Grid holder

Abstract

A grid holder is provided to prevent ice crystals from adhering to the sample. [Solution] The grid holder 10 comprises a holder body 14 that supports a grid 12 on which a sample is fixed in a fixed area, and a cover 15 that is detachably attached to the holder body 14 and, when attached, surrounds the grid 12 together with the holder body 14 with a gap that prevents contact with the sample on the grid 12, thereby forming a storage space for the grid 12, with the part of the cover facing the fixed area being optically transparent.

Description

Technical Field

[0001] The present invention relates to a grid holder.

Background Art

[0002] In structural analysis using a cryo transmission electron microscope, since the three-dimensional structure of a large molecular complex can be visualized, its application range is rapidly expanding. Also, a correlative light-electron microscope (hereinafter referred to as CLEM) method is known, in which the same sample is observed with an optical microscope (for example, a fluorescence microscope) and then with an electron microscope, and the respective observation images are compared and analyzed. This CLEM method is useful for obtaining intracellular space information such as the structure and behavior of specific molecules in cells, the structure of organelles, and their changes. In particular, a sample fixation method by rapid freezing is optimal for preparing a biological sample, and the cryo CLEM method using a rapidly frozen biological sample has attracted attention.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the cryo-CLEM method described above, the sample placed on the grid for the electron microscope is rapidly frozen together with the grid. While maintaining this frozen state, the sample on the grid is sequentially observed using a cryo-fluorescence microscope, thinned using a cryo-focused ion beam apparatus, and observed using a cryo-transmission electron microscope. However, in cryo-fluorescence microscopy, the grid and sample are placed in a dry gas (dry air) and observed while cooling them. During this observation, trace amounts of water contained in the dry air may crystallize and adhere to the surface of the sample as ice crystals. While the adhesion of ice crystals to the sample is not a major problem in cryo-fluorescence microscopy, it poses a problem in electron microscopes, including cryo-transmission electron microscopy, because electrons cannot pass through the crystalline ice containing the ice crystals, making observation impossible. Furthermore, if large crystalline ice forms on the surface of the sample, thinning using a cryo-focused ion beam apparatus becomes difficult.

[0005] Therefore, the present invention aims to provide a grid holder that prevents ice crystals from adhering to a sample. [Means for solving the problem]

[0006] The grid holder of the present invention comprises a holder body that supports a grid on which a sample is fixed in a fixed area, and an optically transparent cover that is detachably attached to the holder body and, when attached, surrounds the grid together with the holder body with a gap that prevents contact with the sample on the grid, thereby forming a space for the grid. The cover has an optically transparent portion that faces the fixed area. [Effects of the Invention]

[0007] According to the present invention, a grid is placed within the storage space formed by the holder body and the cover, and the portion of the cover opposite the fixing area of ​​the grid where the sample is fixed is made optically transparent. This allows for observation of the sample with an optical microscope while preventing ice crystals from adhering to the surface of the cooled (-150°C or below) sample. [Brief explanation of the drawing]

[0008] [Figure 1] This is an exploded perspective view showing the configuration of the grid holder according to the first embodiment. [Figure 2] This is a cross-sectional view showing a cross-section of the grid holder according to the first embodiment. [Figure 3] This is a perspective view showing the grid, C-clip, and auto-grid ring. [Figure 4] This is a cross-sectional view showing the cross-section of the grid. [Figure 5] This flowchart shows the observation procedure using the CLEM method with a grid holder. [Figure 6] This diagram illustrates the simulation results showing the state in which no ice crystals are attached to the optical field of view of the transparent component, and the distribution of light intensity on the grid in that state. [Figure 7] This diagram illustrates the simulation results showing the state in which ice crystals are attached to the optical field of view of the transparent material, and the distribution of light intensity on the grid in that state. [Figure 8] This is a TEM image obtained by observing the fixed area of ​​a grid using a grid holder, after rapid freezing and cryofluorescence observation, with a cryo-transmission electron microscope. [Figure 9] This is a TEM image obtained by observing the fixed area of ​​a grid using a cryo-transmission electron microscope, after rapid freezing and cryofluorescence observation, using a grid holder with the transparent component removed. [Figure 10] This is a TEM image obtained by observing the fixed area of ​​the grid with a cryo-transmission electron microscope after rapid freezing, without prior cryo-fluorescence observation. [Figure 11] This flowchart shows another observation procedure using the CLEM method with a grid holder. [Figure 12] This flowchart shows yet another observation procedure using the CLEM method with a grid holder. [Figure 13] This is a cross-sectional view showing the configuration of a grid holder according to the second embodiment. [Figure 14]This is a cross-sectional view showing an example of a grid holder configuration that holds multiple grids. [Modes for carrying out the invention]

[0009] <First Embodiment> As shown in Figures 1 and 2, the grid holder 10 according to the first embodiment consists of a holder body 14 that supports a grid 12 on which a frozen sample is fixed, and a cover 15 that is attached to the holder body 14 and covers the grid 12. This grid holder 10 is used for observation by cryo-photoelectron correlation microscopy (hereinafter referred to as cryo-CLEM). Cryo-CLEM is a method in which a rapidly frozen sample is observed with a cryo-optical microscope while maintaining its frozen state, then thinned, and observed with a cryo-transmission electron microscope in sequence, and the observed images are compared and analyzed.

[0010] The grid holder 10 prevents ice crystals from adhering to the sample on the grid 12 during observation with a cryo-optical microscope, while allowing observation of the sample through the transparent member 17 provided on the cover 15. By preventing ice crystal adhesion to the sample, thinning and observation with a cryo-transmission electron microscope, which are performed immediately after observation with a cryo-optical microscope, can be preferably carried out. In this example, the case in which a cryo-fluorescence microscope is used as an example of a cryo-optical microscope will be described.

[0011] In this example, the grid 12 is held in the grid holder 10 as an auto-grid 22 with a support 21 attached thereto. The grid 12 is used in a cryo transmission electron microscope. As shown in FIG. 3, the grid 12 has a thin disk shape and consists of a circular (ring-shaped) frame portion 23 and a fixed region 24 formed in a mesh shape inside this frame portion 23. A sample is fixed to the fixed region 24. The material of the grid 12 is, for example, Cu (copper). The diameter of the grid 12 is, for example, about 3 mm, and the thickness is about 0.5 mm. Note that the material of the grid 12 is not limited to Cu, and it may also be Cu / Rh (an alloy composed of rhodium and copper), Au (gold), Ni (nickel), etc.

