Patent Publication Number: US-2022214250-A1

Title: Specimen Pretreatment Method

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims priority to Japanese patent application No. 2021-001504 filed Jan. 7, 2021, the disclosure of which is hereby incorporated by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present invention relates to a specimen pretreatment method. 
     Description of Related Art 
     A section prepared using an ultramicrotome is spread on the surface of distilled water filled in a knife boat. The section spread on the water surface is scooped by a specimen supporting tool such as a mesh grid and supported by the mesh grid. The section supported by the mesh grid can be observed with a scanning electron microscope, a transmission electron microscope, or the like. 
     For example, JP-A-2015-187974 discloses a specimen supporting tool including a silicon substrate and a supporting film formed at an opening of the silicon substrate. The supporting film for supporting a specimen is, for example, a silicon nitride film, a carbon film, or the like. 
     Since the specimen supporting tool disclosed in JP-A-2015-187974 can increase the area of the supporting film, a continuous section in which a plurality of sections are connected can be supported. However, such a specimen supporting tool has a larger thickness than a commonly used mesh grid or the like. For this reason, when a specimen is observed with a large inclination in an electron microscope, the specimen may be behind the substrate. Therefore, it is desirable to use a mesh grid when observing the specimen with a large inclination. As described above, it is necessary to use a specimen supporting tool suitable for the purpose of observation. 
     However, since specimens for electron microscopy such as sections prepared by a microtome are extremely fragile, it is difficult to transfer the specimen between two specimen supporting tools when observing the same specimen for different observation purposes. 
     SUMMARY OF THE INVENTION 
     According to an aspect of the invention, there is provided a specimen pretreatment method for transferring a specimen supported by a first specimen supporting tool to a second specimen supporting tool, the specimen pretreatment method comprising: 
     transferring a specimen supported by the first specimen supporting tool to a film; 
     immersing the film and the specimen on the film in a liquid to dissolve the film; and 
     recovering the specimen from the liquid and supporting the specimen with the second specimen supporting tool. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a plan view schematically illustrating the first specimen supporting tool. 
         FIG. 2  is a cross-sectional view schematically illustrating the first specimen supporting tool. 
         FIG. 3  is an optical micrograph illustrating the first specimen supporting tool supporting a continuous section. 
         FIG. 4  is an optical micrograph illustrating an example of a mesh grid. 
         FIG. 5  is a flowchart illustrating an example of a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 6  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 7  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 8  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 9  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 10  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 11  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 12  is a diagram for explaining a specimen pretreatment method according to an embodiment of the invention. 
         FIG. 13  is an optical micrograph for explaining a specimen pretreatment method. 
         FIG. 14  is an optical micrograph for explaining a specimen pretreatment method. 
         FIG. 15  is an optical micrograph for explaining a specimen pretreatment method. 
         FIG. 16  is an optical micrograph of a continuous section on a supporting film before transferring to a mesh grid. 
         FIG. 17  is an optical micrograph of a continuous section after transferring to a mesh grid. 
         FIG. 18  is a transmission electron microscope image of a section supported by a SiN Window chip. 
         FIG. 19  is a transmission electron microscope image of a section transferred from a SiN Window chip to a mesh grid. 
         FIG. 20  is a transmission electron microscope image of a section supported by a SiN Window chip. 
         FIG. 21  is a transmission electron microscope image of a section transferred from a SiN Window chip to a mesh grid. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     According to an embodiment of the invention, there is provided a specimen pretreatment method for transferring a specimen supported by a first specimen supporting tool to a second specimen supporting tool, the specimen pretreatment method comprising: 
     transferring a specimen supported by the first specimen supporting tool to a film; 
     immersing the film and the specimen on the film in a liquid to dissolve the film; and 
     recovering the specimen from the liquid and supporting the specimen with the second specimen supporting tool. 
     With such a specimen pretreatment method, a specimen can be easily transferred from the first specimen supporting tool to the second specimen supporting tool. In addition, such a specimen pretreatment method can reduce deformation of the specimen and deterioration of image quality when the specimen is observed with an electron microscope. 
