic diagram showing a state in which a replacement gas is being introduced into a sample chamber in an electronic staining apparatus of the present invention having a replacement gas introduction means. FIG. 8 is a schematic diagram of an electronic staining apparatus of the present invention (when a plurality of electronic staining agent introduction means are provided). FIG. 9 is a schematic diagram of an electronic staining apparatus of the present invention (when a plurality of electronic staining agent introduction means are provided, and the electronic staining agent introduction means is composed of an electronic staining agent storage section and an electronic staining agent sublimation chamber).

[0021] The present invention will be described below, but the present invention is not limited to the following embodiments and can be practiced with any modifications.

[0022] 1 shows a schematic diagram of an electronic staining apparatus 1 of the present invention. The electronic staining apparatus 1 of the present invention has a sample chamber 10, an electronic stain introduction means 11, and an exhaust means 12.

[0023] The sample chamber 10 is a space for placing a sample S to be electron stained. During the electron staining process, the sample chamber 10 is filled with electron stain vapor V. The sample chamber 10 is provided with a sample inlet / outlet 15 for inserting and removing the sample S.

[0024] The electronic stain introducing means 11 is a means for introducing electronic stain vapor V into the sample chamber 10. An electronic stain E is stored inside the electronic stain introducing means 11. The electronic stain introducing means 11 is connected to the sample chamber 10 by a pipe, and an opening / closing means 21 such as a valve is provided between the electronic stain introducing means 11 and the sample chamber 10.

[0025] The type of the electronic stain E is not particularly limited, and osmium tetroxide (OsO ４ ), ruthenium tetroxide (RuO ４ ), phosphotungstic acid (H ３ [P(W ３ O １０ ) ４ ]・nH ２ O) can be exemplified.

[0026] The electronic dye E is sublimable, and by opening the opening / closing means 21 while the pressure inside the sample chamber 10 is reduced, electronic dye vapor V (sublimated electronic dye E) flows from the electronic dye introduction means 11 into the sample chamber 10.

[0027] It is desirable to select an appropriate electronic stain E depending on the type of sample S to be measured. For example, osmium tetroxide is known to selectively react with double bonds in unsaturated polymeric materials. Ruthenium tetroxide is also known to crosslink with the amorphous portions of saturated polymeric materials. Phosphotungstic acid is known to react with polyamides and the like.

[0028] As will be described later, the electronic staining apparatus 1 of the present invention may have a plurality of electronic stain introduction means, in which case the same sample S can be stained with a plurality of types of electronic stains E.

[0029] There is no particular limitation on the state in which the electronic stain E is stored inside the electronic stain introducing means 11 .

[0030] For example, the electronic stain E may be sealed in a glass ampoule, the glass ampoule may be stored in the electronic stain introducing means 11, and the glass ampoule may be broken just before using the electronic stain E. When the electronic stain E is osmium tetroxide, this storage method is preferable.

[0031] The electronic stain E may be stored in a cooled state in the electronic stain introduction means 11. The cooling temperature is preferably between -20°C and 4°C. Furthermore, it is preferable that the electronic stain introduction means 11 be capable of being heated to between 25°C and 40°C when the electronic stain E is used. This storage method is preferable when the electronic stain E is ruthenium tetroxide.

[0032] The electronic staining agent introducing means 11 is preferably in the form of a cartridge that is detachable from the electronic staining apparatus 1 and whose interior is kept airtight.

[0033] The exhaust means 12 is a means for exhausting the inside of the sample chamber 10. Examples of the exhaust means 12 include a rotary pump and a reciprocating pump.

[0034] Before electron staining, i.e., before introducing the electron stain vapor V into the sample chamber 10, or after electron staining is completed, the sample chamber 10 is evacuated by the evacuation means 12.

[0035] A pressure gauge (not shown) is installed inside the sample chamber 10 (or in the piping section) so that the pressure inside the sample chamber 10 can be observed. It is desirable that the exhaust means 12 be able to exhaust the inside of the sample chamber 10 to a vacuum level of about 1 to 100 Pa.

[0036] The electronic staining apparatus 1 of the present invention has a pressure changing means for changing the pressure inside the sample chamber 10 during electronic staining. As described above, conventional electronic staining apparatuses such as those described in Patent Documents 3 and 4 perform electronic staining under vacuum or low pressure, which has the drawback of resulting in insufficient staining or requiring a long time for staining.