[0012] As shown in FIG. 3, the support 21 is composed of an auto-grid ring (C clip ring) 21a and a C clip 21b. The grid 12 is fitted into the ring-shaped auto-grid ring 21a, and further, by fitting the C-shaped C clip 21b into the auto-grid ring 21a, the grid 12 is fixed within the auto-grid ring 21a.

[0013] Note that commercially available products can generally be used for the grid 12, the auto-grid ring 21a, and the C clip 21b. Also, the grid 12 may be held in the grid holder 10 alone, or the grid 12 fitted into the auto-grid ring 21a without being fixed by the C clip 21b may be held in the grid holder 10.

[0014] In the fixed region 24, a sample such as a protein (cell), etc. is rapidly frozen, and the water in the sample is frozen in a glassy state, that is, it is fixed in a state where ice crystals are not formed. In this example, as shown in FIG. 4, a thin sample support film 27 (for example, a carbon film) is stretched over the opening (mesh) 25 of the fixed region 24, and the sample is fixed on the sample support film 27. Note that the sample support film 27 in this example has holes (not shown) of about 1 μm formed regularly.

[0015] As shown in FIGS. 1 and 2, the holder body 14 is integrally formed with a support portion 31, a pedestal portion 32, and a connecting portion 33 that connects them. The holder body 14 has a hollow portion 34 formed therethrough in its axial direction (vertical direction in FIG. 1). The hollow portion 34 has a circular cross-section, and its axis coincides with the axis of the holder body 14. The support portion 31, the pedestal portion 32, and the connecting portion 33 in this example are cylindrical with a hollow interior due to the hollow portion 34, and the outer diameter of the connecting portion 33 is the smallest, while the outer diameter of the pedestal portion 32 is the largest. The holder body 14 is made of, for example, a metal such as brass, stainless steel, or titanium, which is preferable from the viewpoints of versatility and thermal conductivity. Also, depending on the application, it may be made of a resin such as PEEK (polyetheretherketone) that can be used at low temperatures.

[0016] In this example, one end of the support portion 31 is a support surface 35 that supports the grid 12. A recess 36 is formed at the center of the support surface 35. The above-described auto-grid 22 is disposed within this recess 36. The auto-grid 22 is disposed within the recess 36 such that the surface of the grid 12 to which the sample is fixed faces the transparent member 17 (above in FIG. 1). The depth of the recess 36 is smaller than the thickness of the auto-grid 22, which in this example is the thickness of the support tool 21 (auto-grid ring 21a). As a result, the auto-grid 22 disposed within the recess 36 protrudes from the recess 36 in its thickness direction (toward the cover 15).

[0017] The inner diameter of the recess 36 is approximately the same as the outer diameter of the autogrid 22. This allows the grid 12 to be positioned and placed on the support surface 35. In addition, one end of the hollow portion 34 is exposed as an opening 37 at the center of the bottom surface of the recess 36. The size of this opening 37 is smaller than the inner diameter of the recess 36 and is approximately the same size as the fixing area 24 of the grid 12. In other words, one end of the hollow portion 34 opens in the area of ​​the support surface 35 opposite the fixing area 24 of the grid 12. As a result, the grid 12 is supported by the support portion 31 with only its outer circumference in contact with the bottom surface of the recess 36, i.e., the fixing area 24 is not in contact with it. By supporting the fixing area 24 in this non-contact state, damage to the sample is less likely to occur, and the autogrid 22 is easier to grasp with tweezers when attaching, detaching, or fixing, improving workability.

[0018] In this example, the autogrid 22 has an outer diameter of 3.5 mm and a thickness of 0.4 mm, while the recess 36 has an inner diameter of 3.6 mm and a depth of 0.3 mm.

[0019] The outer periphery of the grid 12 is the portion of the grid 12, or anything integrated with it or treated as an integral part thereof, that is outside the fixed area 24. In the case of an autogrid 22 in which the grid 12 is attached to a support 21, as in this example, the outer periphery of the grid 12 is at least one of the frame portion 23 of the grid 12, the autogrid ring 21a, and the C-clip 21b. In the case where the grid 12 is fitted into the autogrid ring 21a without the C-clip 21b, the outer periphery of the grid 12 is at least one of the frame portion 23 of the grid 12 and the autogrid ring 21a.

[0020] As described above, the outer diameter of the base portion 32 is larger than the outer diameters of the support portion 31 and the connecting portion 33. Therefore, the holder body 14 can be placed stably in the liquid nitrogen bath with the base portion 32 facing downwards. In addition, when handling the grid holder 10 or the holder body 14, the base portion 32 can be easily manipulated by gripping it from above with a tool or fitting it into a tool.

[0021] A female thread 34a is formed on the inner surface of the hollow portion 34 in the base portion 32. By screwing a male thread formed on the tip of, for example, a rod-shaped jig into this female thread 34a, the jig can be attached to the grid holder 10. This jig can be used to remove the grid holder 10 from the liquid nitrogen bath or to hold it in the sample holder of a cryofluorescence microscope.

[0022] The cover 15 has the transparent member 17 described above and a cylindrical holding portion 41 that holds the transparent member 17. The cover 15 is attached to the holder body 14 by fitting the support portion 31 into the hollow interior of the holding portion 41 and placing the cover 15 over the support portion 31. The transparent member 17 is provided at one end of the holding portion 41 so that the transparent member 17 faces the support surface 35 when the cover 15 is attached to the holder body 14.

[0023] The transparent member 17 is optically transparent. Optical transparency of the transparent member 17 means that it has high transmittance to the light used for observation. In this example, the transparent member 17 of the grid holder 10 used in a cryofluorescence microscope has high transmittance to the excitation light irradiated onto the sample through the objective lens of the cryofluorescence microscope and to the fluorescence emitted from the sample that enters the objective lens.

[0024] The transparent member 17 is flat, and in this example, a circular glass plate is used. The thickness of the transparent member 17 is made thin to take into account the short working distance of the objective lens used in cryofluorescence microscopy observation. For example, the thickness of the transparent member 17 is about 0.1 mm to 1 mm. The size of the transparent member 17 only needs to be large enough to observe the fixed area 24 of the grid 12, and in this example, the diameter is about 5 mm. Note that the shape of the transparent member 17 is not limited to a circle.