     Hereinafter, preferred embodiments of the invention will be described in detail with reference to the drawings. It is noted that the following embodiments do not unduly limit the scope of the invention as stated in the claims. Moreover, all of the components described below are not necessarily essential requirements of the invention. 
     1. Specimen Pretreatment Method 
     1.1. First Specimen Support 
     First, a specimen pretreatment method according to an embodiment of the invention will be described with reference to the drawings. 
       FIG. 1  is a plan view schematically illustrating a first specimen supporting tool  100 .  FIG. 2  is a cross-sectional view schematically illustrating the first specimen supporting tool  100 .  FIG. 2  is a cross-sectional view taken along the line II-II of  FIG. 1 . 
     As illustrated in  FIGS. 1 and 2 , the first specimen supporting tool  100  includes a substrate  102  and a supporting film  104 . 
     The substrate  102  is a semiconductor substrate such as a silicon substrate. The substrate  102  may be exemplified by a variety of substrates such as a ceramic substrate, a glass substrate, a sapphire substrate, a synthetic resin substrate, and the like. The thickness of the substrate  102  is, for example, about several hundred micrometers. The substrate  102  is formed with an opening  103  that penetrates the substrate  102 . The opening  103  is for passing an electron beam in a transmission electron microscope. The planar shape of the opening  103  is, for example, a rectangle having a short side of about 1 mm and a long side of about 2 mm. 
     The supporting film  104  is, for example, a silicon nitride film. The supporting film  104  may be a formvar film, a carbon film, a graphene film, or the like. The region of the supporting film  104  that overlaps with the opening  103  serves as a region for supporting the specimen. 
       FIG. 3  is an optical micrograph illustrating the first specimen supporting tool  100  supporting a continuous section. 
     As illustrated in  FIG. 3 , in the first specimen supporting tool  100 , since the supporting film  104  having a relatively large area can be formed flat, a continuous section can be supported. A continuous section is a series of a plurality of sections cut out continuously by a microtome. By observing each section constituting the continuous section with a transmission electron microscope or a scanning electron microscope, a continuous cross-sectional image (continuous cross-sectional image) can be acquired. By stacking the acquired continuous cross-sectional images, it is possible to perform three-dimensional reconstruction. 
     In a tomography method using continuous cross-sectional images in this way, the resolution in the X and Y directions depends on the resolution of the device used, but the resolution in the Z direction depends on the thickness of the section. 
     Here, a method in which a section is continuously tilted and observed with a transmission electron microscope to obtain a continuous tilt image (continuous tilt image), and the obtained continuous tilt image is used for three-dimensional reconstruction has been known as a tomography method. In the tomography method using this continuous tilt image, the resolution in the X, Y, and Z directions depends on the resolution of the device used. Therefore, the tomography method using the continuous tilt image can improve the resolution in the Z direction as compared with the tomography method using the continuous cross-sectional image. 
     By combining these two tomography methods, a three-dimensional structure of a specimen can be analyzed in more detail. For example, by combining these two tomography methods, it is possible to perform three-dimensional reconstruction of a specific protein in a cell after performing three-dimensional reconstruction of the entire cell. Specifically, after observing a continuous section, acquiring a continuous cross-sectional image and performing three-dimensional reconstruction of the entire cell, a continuous tilt image of one of a plurality of sections constituting the continuous section is acquired and then three-dimensional reconstruction of a specific protein in the cell is performed. 
     However, since the thickness of the substrate  102  of the first specimen supporting tool  100  is large, where the specimen is tilted, the specimen may be behind the substrate  102 . Therefore, it is difficult to acquire a continuous tilt image with the first specimen supporting tool  100 . If the specimen supported by the first specimen supporting tool  100  can be transferred to the second specimen supporting tool capable of acquiring a continuous tilt image, the two tomography methods can be combined. 
     As the second specimen supporting tool, for example, a mesh grid for a transmission electron microscope can be used.  FIG. 4  is an optical micrograph illustrating an example of a mesh grid for a transmission electron microscope. 