[0037] Specific examples of the pressure changing means include a volume changing means 13 for changing the volume of the sample chamber 10 and a pressure pump that can pressurize the inside of the sample chamber 10 .

[0038] When the pressure changing means of the electronic staining apparatus 1 of the present invention is the volume changing means 13, the sample chamber 10 can be pressurized by reducing the volume of the sample chamber 10 while the electronic stain vapor V is introduced into the sample chamber 10. That is, the electronic staining process can be performed with the partial pressure of the electronic stain vapor V in the sample chamber 10 increased. For example, in the electronic staining apparatus 1 of the present invention, the pressure in the sample chamber 10 during electronic staining can be set to 1 atmosphere or more. Therefore, the electronic staining apparatus 1 can sufficiently stain the sample S in a short time.

[0039] 1 shows an example of the volume changing means 13 in the electronic staining apparatus 1 of the present invention. The sample chamber 10 is formed in a cylindrical shape, and the volume changing means 13 is a piston that moves in the axial direction of the sample chamber 10.

[0040] In the electron staining apparatus 1 of the present invention, for example, as shown in Fig. 1, the bottom surface of the sample chamber 10 serves as a stage on which the sample S is placed. By moving the piston up and down, the stage (movable sample stage) on which the sample S is placed moves up and down accordingly.

[0041] In FIG. 1, the volume of the sample chamber 10 can be reduced by moving the volume change means (piston) 13 upward, and the volume of the sample chamber 10 can be increased by moving it downward.

[0042] There are no particular limitations on the drive mechanism for the volume change means (piston) 13, and for example, the volume change means (piston) 13 can be moved in the axial direction of the sample chamber 10 by a worm gear.

[0043] A worm gear is a gear consisting of a worm and a worm wheel, which can achieve a large reduction ratio. The worm and the worm wheel usually intersect at a 90° angle, making them quiet and self-locking.

[0044] Furthermore, when the drive mechanism of the volume changing means 13 in the electronic staining apparatus 1 of the present invention is a worm gear, the worm gear preferably has a double-start screw.

[0045] The volume of the sample chamber 10 in the electronic staining apparatus 1 of the present invention can be changed, and the preferred conditions for the maximum and minimum volumes are as follows. The maximum volume of the sample chamber 10 is the maximum value of the volume of the sample chamber 10 when the sample chamber 10 is empty (when no sample S is placed therein). That is, in the case of the apparatus illustrated in FIG. 1, it is the volume of the sample chamber 10 when the piston is moved as far downward as possible. The minimum volume of the sample chamber 10 is the smallest value of the volume of the sample chamber 10 when the sample chamber 10 is empty. That is, in the case of the apparatus illustrated in FIG. 1, it is the volume of the sample chamber 10 when the piston is moved as far upward as possible.

[0046] The maximum volume is 20 cm ３ It is preferable that the length is 100 cm or more. ３ More preferably, it is 200 cm or more. ３ It is particularly preferable that the maximum volume is 200,000 cm or more. ３ Preferably, it is 100,000 cm or less. ３ More preferably, it is 20,000 cm or less. ３ It is particularly preferred that:

[0047] The ratio of the minimum volume to the maximum volume is preferably 0.001 or more, more preferably 0.003 or more, and particularly preferably 0.01 or more, and is preferably 0.2 or less, more preferably 0.15 or less, and particularly preferably 0.1 or less.

[0048] When the maximum volume, minimum volume and ratio of the sample chamber 10 are within the above ranges, it is easy to obtain advantages such as the device being compact, sufficient space for the sample S being secured, and sufficient pressure being applied inside the sample chamber 10 (which in turn makes the staining process more efficient).

[0049] When the volume changing means 13 is a piston that moves in the axial direction of the sample chamber 10, the piston stroke is preferably 1 mm or more, more preferably 10 mm or more, and particularly preferably 45 mm or more. The piston stroke is preferably 300 mm or less, more preferably 250 mm or less, and particularly preferably 200 mm or less. When the piston stroke is within the above range, it is easy to obtain advantages such as compactness of the device, sufficient space for the sample S, and sufficient pressurization of the sample chamber 10 (which in turn makes the staining process more efficient).

[0050] The outline of the operation when performing electronic staining using the device shown in FIG. 1 will be explained with reference to FIGS.

[0051] First, the sample inlet / outlet 15 of the sample chamber 10 of the electronic staining device 1 is opened, the sample S to be electronically stained is placed in the sample chamber 10, and then the sample inlet / outlet 15 is closed. If necessary, the electronic stain E for staining the sample S is stored inside the electronic stain introduction means 11. The state after these steps are completed is shown in Figure 2.