[0025] The holding portion 41 is made of the same metal as the holder body 14, for example. One end of the holding portion 41 is open in a circular shape, and the transparent member 17 is fitted and fixed into this opening 43. In this example, the transparent member 17 is fixed by fitting a ring member 44 between the holding portion 41 and the transparent member 17. Note that the method of fixing the transparent member 17 to the holding portion 41 is not limited to the above, and for example, the transparent member 17 and the holding portion 41 may be bonded together with an adhesive.

[0026] A ring-shaped annular portion 55 is provided near the opening 43 inside the hollow interior of the holding portion 41. The centers of the opening 43 and the central opening 55a of the annular portion 55 are on the axis of the holding portion 41. The transparent member 17 is positioned and fixed inside the opening 43 with one of its surfaces resting on the surface 55b of the annular portion 55 on one end side (opening 43 side). In other words, the transparent member 17 is fixed in a recess provided at one end of the holding portion 41, and the opening 55a is formed in the center of the bottom surface (surface 55b) of the recess.

[0027] The inner diameter of the opening 55a of the annular portion 55 is the same as or larger than the outer diameter of the fixing area 24 of the grid 12. On the other hand, as described above, the autogrid 22 is positioned in the recess 36 with its thickness protruding from the recess 36. As a result, when the cover 15 is attached to the holder body 14, the autogrid ring 21a, which is the outer circumference of the grid 12, is sandwiched between the other end surface 55c of the annular portion 55 and the bottom surface of the recess 36, fixing the autogrid 22 between the holder body 14 and the cover 15. In addition, the fixing area 24 is exposed in the opening 55a, making it possible to observe the sample on the grid 12 through the transparent member 17. The surface 55b of the annular portion 55 is inclined so that its thickness gradually decreases toward the center in order to prevent vignetting.

[0028] The cover 15 is attached (assembled) to the holder body 14 by screwing the female thread 41a formed on the inner circumferential surface of the holding part 41 into the male thread 31a formed on the outer circumferential surface of the support part 31. The cover 15 can be removed from the holder body 14 by unscrewing the male thread 31a and the female thread 41a. In other words, the cover 15 is detachable from the holder body 14. In this example, a screw is used to maintain the cover 15 in place on the support part 31, but this is not the only option. For example, a magnet may be used to maintain the cover 15 in place on the support part 31, or a bayonet structure may be used.

[0029] By attaching the cover 15 to the holder body 14, a housing space for accommodating the autogrid 22 is formed, enclosed by the holder body 14 and the cover 15. Within this housing space, the autogrid 22 is held in place as described above, with the autogrid ring 21a sandwiched between the bottom surface of the recess 36 and the surface 55c of the annular portion 55. Therefore, within the housing space for the autogrid 22, a gap of at least the thickness of the annular portion 55 is created between the fixed area 24 and the transparent member 17. This creates a gap between the transparent member 17 and the grid 12 that prevents the sample on the fixed area 24 from contacting the transparent member 17. In this example, the thickness of the annular portion 55 is made thin, approximately 0.5 mm, considering the short working distance of the objective lens.

[0030] Furthermore, as described above, the autogrid 22 is positioned within the recess 36, protruding from the recess 36 in its thickness direction. As a result, a gap 58 is formed between the support surface 35 and the surface 55c of the annular portion 55 on the outer circumference of the recess 36 (autogrid 22).

[0031] The holding portion 41 is provided with multiple gas vents 61 that penetrate its peripheral wall. These gas vents 61 allow nitrogen gas, which is produced when liquid nitrogen vaporizes within the containment space, to escape to the outside space, thus connecting the containment space and the outside space. The gas vents 61 open into the gap 58 on the containment space side. That is, the gas vents 61 open at a position on the containment space side facing the side surface (outer peripheral surface) of the autogrid 22. This prevents ice crystals from adhering to the sample by preventing gas from the outside space from entering the containment space through the gas vents 61 from directly heading towards the fixed area 24. In this example, four gas vents 61 are provided at equal intervals in the circumferential direction of the holding portion 41, but this is not limited to this. For example, there may be one to three gas vents 61, or five or more.

[0032] The gas venting holes 61 are formed to be narrow. This allows gas from the outside space to enter the gas venting holes 61 of the holding section 41, which has been cooled to below -150°C, and traps the moisture in that gas by freezing it within the gas venting holes 61. This prevents ice crystals from adhering to the grid 12 and the sample.

[0033] Similarly, the holder body 14 is provided with a gas vent hole 63 that penetrates its peripheral wall. The gas vent hole 63 is provided in the connecting portion 33 and connects the hollow portion 34 to the external space. This gas vent hole 63 releases nitrogen gas, which is vaporized from liquid nitrogen in the hollow portion 34, to the external space. In this example, two gas vent holes 63 are provided at opposing positions on either side of the axis of the holder body 14, but the number and position of the gas vent holes are not limited to this. The connecting portion 33 is thinner than the support portion 31 and the base portion 32, making it easier for the gas vent holes 63 to penetrate, so the gas vent holes 63 are provided in the connecting portion 33, but they may also be provided in the support portion 31 or the base portion 32. The gas vent hole 63, like the gas vent hole 61 described above, is formed to be narrow and traps moisture in the gas from the external space inside. This prevents ice crystals from adhering to the grid 12 exposed in the hollow portion 34. In this example, the inner diameter of the gas vent holes 61 and 63 is 0.7 mm.

[0034] Next, the operation of the above configuration will be explained with reference to Figure 5. Note that the procedure described below is just one example and is not limited to this.

[0035] First, the sample on grid 12 is rapidly frozen (step ST1). This rapid freezing of the sample is performed, for example, in a chamber using a rapid freezer. Inside the chamber, a liquid nitrogen bath containing liquid nitrogen is prepared, and liquefied ethane cooled with liquid nitrogen is prepared in a container placed inside the liquid nitrogen bath. Grid 12 with the sample attached is also prepared. Then, the entire grid 12 is immersed in the liquefied ethane for a short time to rapidly freeze the sample on grid 12.