     The thickness of the mesh grid is about several tens of micrometers. As described above, a tool having a thickness smaller than that of the first specimen supporting tool is used as the second specimen supporting tool. As a result, even if the specimen is tilted, the specimen does not get behind the second specimen supporting tool, and a continuous tilt image can be acquired. 
     1.2. Flow of Pretreatment Method 
       FIG. 5  is a flowchart illustrating an example of a specimen pretreatment method according to an embodiment of the invention.  FIGS. 6 to 12  are diagrams for explaining a specimen pretreatment method according to an embodiment of the invention. 
     1.2.1. Step S 10  of Transferring a Section to a Water-Soluble Film 
     First, a specimen  2  supported by the supporting film  104  of the first specimen supporting tool  100  is transferred to a water-soluble film  10 . 
     First, as illustrated in  FIGS. 6 and 7 , the specimen  2  supported by the supporting film  104  of the first specimen supporting tool  100  is prepared. The specimen  2  is a continuous section and consists of a plurality of sections  3  continuously cut out by a microtome. 
     Next, as illustrated in  FIG. 8 , the water-soluble film  10  is attached to a smooth substrate  12 . The water-soluble film  10  is, for example, a polyvinyl alcohol film. Another water-soluble film may also be used as the water-soluble film  10 . As the smooth substrate  12 , a semiconductor substrate such as a silicon substrate can be used. For example, the water-soluble film  10  can be attached to the smooth substrate  12  using an adhesive tape or the like. 
     Next, as illustrated in  FIG. 9 , the supporting film  104  of the first specimen supporting tool  100  and the specimen  2  on the supporting film  104  are brought into contact with the water-soluble film  10 . Then, the substrate  102  is pushed. As a result, the supporting film  104  is separated from the substrate  102 . Next, the substrate  102  is removed from the water-soluble film  10 . As a result, as illustrated in  FIG. 10 , the specimen  2  can be transferred from the first specimen supporting tool  100  onto the water-soluble film  10 . In this step, the specimen  2  is transferred from the first specimen supporting tool  100  onto the water-soluble film  10  with the supporting film  104  attached to the specimen  2 . 
     In this step, the entire specimen  2  may be transferred from the first specimen supporting tool  100  onto the water-soluble film  10 , or a part of the specimen  2  may be transferred. That is, all the sections constituting the continuous section may be transferred from the first specimen supporting tool  100  to the water-soluble film  10 , or some of the plurality of sections constituting the continuous section may be transferred. 
     1.2.2. Step S 20  of Dissolving the Water-Soluble Film 
     Next, as illustrated in  FIG. 11 , the water-soluble film  10  and the specimen  2  on the water-soluble film  10  are immersed together with the smooth substrate  12  in a petri dish  5  containing pure water  6 . As a result, the water-soluble film  10  is dissolved and the specimen  2  is peeled off. Then, the specimen  2  floats on the water surface of the pure water  6 . As illustrated in  FIG. 11 , the supporting film  104  is attached to the specimen  2  in this step as well. 
     Here, the water-soluble film  10  is dissolved in pure water  6 , but the liquid that dissolves the water-soluble film  10  is not limited to pure water  6 , and may be distilled water, an aqueous solution, or the like. 
     1.2.3. Step S 30  of Supporting the Continuous Section with the Second Specimen Supporting Tool 
     As illustrated in  FIG. 12 , the specimen  2  floating on the water surface of the pure water  6  is recovered, and the specimen  2  is supported by the second specimen supporting tool  200 . 
     The second specimen supporting tool  200  is, for example, a mesh grid, and the specimen  2  floating on the water surface of pure water  6  is scooped by the mesh grid. Then, the specimen  2  scooped by the second specimen supporting tool  200  is dried. As a result, the specimen  2  can be recovered from the water surface of the pure water  6 , and the specimen  2  can be supported by the second specimen supporting tool  200 . 
     In this step, the specimen  2  is supported by the second specimen supporting tool  200  with the supporting film  104  attached to the specimen  2 . The supporting film  104  is attached to the specimen  2  from the time when the specimen  2  is transferred from the first specimen supporting tool  100  to the water-soluble film  10  until the specimen  2  is supported by the second specimen supporting tool  200 . 
