OBSERVATION METHOD EMPLOYING SCANNING ELECTRON MICROSCOPE, AND SAMPLE HOLDER FOR THE SAME

Abstract

Disclosed is a method with which it is possible to observe a biological sample in a living state, without significant restrictions due to the properties of the sample or due to a structural body accommodating the sample. To observe a target sample on a sample stage using a SEM including a radiation source for electron beam irradiation, the method includes: a step of bringing an insulating and electron-permeable thin film into contact with the target sample in such a way as to follow a surface on the radiation source side of the target sample, and sealing the target sample in a gap between the film and the sample stage; and a step of radiating an electron beam onto the target sample from the radiation source through the film.

Claims

1. A method for observing a target sample on a sample stage using a scanning electron microscope comprising a radiation source for electron beam irradiation, the method comprising: a sealing step of bringing an insulating and electron-permeable thin film into contact with the target sample in such a way that the thin film follows a surface on the radiation source side of the target sample, and sealing the target sample in a gap between the thin film and the sample stage; and a radiating step of radiating an electron beam onto the target sample from the radiation source through the thin film.

2. The method according to claim 1, wherein the target sample coexists with a fluid material or is in a hydrated state.

3. The method according to claim 1, wherein the observation is carried out by detecting a secondary electron emitted from the target sample or the thin film at a time of the electron beam irradiation.

4. The method according to claim 1, wherein the thin film is capable of being elastically deformed according to movement of the target sample.

5. The method according to claim 4, wherein, using a relative displacement between two or more points on the thin film, a stress caused between corresponding points on the target sample is measured.

6. The method according to claim 1, further comprising a marker disseminating step of disseminating a marker to a surface of the thin film opposite to the target sample side before the radiating step.

7. The method according to claim 6, further comprising an analysis step of analyzing astigmatism of the marker after the radiating step, wherein the marker has a known shape.

8. A sample holder for a scanning electron microscope, comprising: a sample stage capable of supporting a target sample such that the target sample faces a radiation source for electron beam irradiation of the scanning electron microscope; an insulating and electron-permeable thin film; and means for sealing the target sample in a gap between the thin film and the sample stage such that the thin film is brought into contact with the target sample in such a way that the thin film follows a surface on the radiation source side of the target sample.

9. The sample holder according to claim 8, further comprising adjusting means for adjusting a tension applied to the thin film.

10. The sample holder according to claim 9, wherein the adjusting means comprises one or two or more spacers disposed between the target sample and the sample stage.

11. The sample holder according to claim 10, wherein the spacers comprise a plurality of spacers, and wherein at least one of the spacers comprises an elastic material.

12. The sample holder according to claim 8, wherein the thin film comprises a polyimide.

13. The sample holder according to claim 12, wherein the thin film has a thickness of 100 nm or more and 5 μm or less.

14. The sample holder according to claim 8, wherein the sample stage comprises a conductive material.

15. The sample holder according to claim 8, wherein the sealing means comprises: a sealing member present between the thin film and the sample stage and capable of being elastically deformed in a direction orthogonal to a surface of the sample stage on a thin film side; a rigid pressing member sandwiching the thin film with the sealing member; and a fixing member fixing the pressing member to the sample stage.

16. The sample holder according to claim 15, wherein the pressing member extends outward beyond the sealing member, and wherein the fixing member comprises a bolt configured to pass through a portion of the pressing member extending outward and be screwed into the sample stage without interfering with the sealing member.

17. The sample holder according to claim 8, wherein the sealing means comprises an adhesive bonding a periphery of the thin film to the sample stage.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a cross-sectional view in the vertical direction showing a sample chamber of a scanning electron microscope according to Embodiment 1.

[0043] FIG. 2 is a cross-sectional view in the vertical direction showing a sample chamber of a scanning electron microscope according to Embodiment 2.

[0044] FIG. 3 is an SEM micrograph of fluorescent microspheres with which a thin film has been brought into contact in accordance with Embodiment 1.

[0045] FIG. 4 is an SEM micrograph of a blood tissue with which the thin film has been brought into contact in accordance with Embodiment 1.

[0046] FIG. 5 is an SEM micrograph of a section of cardiac muscle fibers with which a thin film has been brought into contact in accordance with Embodiment 1.

[0047] FIG. 6 is an SEM micrograph of a section of collagen fibers with which a thin film has been brought into contact in accordance with Embodiment 1.

[0048] FIG. 7 is an SEM micrograph of a semiconductor chip with which a thin film has been brought into contact.

[0049] FIG. 8 is an SEM micrograph of a semiconductor chip with which another thin film has been brought into contact.

[0050] FIG. 9 includes a SEM micrograph of a section of cardiac muscle fibers with which the thin film has been brought into contact, and a graph showing changes of the strain over time.