[0036] After rapidly freezing the sample, the grid 12 is moved into the liquid nitrogen in the liquid nitrogen bath. Then, in the liquid nitrogen, the grid 12 is fixed to the support 21 using a predetermined tool (step ST2). That is, the grid 12 is fitted into the auto-grid ring 21a and fixed with the C-clip 21b to form the auto-grid 22.

[0037] Furthermore, the procedure for rapidly freezing the sample and the procedure for fixing the grid 12 to the support 21 can be the same as the conventional procedure for observation with a cryo-transmission electron microscope.

[0038] Next, the auto grid 22 is held in the grid holder 10 (step ST3). For this purpose, the holder body 14 and the cover 15 are separated and immersed in liquid nitrogen in a liquid nitrogen bath to cool them down. For example, a plate-shaped jig with multiple holes is placed inside the liquid nitrogen bath for fixing the grid holder 10. By fitting the base portion 32 into one of the multiple holes, the holder body 14 is fixed in a position with the support surface 35 facing upwards.

[0039] After the holder body 14 and cover 15 have finished cooling (the liquid nitrogen has stopped boiling), the autogrid 22 is placed in the recess 36 of the holder body 14 in liquid nitrogen, for example, using cooled tweezers. At this time, the autogrid 22 is placed in the recess 36 so that the surface of the grid 12 on which the sample is fixed is facing upwards (opposite to the bottom surface of the recess 36). Within the recess 36, the autogrid 22 is positioned such that the autogrid ring 21a of the outer circumference of the grid 12 is in contact with the bottom surface of the recess 36, while the fixing area 24 is on the opening 37 and is not in contact with the bottom surface of the recess 36.

[0040] Next, using a designated tool, the female thread 41a of the cover 15 is screwed onto the male thread 31a of the support part 31, and the cover 15 is attached to the support part 31 of the holder body 14 by placing it over the support part 31. By attaching the cover 15 to the support part 31 in this way, the surface 55c of the annular part 55 of the cover 15 comes into contact with the auto grid ring 21a of the auto grid 22 in the recess 36, and the auto grid ring 21a is sandwiched between the annular part 55 and the recess 36, thereby fixing the auto grid 22.

[0041] As described above, the fixing of the autogrid 22 creates a gap between the surface of the fixed area 24 of the grid 12 and the transparent member 17 that is at least the thickness of the annular portion 55. As a result, the sample on the fixed area 24 of the grid 12 can be kept in non-contact with the transparent member 17, and the cover 15 can be assembled to the support portion 31. Furthermore, since the work is performed in liquid nitrogen at this time, no ice crystals adhere to the sample or the transparent member 17, etc.

[0042] For example, the autogrid 22 may be temporarily stored in a grid case in liquid nitrogen, and then removed from the grid case when it is to be held in the grid holder 10. This method is useful when the transfer time from the rapid freezer to the cryo-optical microscope is long, or when rapid freezing is performed on samples with multiple grids 12, and these grids 12 are sequentially replaced and held in the grid holder 10 for fluorescence observation.

[0043] The grid holder 10, which holds the autogrid 22, is removed from the liquid nitrogen using a jig and placed in a sample holder fixed to an electric stage for introduction into the cryofluorescence microscope (step ST4). Here, the grid holder 10 is inverted in liquid nitrogen so that the female thread 34a of the base portion 32 is facing upwards, and the grid holder 10 is fixed in place with the cover 15 inserted into another hole in the plate mentioned above. After this, the male thread of a rod-shaped jig, which has been cooled in liquid nitrogen beforehand, is screwed into the female thread 34a, and the rod-shaped jig is attached to the grid holder 10. Then, using this jig, the grid holder 10 is removed from the liquid nitrogen and inserted into the sample holder in the cryostat (cooling bath). The sample holder is designed to hold the base portion 32 from all sides, and the grid holder 10 is fixed to the sample holder. The sample holder holding the grid holder 10 is set in the observation position inside the cryofluorescence microscope by the movement of the electric stage (step ST5). At the observation position, the grid holder 10 is set so that the transparent member 17 faces the objective lens of the cryofluorescence microscope. The grid holder 10 is then cooled while being held in the sample holder and subjected to fluorescence observation in dry air (step ST6).

[0044] Incidentally, when the grid holder 10 is removed from the liquid nitrogen, the liquid nitrogen inside the grid holder 10 vaporizes due to the rise in temperature. For example, in the containment space where the grid 12 is placed, the liquid nitrogen in the gap between the transparent member 17 and the grid 12 vaporizes. When the liquid nitrogen vaporizes, the pressure inside the containment space increases, and this pressure increase causes the liquid nitrogen inside the containment space and the vaporized nitrogen gas to escape to the outside space through the gap created between the autogrid 22 and the annular portion 55 and recess 36 of the grid holder 10, via the vent holes 61 and 63. In addition, some of the nitrogen gas enters the hollow portion 34 through a pinhole in the sample support membrane 27 of the autogrid 22 and escapes to the outside space through the vent hole 63. Therefore, the pressure between the containment space and the hollow portion 34 hardly rises, and does not damage the sample support membrane 27 of the grid 12 or the sample frozen on its surface. Furthermore, when observing with a cryo-fluorescence microscope, all of the liquid nitrogen inside the containment space has vaporized, so the liquid nitrogen does not affect the fluorescence observation.

[0045] When removing the grid holder 10 from liquid nitrogen, the male screw of the jig is screwed into the female screw 34a of the base portion 32, but after fixing the grid holder 10 to the sample holder, the screw is released. When the jig is screwed in, the end of the hollow portion 34 of the grid holder 10 on the base portion 32 side is closed, but the liquid nitrogen remaining in the hollow portion 34 and the nitrogen gas that has vaporized from it are released into the outside space through the gas vent hole 63. Therefore, the pressure in the hollow portion 34 hardly rises, and does not damage the sample support film 27 of the grid 12 or the sample frozen on its surface.

[0046] In cryofluorescence microscopy, the grid holder 10 is placed in dry air, and the sample on the grid 12 is subjected to fluorescence observation. Therefore, small amounts of moisture contained in the dry air may adhere to the outer surface of the grid holder 10 as ice crystals. However, since the sample is not directly exposed to the dry air, ice crystals do not adhere to the sample.