     The mesh grid used as the second specimen supporting tool  200  is a mesh grid for a transmission electron microscope. The mesh grid is a mesh-shaped (mesh-like) metal plate. The material of the mesh grid is, for example, a metal such as copper, stainless steel, molybdenum, platinum, or the like. 
     By the above steps, the specimen  2  can be transferred from the first specimen supporting tool  100  to the second specimen supporting tool  200 . 
     2. Operational Effect 
     A specimen pretreatment method according to an embodiment of the invention includes the step S 10  of transferring the specimen  2  supported by the first specimen supporting tool  100  to the water-soluble film  10 , the step S 20  of immersing the specimen  2  on the water-soluble film  10  and the water-soluble film  10  in pure water  6  to dissolve the water-soluble film  10 , and the step S 30  of recovering the specimen  2  from the pure water  6  and supporting the specimen  2  with the second specimen supporting tool  200 . For this reason, the specimen  2  can be easily transferred from the first specimen supporting tool  100  to the second specimen supporting tool  200 . Therefore, for example, the specimen  2  can be transferred from the first specimen supporting tool  100  including the substrate  102  and the supporting film  104  illustrated in  FIG. 3  to the mesh grid (second specimen supporting tool  200 ) illustrated in  FIG. 4 . 
     As a result, a continuous cross-sectional image and a continuous tilt image of the same specimen  2  can be acquired. Therefore, for example, it is possible to perform three-dimensional reconstruction of the entire cell by a tomography method using a continuous cross-sectional image, and then perform three-dimensional reconstruction of a specific protein in the cell by a tomography method using a continuous tilt image. 
     Further, in the specimen pretreatment method according to the present embodiment, as illustrated in “4. Experimental Example” described later, it is possible to reduce the deformation of the specimen  2  and the deterioration of image quality when observing the specimen with an electron microscope. 
     Here, it is also conceivable to transfer the specimen  2 , for example, by using a mesh grid equipped with a supporting film as the second specimen supporting tool  200  and bringing the specimen  2  supported by the first specimen supporting tool  100  into direct contact with the supporting film of the second specimen supporting tool  200 . However, a thin formvar film or the like is used as the supporting film used for the specimen supporting tool so as not to interfere with the observation with a transmission electron microscope. Therefore, even if the specimen  2  supported by the first specimen supporting tool  100  is brought into direct contact with the supporting film of the second specimen supporting tool  200 , the supporting film is broken and cannot support the specimen  2 , or the supporting film is ruptured or wrinkled. 
     In the step S 10  of transferring the specimen  2  to the water-soluble film  10 , the specimen  2  is brought into contact with the water-soluble film  10  and the specimen  2  is transferred to the water-soluble film  10 . Therefore, it is possible to reduce the deformation of the specimen  2  and the deterioration of image quality when observing the specimen with an electron microscope. 
     In the step S 10  of transferring the specimen  2  to the water-soluble film  10 , the specimen  2  is transferred to the water-soluble film  10  in a state where the supporting film  104  is attached to the specimen  2 , and in the state S 30  of supporting the specimen  2  with the second specimen supporting tool  200 , the specimen  2  is supported by the second specimen supporting tool  200  with the supporting film  104  being attached to the specimen  2 . In this way, the specimen  2  can be transferred from the first specimen supporting tool  100  to the second specimen supporting tool  200  with the supporting film  104  being attached to the specimen  2 . In this way, in the specimen pretreatment method according to the present embodiment, since the specimen  2  is supported by the supporting film  104 , the deformation of the specimen  2  can be reduced. 
     Further, the supporting film  104  is a silicon nitride film. Since the silicon nitride film is less likely to wrinkle and tear, it does not interfere with the observation of the specimen  2  even when the supporting film  104  is attached to the specimen  2 . 
     In the step S 30  of supporting the specimen  2  with the second specimen supporting tool  200 , the specimen  2  floating on the pure water  6  is scooped with the second specimen supporting tool  200 . Therefore, the specimen  2  can be easily supported by the second specimen supporting tool  200 . 