[0051] FIG. 10 schematically illustrates the action of the present invention.

[0052] FIG. 11 schematically illustrates the action of the conventional art disclosed in Non-Patent Document 1.

[0053] FIG. 12 is a SEM micrograph of a thin film of a sample holder on which heart has been sealed and a marker disseminated over the thin film.

[0054] FIG. 13 is a graph showing the locus of the marker.

[0055] FIG. 14 includes SEM micrographs capturing changes over time of a calcium phosphate crystal separated inside a fluid material in a state where a thin film has been brought into contact in accordance with Embodiment 1.

MODE FOR CARRYING OUT THE INVENTION

Embodiment 1

[0056] As shown in FIG. 1, a sample holder 1 for a scanning electron microscope according to the first embodiment includes a sample stage 2 made of a conductive material, specifically aluminum, a thin film 3 made of an insulating and electron-permeable organic polymer, a waterproof sealing member 4 that is made of an elasticity material and has a ring shape or a circular cylindrical shape, a disc-shaped spacer 5 made of an elastic material, an annular pressing member 6 made of a rigid material, and a plurality of bolts 7 corresponding to fixing members. Any of these parts can be combined together and separated from one another regardless of whether the timing is before or after use.

[0057] The sealing member 4 has an outer circumference located sufficiently inside the edges of the sample stage 2. The thin film 3 has a size enough to close one opening of the sealing member 4 and to extend outward beyond the outer circumference of the sealing member 4. The spacer 5 has an outer diameter enough to prevent contact with the inner circumference of the sealing member 4 and a height sufficiently smaller than the height of the sealing member 4. The pressing member 6 has an inner circumference having the same center and the same outer diameter as the inner circumference of the sealing member 4, and the outer edges protrude from the sealing member 4. Tapped holes (not shown) that are engaged with the bolts 7 are provided at positions on the upper surface of the sample stage 2, the positions protruding from the sealing member 4, and through holes are provided at the corresponding positions of the pressing member 6.

[0058] When the sample holder 1 is used, the sealing member 4 is put almost at the center of the sample stage 2, and the spacer 5 having appropriate thickness and elasticity according to the properties of a sample 8 is put inside the sealing member 4. The sample 8 is put on the spacer 5, and the periphery of the sample 8 is filled with a fluid material 9 such as an aqueous solution. The thin film 1 is put on the sealing member 8 to close the opening of the sealing member 8, the pressing member 6 is then put, and the bolts 7 are inserted into the through holes of the pressing member 6 and engaged with the tapped holes of the sample stage 2. By engaging the bolts 7, the sealing member 4 and the spacer 5 are compressed, and the thin film 3 is lightly pressed against the sample 8 and follows the shape of the upper surface of the sample 8. The surplus fluid material 9 oozes out of the gap between the sealing member 4 and the thin film 3 or the sample stage 2. Since the sealing member 4 is flexibly in contact with the thin film 3 pressed by the pressing member 6, the periphery of the sample 8 is kept fluid-tight.

[0059] The above operations may be carried out outside the sample chamber of the scanning electron microscope, such as in the air at normal temperature. The sample holder 1 on which the sample 8 and the fluid material 9 have been sealed is put in the sample chamber such that the sample 8 faces a radiation source 10 for electron beam irradiation across a lens. The sample stage 2 is electrically grounded. A secondary electron detector 12 is installed above the sample holder 1 at such a position as not to obstruct the path of an electron beam 11 radiated from the radiation source 10. There is no member other than the detector 12 up to the lens above a portion of the thin film 3 surrounded by the inner circumference of the sealing member 4.

[0060] When the electron beam 11 is radiated from the radiation source 10 in this state, incident electrons that are not applied to the sample 8 and backscattered electrons flow through the fluid material 9 and the sample stage 2 to the ground. Accordingly, either of the sample 8 and the thin film 3 will not become charged. On the other hand, since the thin film 3 has insulating and electron-permeable properties, secondary electrons 13 emitted from the surface of the sample 8 and the thin film 3 are detected by the detector 12. The thin film 3 has projections and depressions following the surface of the sample 8, and follows the movement of the sample 8, so that the intensity of the secondary electron 13 to be detected varies depending on the angle of the surface of the sample 8 or the thin film 3 with the incident electron beam. Accordingly, it is possible to measure the microstructure and the direction and amount of the displacement of the sample 8.

Embodiment 2

[0061] A sample holder 14 for a scanning electron microscope according to the second embodiment differs from that in the first embodiment in that none of the sealing member 4, the pressing member 6, and the bolts 7 are included. An adhesive 15 is included instead as shown in FIG. 2.