[0047] Although a small amount of ice crystals may adhere to the outer surface of the transparent member 17, this small amount of ice crystal adhesion to the transparent member 17 has virtually no effect on fluorescence observation using a cryofluorescence microscope. Figure 6 shows the results of a simulation of the light intensity distribution on the grid 12, assuming that no ice crystals are attached to the transparent member 17. In Figure 6, the upper image shows the optical field of view at the position of the transparent member 17, the middle image shows the light intensity distribution of the excitation light irradiation spot at the focal plane of the cryofluorescence microscope objective lens corresponding to the sample support film 27 of the grid 12, and the lower graph shows the light intensity in the radial direction of the irradiation spot. Furthermore, Figure 7 shows the results of a simulation of the light intensity distribution on the grid 12 when ice crystals are attached to the transparent member 17 and are present in the optical field of view, similar to Figure 6.

[0048] As can be seen from the light intensity distribution and light intensity graph shown in Figures 6 and 7, when ice crystals are attached to the transparent member 17 compared to when no ice crystals are attached, the light intensity decreases slightly, but not to a level that would affect fluorescence observation. Therefore, it can be seen that even if ice crystals are attached to the transparent member 17 of the grid holder 10, it does not substantially affect the fluorescence observation of the sample.

[0049] Furthermore, it was confirmed that ice crystals had formed inside the gas vent holes 61 and 63 of the grid holder 10 after fluorescence observation. Therefore, it was confirmed that trace amounts of moisture in the dry air were being captured as ice crystals in the gas vent holes 61 and 63.

[0050] After the fluorescence observation is complete, the autogrid 22 is placed in the grid case and moved (step ST7). To do this, first the sample holder is removed from the fluorescence microscope and immersed in liquid nitrogen in the liquid nitrogen bath. The grid case is then immersed in the liquid nitrogen. In the liquid nitrogen, the grid holder 10 is removed from the sample holder, then the cover 15 is removed from the holder body 14 of the grid holder 10 to take out the autogrid 22, and the autogrid 22 is placed in the grid case containing liquid nitrogen. Since the operation of removing the autogrid 22 and placing it in the grid case is performed in liquid nitrogen, no ice crystals will adhere to the sample on the grid 12. After this, the grid case is removed from the liquid nitrogen and moved.

[0051] Next, the autogrid 22 is held in the FIB holder in order to thin the sample using a cryo-FIB (focused ion beam) (step ST8). In this example, since the sample is thinned using a cryo-FIB-SEM, which combines FIB and SEM (scanning electron microscope), a FIB-SEM holder is used as the FIB holder. The grid case that was moved above is immersed in liquid nitrogen. The FIB holder is then immersed in the liquid nitrogen. The autogrid 22, which was removed from the grid case, is held in the FIB holder while still in the liquid nitrogen.

[0052] The FIB holder is removed from the liquid nitrogen and set in the cryo-FIB-SEM (step ST9), and the sample is thinned (step ST10). The autogrid 22 set in the cryo-FIB-SEM is cooled by the cooling mechanism and loaded into the cryo-FIB-SEM processing chamber while maintaining a frozen state, and the sample in the autogrid 22 is thinned for transmission electron microscopy while remaining frozen under high vacuum. Because thinning is performed under high vacuum, no ice crystals adhere to the sample.

[0053] After thinning, the FIB holder is removed from the cryo-FIB-SEM and immersed in liquid nitrogen. The grid case is also immersed in the liquid nitrogen. While in the liquid nitrogen, the autogrid 22, on which the thinned sample is fixed, is removed from the FIB holder and placed in the grid case. The grid case is then removed from the liquid nitrogen and moved to the cryo-transmission electron microscope (step ST11).

[0054] Then, the grid case and the TEM (transmission electron microscope) holder are immersed in liquid nitrogen, and the autogrid 22 is removed from the grid case in the liquid nitrogen and held in the TEM holder (step ST12).

[0055] The TEM holder holding the autogrid 22 is set in the cryotransmission electron microscope (step ST13). The set TEM holder is cooled by the cooling mechanism of the cryotransmission electron microscope. After the TEM holder set in the cryotransmission electron microscope is loaded into the observation position, a high vacuum is created. Then, under high vacuum, the thinned sample on the autogrid 22 is irradiated with an electron beam to acquire a TEM image (step ST14). In steps ST12 to ST14, the autogrid 22 is handled under a liquid nitrogen atmosphere or high vacuum, so no ice crystals adhere to the sample.

[0056] Furthermore, the procedure from removing the autogrid 22 from the grid holder 10 after fluorescence observation to observing it with a cryotransmission electron microscope can be the same as the conventional procedure for thinning the grid 12 on which the rapidly frozen sample is fixed and observing it with a cryotransmission electron microscope.

[0057] As described above, observation is performed from cryofluorescence microscopy to cryotransmission electron microscopy. However, since the grid 12 is held in the grid holder 10 during fluorescence observation, ice crystals do not adhere to the sample. Therefore, thinning by cryoFIB-SEM and observation by cryotransmission electron microscopy can be performed without any problems. As a result, observation by the cryoCLEM method can be performed efficiently. In addition, it is possible to screen for samples suitable for thinning by observation with cryofluorescence microscopy, and observation can be performed efficiently.

[0058] Figure 8 shows a TEM image obtained by observing the fixed region 24 of the grid 12, which has undergone fluorescence observation using a cryo-fluorescence microscope, with a cryo-transmission electron microscope using the grid holder 10 described above. In this observation, the sample was rapidly frozen without adhering to the fixed region 24, and the sectioning process was omitted. In the TEM image of Figure 8, the grid-like black lines are a part of the grid 12 of the fixed region 24, and the rectangular white area is the sample support film 27 on the opening (mesh) 25 of the fixed region 24. The black shadows in this white area (sample support film 27) are ice crystals adhering to the sample support film 27.

[0059] Furthermore, using a grid holder with the transparent member 17 removed, Figure 9 shows a TEM image of the fixed region 24 of the grid 12 after fluorescence observation using a cryo-transmission electron microscope, following the same procedure as when using grid holder 10. In addition, Figure 10 shows a TEM image of the fixed region 24 of the grid 12 after rapid freezing using the conventional procedure, without undergoing fluorescence observation, using a cryo-transmission electron microscope.