[0052] When placing the sample S, it is easier to perform the work if the volume changing means (piston) 13 is moved upward, as this shortens the distance between the sample inlet / outlet 15 and the location where the sample is placed.

[0053] 2, with the volume changing means (piston) 13 moved downward, the exhaust means 12 is operated, the opening and closing means 22 is opened, and the inside of the sample chamber 10 is evacuated. When the sample chamber reaches a vacuum level of about 1 Pa to 100 Pa, the opening and closing means 22 is closed.

[0054] Next, by opening the opening / closing means 21 and, if necessary, breaking the glass ampoule in which the electronic stain E is sealed, the electronic stain E in the electronic stain introducing means 11 sublimes, and electronic stain vapor V flows into the sample chamber 10 (FIG. 3). As a result, the pressure inside the sample chamber 10 becomes approximately 100 Pa to 5000 Pa.

[0055] When the pressure in the sample chamber 10 falls within the above range, it can be said that a sufficient amount of electronic stain vapor V is present in the sample chamber 10, and the opening / closing means 21 is closed to isolate the sample chamber 10 from the electronic stain introducing means 11. Alternatively, the opening / closing means 21 may be designed to close automatically after a certain time has elapsed since it was opened.

[0056] When the sample chamber 10 is filled with the electronic dye vapor V and is closed, the volume of the sample chamber 10 is reduced by moving the volume changer (piston) 13 upward (FIG. 4). This increases the pressure in the sample chamber 10 (the partial pressure of the electronic dye vapor V), allowing the sample S to be efficiently electronically stained.

[0057] When the volume changing means (piston) 13 is moved upward to its maximum extent, the pressure in the sample chamber 10 can be increased to a maximum of approximately 6.7 atmospheres (0.67 MPa). By adjusting the amount of movement of the volume changing means (piston) 13, the pressure in the sample chamber 10 during electron staining can be adjusted. The desired pressure is selected depending on the type of sample S.

[0058] In the electronic staining apparatus 1 of the present invention, the pressure in the sample chamber 10 during electronic staining can be, for example, 0.5 atmospheres or more, 0.7 atmospheres or more, or 1 atmosphere or more. The pressure can also be, for example, 6.7 atmospheres or less, 4 atmospheres or less, 2.5 atmospheres or less, 2 atmospheres or less, or 1.5 atmospheres or less.

[0059] The electronic staining apparatus 1 of the present invention can change the pressure in the sample chamber 10 during the electronic staining process. For example, during the electronic staining process, it is possible to change the pressure in the sample chamber 10 from positive pressure to negative pressure, or from negative pressure to positive pressure.

[0060] In the present invention, the pressure in the sample chamber 10 can be changed very easily during the electron staining process by simply moving the volume change means (piston) 13 upward or downward. Therefore, in the present invention, the electron staining conditions can be easily changed depending on the characteristics of the sample S, making it easy to improve the quality of the electron staining.

[0061] After a desired time has elapsed, the electronic staining process is completed, and the process moves to a purification process in which the electronic stain vapor V is removed (exhausted) from the sample chamber 10. A preset alarm may be set to notify the operator of the time when the electronic staining process is complete.

[0062] In the present invention, the time required for the electron staining process is approximately 10 to 20 minutes for a 100-nm section of a polymer sample. When using an apparatus for performing electron staining under reduced pressure, such as the apparatuses described in Patent Documents 3 and 4, the electron staining process can take several hours. In other words, the electron staining apparatus 1 of the present invention can perform the electron staining process in a significantly shorter time. Furthermore, in the present invention, sufficient staining effects can be obtained even when the electron staining process is performed in such a short time. Note that the "time required for the electron staining process" refers to the time required from the start of the inflow of the electron stain vapor V into the sample chamber 10 by opening the gate 21 to the start of the removal (exhaust) of the electron stain vapor V from the sample chamber 10 by opening the gate 22, as described below.

[0063] The cleaning process is a process for removing the electron staining agent vapor V remaining in the sample chamber 10 before the electron-stained sample S is removed from the sample chamber 10. At this time, the electron staining agent vapor V remaining inside the sample S is also removed (degassing is performed). As mentioned above, the electron staining agent vapor V is harmful to the human body, so the cleaning process is performed to ensure the safety of the operator.