     The specimen  2  supported by the first specimen supporting tool  100  is a continuous section in which a plurality of sections  3  continuously cut out by a microtome are connected, and in the step S 30  in which the specimen  2  is supported by the second specimen supporting tool  200 , the specimen  2  is supported, a part of the continuous section is recovered and a part of the continuous section is supported by the second specimen supporting tool  200 . Therefore, after observing the continuous section supported by the first specimen supporting tool  100 , acquiring a continuous cross-sectional image and performing three-dimensional reconstruction, the continuous section can be transferred from the first specimen supporting tool  100  to the second specimen supporting tool  200 , a continuous tilt image of one section  3  of the plurality of sections  3  constituting the continuous section supported by the second specimen supporting tool  200  can be acquired, and three-dimensional reconstruction can be performed. 
     In the step S 10  of transferring the specimen  2  to the water-soluble film  10 , the substrate  102  is removed. Therefore, the specimen  2  can be supported by the second specimen supporting tool  200  in a state where the substrate  102  is removed. 
     In the specimen pretreatment method according to the present embodiment, the second specimen supporting tool  200  is a mesh grid, and the thickness of the mesh grid is smaller than the thickness of the substrate  102 . Therefore, by transferring the specimen  2  to the second specimen supporting tool  200 , even if the specimen  2  is greatly tilted, the specimen  2  does not get behind the second specimen supporting tool  200 . Therefore, in the specimen pretreatment method according to the present embodiment, a continuous tilt image can be acquired. 
     3. Modification Example 
     In the above-described embodiment, the specimen  2  is transferred to the water-soluble film  10 , but the film to which the specimen  2  is to be transferred is not limited to the water-soluble film. For example, the film to which the specimen  2  is to be transferred may be a film that is soluble in an organic solvent such as ethanol. In this case, in the step S 20  of dissolving the film, the liquid for dissolving the film is an organic solvent, and the film and the specimen  2  on the film are immersed in the organic solvent. 
     In the above-described embodiment, the case where the first specimen supporting tool  100  includes the substrate  102  and the supporting film  104 , and the second specimen supporting tool  200  is a mesh grid has been described, but such combination of the first specimen supporting tool  100  and the second specimen supporting tool  200  is not limiting. 
     4. Experimental Example 
     An experimental example is shown below, and the invention will be described in more detail. The invention is not limited to the following experimental example. 
     4.1. Specimen Pretreatment 
       FIGS. 13 to 15  are optical micrographs for explaining a specimen pretreatment method. First, as illustrated in  FIG. 13 , a water-soluble film was attached to a smooth substrate. As the water-soluble film, a polyvinyl alcohol film (water-soluble poval film) having a thickness of 18 μm was used. A 2-inch silicon wafer was used as the smooth substrate. The thickness of the silicon wafer was set to 0.2 mm. 
     Next, a specimen supporting tool (hereinafter referred to as “SiN Window chip”) in which a supporting film was a silicon nitride film, the size of the supporting film (that is, the size of the opening) was 1.0 mm×2.0 mm, and the thickness of the supporting film was 30 nm was prepared as the first specimen supporting tool. Then, the specimen was placed on the supporting film of the SiN Window chip. As a specimen, a continuous section having a section thickness of 70 nm, which was prepared by cutting a resin-embedded Paramecium with a microtome, was used. 
     Next, as illustrated in  FIG. 14 , the SiN Windows chip was placed on a smooth substrate to which a water-soluble film was attached, with the supporting film on the bottom and the substrate on the top. 
     Next, the substrate of the SiN Window chip was pressed with a finger to transfer the supporting film and the continuous section to the water-soluble film. Then, the substrate was removed. As a result, as illustrated in  FIG. 15 , the continuous section and the supporting film were placed on the water-soluble film. 
     Next, the water-soluble film and the continuous section and the supporting film on the water-soluble film were immersed together with the smooth substrate in a petri dish containing pure water. As a result, the water-soluble film was dissolved and the continuous section was peeled from the water-soluble film. The peeled-off continuous section floated on the water surface with the supporting film attached to the continuous section. 
     Next, the second specimen supporting tool was prepared. As the second specimen supporting tool, a Cu mesh grid having a thickness of 70 μm was used. 