[0060] Figures 8 to 10 show that when the transparent member 17 is removed and fluorescence observation is performed, i.e., when the grid holder 10 is not used, a considerable amount of ice crystals adhere to the sample support film 27. However, when the grid holder 10 is used, the amount of ice crystal adhesion is about the same as when cryofluorescence observation has not been performed, and is considerably less. Therefore, it can be seen that the grid holder 10 configured as described above is effective in preventing ice crystal adhesion during fluorescence observation under dry air conditions using a cryofluorescence microscope.

[0061] The procedure shown in Figure 11 involves obtaining an autogrid 22 after rapid freezing of the sample (steps ST1, ST2), thinning the sample using cryo-FIB-SEM (steps ST8-ST10), followed by fluorescence observation with a cryo-fluorescence microscope (steps ST3-ST6), and then electron microscopy observation with a cryo-transmission electron microscope (steps ST11-ST14), in this order. In this example procedure, the sample (cells) thinned by cryo-FIB-SEM is held in a grid holder 10 and fluorescence observation is performed in dry air using a cryo-fluorescence microscope, but no ice crystals form on the sample. Therefore, the thinned sample used for fluorescence observation can be used directly for observation with a cryo-transmission electron microscope.

[0062] The procedure shown in Figure 12 involves obtaining an autogrid 22 after rapid freezing of the sample (steps ST1, ST2), fluorescence observation of the sample using a cryo-fluorescence microscope (steps ST3-ST6), thinning using a cryo-FIB-SEM (steps ST8-ST10), fluorescence observation of the thinned sample using a cryo-fluorescence microscope (steps ST3-ST6), and electron microscopy observation using a cryo-transmission electron microscope (steps ST11-ST14), in this order. That is, fluorescence observation is performed on both the sample before and after thinning using a grid holder 10. No ice crystals adhere to the sample after fluorescence observation in any case. This procedure allows for screening of samples suitable for thinning by fluorescence observation of the sample before thinning, and enables more efficient comparison and analysis of the fluorescence observation images and electron microscopy observation images of the thinned sample.

[0063] <Second Embodiment> The second embodiment is characterized by the flat shape of the holder body and cover of the grid holder. Except as described below, it is the same as the first embodiment, and substantially identical components are denoted by the same reference numerals, and detailed descriptions are omitted.

[0064] As shown in Figure 13, the grid holder 100 according to the second embodiment consists of a holder body 114 that supports a grid 12 on which a frozen sample is fixed, and a cover 115 that is attached to the holder body 114 and covers the grid 12. The holder body 114 and the cover 115 are flat plates of approximately the same size. The grid holder 100 is assembled so that one side of the holder body 114 and the cover 115 are aligned with each other, and the auto-grid 22 is held in place by being sandwiched in the housing space formed between them, and the sample on the grid 12 can be observed by fluorescence through a transparent member 17 provided on the cover 115.

[0065] The holder body 114 includes, for example, a circular, flat support plate 131 and a cover member 132 attached to the support plate 131. The side of the support plate 131 on which the cover 115 is attached is the support surface 131a, and a recess 36 is provided in the center of the support surface 131a. The support plate 131 also has a through hole 134 that penetrates in the thickness direction, and one end of the through hole 134 opens in a circular shape in the center of the bottom surface of the recess 36. The inner diameter of the recess 36 is larger than the inner diameter of the through hole 134. The inner diameter of the through hole 134 is approximately the same as the inner diameter of the fixing area 24 of the grid 12.

[0066] A recess 136 is formed on the surface 131b of the support plate 131 opposite to the support surface 131a, and the other end of the through hole 134 opens in the center of the bottom surface of this recess 136. Therefore, the bottom of the recesses 36 and 136 is an annular portion 135 with an opening in the center. The inner diameter of the recess 136 is larger than the inner diameter of the through hole 134. The lid member 132 is fitted and fixed inside this recess 136. In this example, the support plate 131 and the lid member 132 are fixed with adhesive, but the method of fixing the lid member 132 is not limited to this.

[0067] The lid member 132 is flat, and in this example, the same circular glass plate as the transparent member 17 is used. This lid member 132 is provided to prevent ice crystals from entering from the surface 131b side. Note that the material of the lid member 132 is not limited to a glass plate; any material that can prevent ice crystals from entering may be used. Also, the recess 136 and the lid member 132 may be omitted. In this case, the through hole 134 can be replaced with a bottomed hole.

[0068] The cover 115 has a transparent member 17 and a flat retaining plate 141 that holds the transparent member 17. The retaining plate 141 is a flat circular plate with an outer diameter approximately the same as that of the support plate 131. The retaining plate 141 has a recess 146 in the center of the surface 141a on the side to which the support plate 131 is attached. The inner diameter of the recess 146 is approximately the same as the outer diameter of the autogrid 22, and its depth is the same as the height of the autogrid 22 protruding from the recess 36. As a result, when the cover 115 is attached to the holder body 114 so that the surface 141a is aligned with the support surface 131a, the autogrid 22 is housed in the space formed by the recess 36 and the recess 146, and the outer periphery of the autogrid 22 is clamped by the bottom surfaces of the recess 36 and the recess 146.

[0069] A recess 147 is formed on the surface 141b of the retaining plate 141 opposite to surface 141a, and the transparent member 17 is fixed to this recess 147 with adhesive. A through hole 148 is formed in the retaining plate 141, penetrating in the thickness direction, and opens at the center of the bottom surface of recesses 146 and 147. That is, the bottom of recesses 146 and 147 is an annular portion 149 with an opening in the center. The sample on the grid 12 is observed through the transparent member 17 and the through hole 148.

[0070] Multiple positioning pins 151 are embedded in the support surface 131a of the support plate 131, and positioning holes 152 are formed in the surface 141a of the retaining plate 141 at positions corresponding to the positioning pins 151. The cover 115 is assembled to the holder body 114 by inserting the positioning pins 151 into the positioning holes 152, thereby aligning the central axes of the recesses 36 and 146, and the auto grid 22 is housed in the space formed by the recesses 36 and 146.