[0064] In the purification step, the exhaust means 12 is operated, the opening / closing means 22 is opened, and the electronic stain vapor V remaining inside the sample chamber 10 is removed (FIG. 5). Since the electronic stain vapor V and the electronic stain E are harmful to the human body, a filter for collecting the electronic stain E may be provided midway in the piping from the exhaust means 12 to the outside of the system to prevent their dispersion.

[0065] The electronic staining apparatus 1 of the present invention may have a replacement gas introduction means 14 for introducing a replacement gas into the sample chamber 10. By having the replacement gas introduction means 14, the electronic stain vapor V can be sufficiently removed from the inside of the electronic staining apparatus 1 during the cleaning step. Furthermore, the time required for the cleaning step can be shortened.

[0066] 6 shows a schematic diagram of the electron staining apparatus 1 of the present invention having a replacement gas introduction means 14. The replacement gas introduction means 14 is connected to the sample chamber 10 via an opening / closing means 24. In the cleaning step, the opening / closing means 24 is opened to introduce replacement gas from the replacement gas introduction means 14 into the sample chamber 10.

[0067] In the cleaning process, the electronic stain vapor V remaining inside the sample chamber 10 is exhausted by the exhaust means 12 together with the replacement gas introduced from the replacement gas introduction means 14, and is removed from the inside of the sample chamber 10. By introducing the replacement gas into the sample chamber 10, the electronic stain vapor V is less likely to adhere to the wall surfaces, and the electronic stain vapor V is more easily removed (exhausted) from the inside of the sample chamber 10.

[0068] Although the electronic stain vapor V and the electronic stain E are harmful to the human body, they are not highly reactive with air, and therefore the replacement gas introduced from the replacement gas introduction means 14 may be air. From a cost perspective, air is preferable as the replacement gas. Furthermore, as will be described later, the air introduced from the replacement gas introduction means 14 does not need to be compressed, and the replacement gas introduction means 14 may be, and preferably is, simply a pipe connected to the electronic staining device 1. However, the use of compressed air or an inert gas such as nitrogen, argon, helium, neon, or xenon as the replacement gas is not excluded from the scope of the present invention.

[0069] FIG. 7 shows the state in which the electronic staining apparatus 1 of the present invention having the replacement gas introducing means 14 has completed electronic staining.

[0070] As in the case of the electronic staining apparatus 1 not having the replacement gas introduction means 14 described above, in the purification process after the completion of the electronic staining process, first, the exhaust means 12 is activated, the opening / closing means 22 is opened, and the electronic stain vapor V remaining inside the sample chamber 10 is removed.

[0071] When the pressure inside the sample chamber 10 drops to a certain level (for example, to about 100 to 500 Pa), the opening and closing means 24 is opened and replacement gas G is introduced into the sample chamber 10 from the replacement gas introduction means 14 (FIG. 8).

[0072] The replacement gas G introduced into the sample chamber 10 is removed by the exhaust means 12 together with the small amount of electronic dye vapor V remaining in the sample chamber 10 .

[0073] In the cleaning process, the volume changing means (piston) 13 may be moved up and down several times, which facilitates the removal of residual electron stain vapor V that has adhered to the wall surface of the sample chamber 10.

[0074] In the cleaning process, the exhaust means 12 is kept in operation, and the sample chamber 10 is evacuated to about 100 Pa, after which the opening and closing means 22 is closed and the opening and closing means 24 is opened to introduce the replacement gas G. In the cleaning process, it is desirable to repeat the above-described operations of evacuating the sample chamber 10 and introducing the replacement gas G into the sample chamber 10 multiple times.

[0075] After the purification step is completed, the opening and closing means 22 and the opening and closing means 24 are closed, and the exhaust means 12 is stopped. Thereafter, the electron-stained sample S is taken out from the sample inlet / outlet 15, and the sample S is subjected to observation with an electron microscope.

[0076] In the present invention, the time required for the purification step is approximately 10 to 60 minutes. Note that the "time required for the purification step" refers to the time required from the start of removal (exhaust) of the electronic dye vapor V from inside the sample chamber 10 by operating the exhaust means 12 and opening the opening / closing means 22 until the closing of the opening / closing means 22.

[0077] The electronic staining apparatus of the present invention may be provided with a leakage prevention space around the sample chamber 10 to prevent leakage of the electronic stain vapor V, and the leakage prevention space may be evacuated. This configuration further ensures the safety of the operator.