     Next, the continuous section floating on the surface of the water was scooped with the mesh grid. Then, the continuous section scooped with the mesh grid was dried. Through the above steps, the continuous section was transferred from the SiN Window chip to the mesh grid. 
     The supporting film was attached to the continuous section from the transfer of the continuous section from the SiN Windows chip to the water-soluble film until the continuous section was supported by the mesh grid. 
       FIG. 16  is an optical micrograph of the continuous section before transferring to the mesh grid.  FIG. 17  is an optical micrograph of continuous sections after transferring to a mesh grid. 
     As illustrated in  FIGS. 16 and 17 , the continuous section to which the supporting film was attached could be placed on the mesh grid. In  FIG. 17 , a part of the continuous section is placed on the mesh grid. 
     Through the above steps, the continuous section could be transferred from the SiN Window chip to the mesh grid. 
     4.2. Observation Results 
     Next, the section transferred from the SiN Window chip to the mesh grid by the above pretreatment method was observed with a transmission electron microscope. As a transmission electron microscope, JEM-1400 Flash manufactured by JEOL Ltd. was used. The observation was performed with the acceleration voltage set to 120 keV. 
       FIG. 18  is a transmission electron microscope image (TEM image) acquired by observing the section supported by a SiN Windows chip with a transmission electron microscope.  FIG. 19  is a TEM image acquired by observing the section transferred from the SiN Windows chip to the mesh grid by the above pretreatment method with a transmission electron microscope. The TEM image of  FIG. 18  is obtained by observing one of a plurality of sections constituting the continuous section. Further, the TEM image of  FIG. 19  is obtained by observing the same section as the section of  FIG. 18 . The image of the same Paramecium is captured in the TEM image of  FIG. 18  and the TEM image of  FIG. 19 . 
     In  FIGS. 18 and 19 , the lengths of cracks in Paramecium were compared. In  FIGS. 18 and 19 , two cracks (crack A and crack B) are included. 
     In the TEM image of  FIG. 18 , the length of the crack A was 97.880 pixel. Meanwhile, in the TEM image of  FIG. 19 , the length of the crack A was 97.268 pixel. Further, in the TEM image of  FIG. 18 , the length of the crack B was 83.267 pixel. Meanwhile, in the TEM image of  FIG. 19 , the length of the crack B was 83.156 pixel. 
     In this way, even when the sections were transferred from the SiN Window chip to the mesh grid, there was almost no change in the length of both crack A and crack B. Therefore, it was found that even when the sections were transferred from the SiN Window chip to the mesh grid by the above pretreatment method, there was almost no deformation of the sections. 
       FIG. 20  is a TEM image acquired by observing a section supported by the SiN Window chip with a transmission electron microscope.  FIG. 21  is a TEM image acquired by observing the section transferred from the SiN Windows chip to the mesh grid by the above pretreatment method with a transmission electron microscope. The TEM image of  FIG. 20  is obtained by observing one of a plurality of sections constituting the continuous section. Further, the TEM image of  FIG. 21  is obtained by observing the same section as the section of  FIG. 20 . 
     As illustrated in  FIGS. 20 and 21 , it was found that there was no deterioration in the image quality of the TEM image caused by the transfer of the section from the SiN Window chip to the mesh grid by the above pretreatment method. 
     The invention is not limited to the above-described embodiments, and various modifications can be made. For example, the invention includes configurations that are substantially the same as the configurations described in the embodiments. Substantially same configurations means configurations that are the same in function, method, and results, or configurations that are the same in objective and effects, for example. The invention also includes configurations in which non-essential elements described in the embodiments are replaced by other elements. The invention also includes configurations having the same effects as those of the configurations described in the embodiments, or configurations capable of achieving the same objectives as those of the configurations described in the embodiments. The invention further includes configurations obtained by adding known art to the configurations described in the embodiments. 
     Some embodiments of the invention have been described in detail above, but a person skilled in the art will readily appreciate that various modifications can be made from the embodiments without materially departing from the novel teachings and effects of the invention. Accordingly, all such modifications are assumed to be included in the scope of the invention.