[0071] During observation with a cryofluorescence microscope, the grid holder 100 is cooled in a low-temperature heat bath, for example, at about 77K, on ​​an electric stage, and the sample is cooled via the support plate 131. This low-temperature heat bath consists of, for example, liquid nitrogen stored in an insulated container and a metal body, and the sample is cooled by bringing the support plate 131 into contact with the surface of the metal body exposed from the liquid nitrogen. For this reason, it is preferable that the support plate 131 be made of a material with good thermal conductivity. Also, in this example, as will be described later, the support plate 131 is attracted to a magnet 154 provided on the holding plate 141, so the support plate 131 is made of a material that is attracted to magnets. In this example, the support plate 131 is made of brass. On the other hand, it is preferable that the holding plate 141 be a non-magnetic material so as not to be magnetized, and in this example it is made of stainless steel. Note that the method for cooling the grid holder 100 is not limited to the above.

[0072] The holder body 114 and the cover 115 are held in place by magnetic force, allowing the cover 115 to be easily attached to the holder body 114. The retaining plate 141 has multiple magnets 154 that penetrate a portion of it in the thickness direction. These magnets 154 are attracted to the support plate 131, which maintains the cover 115 attached to the holder body 114.

[0073] In this example, two positioning holes 152, two positioning pins 151, and two magnets 154 are provided, and they are positioned point-symmetrically with respect to the centers of recesses 36 and 146, but the number and positions of these are not limited to these. Also, positioning pins 151 may be provided on the holding plate 141 and positioning holes 152 on the support plate 131. The positioning holes 152 may or may not penetrate as long as they allow for positioning and enable close contact between the support surface 131a and surface 141a. Furthermore, the magnets 154 may be provided on the holder body 114, or on both the holder body 114 and the cover 115.

[0074] By attaching the cover 115 to the holder body 114, a storage space is formed surrounded by the holder body 114 and the cover 115. Specifically, the storage space is the space between the transparent member 17 and the lid member 132, consisting of recesses 36, 146 and through holes 134, 148. Within this storage space, as described above, the autogrid 22 is fixed by the autogrid ring 21a, which is the outer circumference of the grid 12, being sandwiched between the bottom surfaces of the recesses 36 and 146, i.e., the annular portion 135 and the annular portion 149. Therefore, when the autogrid 22 is fixed within the storage space, a gap equal to the thickness of the annular portions 135 and 149 is formed between the fixed area 24 of the grid 12 and the transparent member 17 and the lid member 132. This creates a gap between the sample fixed in the fixed area 24 and the transparent member 17.

[0075] In this example, the thickness of the support plate 131 and the retaining plate 141 is 1 mm, and the thickness of the annular portions 135 and 149 is 0.2 mm. Furthermore, for the autogrid ring 21a with a thickness of 0.4 mm and an outer diameter of 3.5 mm, the depth of the recess 36 is 0.3 mm and its inner diameter is 3.6 mm, and the diameter of the transparent member 17 and the lid member 132 is 3 mm and their thickness is 0.17 mm. Note that the shape of the support plate 131 and the retaining plate 141 is not limited to circular.

[0076] The grid holder 100 is assembled, for example, in the same manner as in the first embodiment, by attaching the cover 115 to the holder body 114 on which the autogrid 22 is placed in the recess 36, while in liquid nitrogen in a liquid nitrogen bath. In this assembled state, it is used for observation with a cryofluorescence microscope. Since the autogrid 22 is not directly exposed to the dry gas in the cryofluorescence microscope, no ice crystals adhere to the sample. In addition, although a small amount of ice crystals may adhere to the outer surface of the transparent member 17, it does not affect fluorescence observation.

[0077] When assembling the grid holder 100, liquid nitrogen is placed inside the containment space. In this example, the airtightness between the holder body 114 and the cover 115 is not high, allowing the nitrogen gas produced by the vaporization of the liquid nitrogen to escape from the containment space to the outside space. Since the cover 115 is attached to the holder body 114 by magnets 154, the airtightness between the support surface 131a and surface 141a is not high. Therefore, when the grid holder 100 is removed from the liquid nitrogen, the nitrogen gas produced by the vaporization of liquid nitrogen inside the containment space can escape to the outside space through the gap between the support surface 131a and surface 141a. On the other hand, since the grid holder 100 itself is below -150°C, even if dry air from the outside space enters between the support surface 131a and surface 141a, the moisture contained in the dry air is frozen and trapped on the support surface 131a and surface 141a. Therefore, moisture from the outside space does not reach the grid 12 held in the grid holder 100, preventing ice crystals from adhering to the sample.

[0078] Furthermore, when using the grid holder 100, the procedure from freezing the sample to observation with a cryo-electron microscope is the same as in the first embodiment, except that the assembly of the cover 115 to the holder body 114 and the setting method and cooling method in the cryo-fluorescence microscope are different.

[0079] Figure 14 shows an example of a grid holder 200 that holds multiple grids 12. In this grid holder 200, multiple recesses 36 are formed, for example, at equal intervals on the support surface 231a of the support plate 231 of the holder body 214, on the side to which the cover 215 is attached, and a grid 12 can be placed in each recess 36. The support plate 231 also has multiple through holes 134 that penetrate in the thickness direction, and one end of each through hole 134 opens in a circular shape at the center of the bottom surface of each recess 36. A lid member 132 is fixed to the surface 231b of the support plate 231 opposite to the support surface 231a so as to close the other end of each through hole 134. This lid member 132 is installed to prevent ice crystals from entering from the surface 231b side. Note that the multiple recesses 36 do not necessarily have to be at equal intervals, and their number and spacing can be arbitrarily determined.

[0080] The retaining plate 241 of the cover 215 is provided with multiple through holes 148. A transparent member 17 is fixed to the side 241b of the retaining plate 241 opposite to the side 241a to which the holder body 214 is attached, covering one end of each through hole 148. The grid holder 200 allows for fluorescence observation of multiple samples on grids 12 simultaneously or sequentially through the transparent member 17 and the through holes 148.

[0081] Although the transparent member 17 and the lid member 132 are fixed with adhesive, the method of fixing them is not limited to those described above.

[0082] In this example, the depth of each recess 36 is greater than the thickness of the autogrid ring 21a. Also, the inner diameter of the recess 36 is slightly larger than the outer diameter of the autogrid ring 21a. When the cover 215 is attached to the holder body 214 so that the surface 241a is aligned with the support surface 231a, spaces consisting of the through holes 134, 148 and each recess 36 are formed between the transparent member 17 and the lid member 132 as storage spaces. Each autogrid 22 is housed in the recess 36 of each storage space without being fixed. That is, each autogrid 22 fits loosely into the recess 36 and is held in the grid holder 200 without being pinched between the holder body 214 and the cover 215.