[0078] 9, the electronic staining apparatus 1 of the present invention may have multiple electronic stain introduction means 11a, 11b, and may be capable of introducing multiple types of electronic stain vapor into the sample chamber 10. The electronic stain introduction means 11a, 11b are connected to the sample chamber 10 via opening and closing means 21a, 21b, respectively.

[0079] The electronic stain introducing means 11a and the electronic stain introducing means 11b store different types of electronic stains Ea and Eb. For example, the electronic stain introducing means 11a stores osmium tetroxide, and the electronic stain introducing means 11b stores ruthenium tetroxide.

[0080] Depending on the type of specimen S to be observed, it may be useful to stain the specimen S with multiple electronic stains Ea and Eb. By using the electronic staining device 1 of the present invention, which has multiple electronic stain introduction means 11a and 11b, electronic staining of such specimen S can be efficiently performed.

[0081] In the electronic staining apparatus 1 of the present invention, first, the vapor of the electronic stain Ea is introduced into the sample chamber 10 to perform staining, and after the vapor of the electronic stain Ea is removed (exhausted) from the sample chamber 10, the vapor of the electronic stain Eb is introduced into the sample chamber 10 to perform staining. In other words, staining with multiple types of electronic stain vapor V can be performed sequentially.

[0082] In addition, in the electronic staining device 1 of the present invention, vapors of multiple types of electronic stains (vapors of electronic stain Ea and vapors of electronic stain Eb) can be introduced into the sample chamber 10 simultaneously, making it possible to simultaneously stain with multiple types of electronic stains.

[0083] As shown in Fig. 10, the electronic dye introduction means (11a, 11b) in the electronic staining apparatus 1 of the present invention may be composed of electronic dye storage sections (16a, 16b) and electronic dye sublimation chambers (17a, 17b). The electronic dye sublimation chambers (17a, 17b) are connected to the electronic dye storage sections (16a, 16b) via opening / closing means (25a, 25b).

[0084] The electronic dye sublimation chambers (17a, 17b) are sections for temporarily storing the electronic dye vapor V before it is introduced into the sample chamber 10. When the electronic staining process is performed, the electronic dye vapor is introduced into the sample chamber 10 from the electronic dye sublimation chambers (17a, 17b).

[0085] Prior to the electronic staining process, when the sample chamber 10 is evacuated by the exhaust means 12, the electronic stain sublimation chambers (17a, 17b) are also evacuated at the same time. That is, the exhaust is performed with the opening / closing means 21a and 21b open and the opening / closing means 25a and 25b closed.

[0086] After exhaust is complete, the opening / closing means 21a and 21b are closed, and in that state the opening / closing means 25a and 25b are opened, whereby the sublimated electronic dye vapor V moves from the electronic dye storage section (16a, 16b) to the electronic dye sublimation chamber (17a, 17b).

[0087] A pressure gauge (not shown) is installed inside the electronic dye sublimation chambers (17a, 17b) (or in the piping), and when the pressure inside the electronic dye sublimation chambers (17a, 17b) has risen sufficiently, the opening and closing means 25a and 25b are closed, thereby filling (storing) the electronic dye vapor V in the electronic dye sublimation chambers (17a, 17b).

[0088] Next, by opening the opening / closing means 21a and 21b, multiple types (two types) of electronic dye vapor V are introduced from the electronic dye sublimation chambers (17a, 17b) into the sample chamber 10. In this state, by operating the volume changing means (piston) 13, staining with multiple types of electronic dye vapor V is performed simultaneously.

[0089] The purification process after the completion of the electronic staining process is carried out with the opening and closing means 21a and 21b open, and not only the electronic stain vapor V remaining inside the sample chamber 10, but also the electronic stain vapor V remaining in the electronic stain sublimation chambers (17a, 17b) are simultaneously removed (exhausted).

[0090] As described above, the electronic staining agent introduction means 11 may be provided as a cartridge that can be detached from the electronic staining apparatus 1. When multiple types of electronic staining agent vapor V are simultaneously introduced into the sample chamber 10, if the electronic staining agent introduction means 11 (cartridge) is directly connected to the sample chamber 10, there is a risk of the cartridges becoming contaminated with each other. By providing the electronic staining agent sublimation chambers (17a, 17b) as described above, it is possible to prevent cartridge contamination by keeping the opening / closing means 25a and 25b always closed during operations after the electronic staining agent sublimation chambers (17a, 17b) are filled (stored) with the electronic staining agent vapor V.