[0083] In this example, the thicknesses of the support plate 231 and the retaining plate 241 are 2 mm and 1 mm, respectively, and the thicknesses of the transparent member 17 and the lid member 132 are 0.17 mm and 0.17 mm, respectively. Furthermore, for an autogrid ring 21a with a thickness of 0.5 mm and an outer diameter of 3.5 mm, the depth of the recess 36 is 1 mm and the inner diameter is 4 mm. Note that while the support plate 231 and the retaining plate 241 in this example are circular flat plates, they are not limited to a circular shape. In this example, the distance between adjacent recesses 36 is 6 mm.

[0084] In this example, the grid holder 200 holds the grid 12 without fixing it within the containment space as described above. However, even without fixing the grid 12 within the autogrid ring 21a with the C-clip 21b, the grid 12 can be placed within the autogrid ring 21a with sufficient precision for observation. For example, in fluorescence observation, samples (cells) in good condition or suitable for thinning can be screened, and only the high-quality grids 12 to be used in the next step can have the C-clip 21b added and be clipped into the autogrid ring 21a, becoming an autogrid 22 that can be used for thinning or electron microscopy observation of the sample. If the C-clip 21b is fitted into the autogrid ring 21a, it cannot be retrieved and reused is difficult. However, by using the grid holder 200 and performing thinning or electron microscopy observation in this manner, even if low-quality grids 12 are found, they can be reused without consuming the expensive consumables, the autogrid ring 21a and C-clip 21b. For similar reasons, the grid 12 may be held alone without being fixed within the containment space of the grid holder 200.

[0085] As described above, since the grid holder 200 has multiple recesses 36, samples on multiple grids 12 can be observed for fluorescence simultaneously or sequentially without changing the grid holder. Therefore, samples on multiple grids 12 can be observed for fluorescence efficiently.

[0086] Furthermore, even when holding a single grid 12, as in grid holders 10 and 100, it is also possible to fit the grid 12 into the auto grid ring 21a without attaching the C clip 21b, or to hold only the grid 12 in the grid holder. Also, when holding multiple grids 12, as in grid holder 200, the outer circumference of the grid 12 may be clamped and fixed, as in grid holder 100.

[0087] In the grid holder 200 described above, the ends of each through-hole 134 and 148 are covered and sealed with a single transparent member 17 and a cover member 132, respectively. However, a transparent member 17 and a cover member 132 may be provided for each through-hole 134 and 148. In this case, recesses may be provided in the support plate 231 and the holding plate 241 for fitting and fixing each transparent member 17 and cover member 132. [Explanation of Symbols]

[0088] 10, 100, 200 grid holders 12 grid 14, 114, 214 Holder body 15, 115, 215 Cover 17 Transparent component 21a Autogrid ring 21b C clip 22 Autogrid 24 Fixed area 31 Support part 35 Support surface 36, 136 recesses 41 Holding part 61, 63 Gas vent holes 131, 231 Support plate 131a, 231a Support surface 141, 241 Holding plate 151 Positioning pins 152 Positioning holes 154 Magnets

Claims

1. A holder body that supports a grid on which the sample is fixed in a fixed area, A cover is detachably attached to the holder body, and when attached, surrounds the grid together with the holder body with a gap that prevents contact with the sample on the grid, thereby forming a storage space for the grid, the part of which is opposite the fixed area is optically transparent, A grid holder equipped with a grid holder.

2. The cover is attached to the holder body by screwing it in. The grid holder according to claim 1.

3. The holder body has a cylindrical support portion with a support surface at one end for supporting the grid, The cover has a cylindrical holding portion and an optically transparent member fixed to one end of the holding portion, and is mounted with the support portion inserted into the holding portion, with the transparent member facing the fixed area. The grid holder according to claim 1.

4. The support portion has male threads formed on its outer surface. The retaining portion has a female thread formed on its inner circumferential surface that engages with the male thread, and is attached to the support portion by screwing the female thread and the male thread together. The grid holder according to claim 3.

5. The cover has a first gas vent hole that connects the containment space to the external space and penetrates the retaining portion for releasing gas from the containment space. The grid holder according to claim 3 or 4.

6. The first gas vent hole opens at a position on the support surface facing the side surface of the grid, The grid holder according to claim 5.

7. The holder body is cylindrical in shape, having a hollow portion with one end open in the region of the support surface opposite to the fixing region. The grid holder according to claim 3 or 4.

8. The holder body has a second gas vent hole that connects the hollow portion to the external space and allows gas to escape from the hollow portion. The grid holder according to claim 7.

9. The holder body has a recess on the support surface in which the grid is arranged and positioned, The grid holder according to claim 3.

10. The cover is attracted to the holder body by a magnet. The grid holder according to claim 1.

11. The magnet is provided on one of the holder body and the cover, and by attracting the other of the holder body and the cover, it maintains the cover in a state where it is attached to the holder body. The grid holder according to claim 10.

12. The aforementioned cover is An optically transparent member opposite to the fixed region, A retaining plate that holds the transparent member and It has, The holder body is mounted on the support surface that supports the grid, The grid holder according to claim 11.

13. The holder body has one or more recesses on the support surface, in which the grid is arranged and positioned. The grid holder according to claim 12.

14. The holder body has one or more recesses on the support surface in which the grid is arranged, The grid holder according to claim 12, wherein the grid is loosely fitted into the recess.

15. The holder body is a flat plate with the support surface as one of its surfaces. The grid holder according to claim 13 or 14.

16. The recess is provided with a through hole that penetrates the holder body and has one end opening into the recess. The grid holder according to claim 15.

17. The other end of the through hole is covered by a cover member. The grid holder according to claim 16.

18. The cover, when installed, has an annular portion that clamps the outer periphery of the grid between itself and the support surface of the holder body. The grid holder according to claim 1.

19. The transparent member is glass. The grid holder according to claim 3 or 12.

20. The lid member is made of glass. The grid holder according to claim 17.

21. The aforementioned grid is a grid for an electron microscope. The grid holder according to claim 1.

Images