[0091] In addition, in FIG. 10, an electronic dyeing apparatus having a plurality of electronic dye introduction means, each of which is composed of an electronic dye storage section and an electronic dye sublimation chamber, has been described. However, even in the case where only one electronic dye introduction means is provided, the electronic dye introduction means may be composed of an electronic dye storage section and an electronic dye sublimation chamber.

[0092] The volume of the electronic dye sublimation chamber is 10 cm ３ It is preferable that the length is 20 cm or more. ３ More preferably, it is 30 cm or more. ３ It is particularly preferable that the volume is 10,000 cm or more. ３ Preferably, it is 5000 cm or less. ３ More preferably, it is 1000 cm or less. ３ It is particularly preferable that the volume is equal to or larger than the lower limit. If the volume is equal to or larger than the upper limit, a sufficient amount of electronic dye vapor V can be stored. If the volume is equal to or smaller than the upper limit, the device can be easily made compact.

[0093] The specimen that has been subjected to electron staining using the electron staining device of the present invention is then observed using an electron microscope. There are no particular limitations on the type of electron microscope, and SEM (scanning electron microscope), TEM (transmission electron microscope), or STEM (scanning transmission electron microscope) can be used.

[0094] REFERENCE SIGNS LIST 1 Electron staining apparatus 10 Sample chamber 11 Electron stain introduction means 11a Electron stain introduction means 11b Electron stain introduction means 12 Exhaust means 13 Volume change means 14 Replacement gas introduction means 15 Sample inlet / outlet 16a Electron stain storage section 16b Electron stain storage section 17a Electron stain sublimation chamber 17b Electron stain sublimation chamber 21 Opening / closing means 21a Opening / closing means 21b Opening / closing means 22 Opening / closing means 24 Opening / closing means 25a Opening / closing means 25b Opening / closing means S Sample E Electron stain Ea Electron stain Eb Electron stain V Electron stain vapor G Replacement gas

Claims

1. An electron staining apparatus having a sample chamber for placing a sample to be electron stained, an electron staining agent introducing means for introducing electron staining agent vapor into the sample chamber, and an exhaust means for exhausting the air from the sample chamber, characterized in that the electron staining apparatus also has a pressure changing means for changing the pressure in the sample chamber when electron staining is performed.

2. The electron staining apparatus according to claim 1, wherein said pressure changing means is a volume changing means for changing the volume of said sample chamber.

3. An electronic staining apparatus according to claim 2, wherein the ratio of the minimum volume of the sample chamber to the maximum volume is 0.001 or more and 0.2 or less.

4. An electron staining apparatus according to claim 2, wherein said sample chamber is formed in a cylindrical shape, and said volume changing means is a piston that moves in the axial direction of said sample chamber.

5. The electronic staining apparatus according to claim 4, wherein the piston stroke is 1 mm or more and 300 mm or less.

6. The electronic staining apparatus according to claim 4, wherein the piston is moved in the axial direction by a worm gear.

7. An electronic staining apparatus according to claim 6, wherein said worm gear has a double-start thread.

8. The electronic staining apparatus according to claim 1, wherein the pressure in the specimen chamber during electronic staining can be set to 1 atmosphere or more.

9. An electron staining apparatus according to any one of claims 1 to 8, further comprising a replacement gas introducing means for introducing a replacement gas into said sample chamber.

10. An electronic staining apparatus according to claim 9, wherein said replacement gas is air.

11. An electron staining apparatus according to any one of claims 1 to 8, which has a plurality of electron stain introduction means and is capable of introducing a plurality of types of electron stain vapor into said sample chamber.

12. The electronic staining apparatus according to claim 11, wherein vapors of a plurality of types of electronic stains can be introduced into the sample chamber simultaneously, and staining with a plurality of types of electronic stains can be performed simultaneously.

13. An electronic staining apparatus as described in any one of claims 1 to 8, wherein the electronic stain introduction means is composed of an electronic stain storage section in which the electronic stain is stored and an electronic stain sublimation chamber connected to the electronic stain storage section via an opening / closing means, and electronic stain vapor is introduced from the electronic stain sublimation chamber into the sample chamber.

14. An electronic staining apparatus as described in any one of claims 1 to 8, wherein a leakage prevention space is provided around the sample chamber to prevent leakage of the electronic stain vapor, and the leakage prevention space can be evacuated.

15. An electron staining apparatus according to any one of claims 1 to 8, wherein the pressure in the specimen chamber can be changed during the electron staining process.

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