[0062] When the sample holder 14 is used, the spacer 5 is put almost at the center of the sample stage 2, and the adhesive 15 is applied to the periphery of the thin film 3. Before the adhesive 15 becomes dry, the sample 8 moistened by the fluid material 9 is put on the spacer 5, the sample 8 is covered with the thin film 3 while applying a tension to the thin film 3 with the surface to which the adhesive 15 has been applied facing downward, and the periphery of the thin film 3 is pressed against the sample stage 2. After fixing of the thin film 3 to the sample stage 2 has been completed, the sample holder 14 is put in the sample chamber. The procedure after that may be the same as in the first embodiment.

EXAMPLE 1

[0063] A sample was sealed on the sample holder of the first embodiment and put in a sample chamber of a field emission scanning electron microscope in which the pressure of the sample chamber was about 1 Pa, and observation was carried out. Details are described below.

[0064] Biphenyltetracarboxylic acid dianhydride (U-Varnish-S 1001) manufactured by Ube Industries, Ltd. was provided as the polyimide precursor. The polyimide precursor was thinly applied to a substrate made of optical glass BK7 and having a thickness of 0.17 mm by spin coating, and the substrate was heated in an electric furnace at a temperature of 450° C. for 20 minutes, followed by peeling the resultant coat from the substrate in water to provide a polyimide thin film having a size of 32×24 mm and a thickness of 300 nm, which was regarded as the thin film 3.

[0065] A product obtained by adding 10% of newborn calf serum, 100 unit/ml of penicillin, and 100 μg/ml of streptomycin to a 1:1 mixed culture medium of Dulbecco's Modified Eagle's Medium and Ham's F12 Nutrient Mixture was used as the fluid material 9. A plurality of fluorescent beads that were made of polystyrene and had a density of 1.05 g/cm.sup.3 and an average particle diameter of 1 μm manufactured by Polysciences Inc. were used as the sample 8.

[0066] An O-ring made of silicone rubber (JIS material code: 4C) conforming to JIS Standard P11 and having an inner diameter of 10.8 mm and a thickness of 2.4 mm was used as the sealing member 4 such that its center line coincided with the center of the radiation field. The spacer 5 was made of melamine sponge, and the pressing member 6 was made of a stainless steel plate. Four bolts 7 were disposed evenly in the circumferential direction.

[0067] FIG. 3 shows the result of observation of the sample 8 by detecting the secondary electrons 13 with the detector 12. An independent fluorescent bead is shown in the upper left portion of the image, an aggregate of four fluorescent beads is shown in the upper right portion, and an aggregate of three fluorescent beads is shown in the lower left portion.

EXAMPLE 2

[0068] A blood tissue of a mouse was used as the sample 8 instead of the fluorescent beads. FIG. 4 shows the result of observation of the sample 8 carried out in the same way as in Example 1 except for the above point. A circular object occupying an area of about ⅔ of the screen is shown at the center of the image and is identified as a red blood cell. Other vesicles and granules derived from the blood tissue are observed on and around the red blood cell.

EXAMPLE 3

[0069] The heart extracted from a mouse was used as the sample 8 instead of the fluorescent beads and sealed on the sample holder 1. A region of a section of the heart tissue in which the sections of the cardiac muscle fibers were oriented in the direction toward the radiation source 10 was searched out and observed. FIG. 5 shows the result of observation of the sample 8 carried out in the same way as in Example 1 except for the above point. It is possible to observe that each bundle of cardiac myofibrils having a diameter of about 1 μm densely gathered to have a hexagonal or quadrangular shape.

EXAMPLE 4

[0070] The heart extracted from a mouse was used as the sample 8 instead of the fluorescent beads and sealed on the sample holder 1. A region of a section of the heart tissue in which the sections of collagen fibers were oriented in the direction toward the radiation source 10 was searched out and observed. FIG. 6 shows the result of observation of the sample 8 carried out in the same way as in Example 1 except for the above point. It is possible to observe that collagen microfibrils having diameters of about 20 to 60 nm that are smaller than 200 nm, which is the resolution of an optical microscope and the length of the scale bar, form bundles having diameters of 400 to 800 nm, and the bundles gather to form collagen fibers.

EXAMPLE 5

[0071] A thermoplastic polyimide varnish (Q-AD-X0516) manufactured by PI R&D Co., Ltd. was provided as the polyimide precursor. The polyimide precursor was thinly applied to a substrate made of optical glass BK7 and having a thickness of 0.17 mm by spin coating at 6,000 rpm, and the substrate was heated on a hot plate at a temperature of 200° C. for 3 minutes, followed by peeling the resultant coat from the substrate in water to provide a polyimide thin film having a size of 32×24 mm and a thickness of 4 μm. A polyimide thin film having a thickness of 1.5 μm was provided in the same way as described above except that a spin speed of 8,000 rpm was employed in the spin coating instead of a spin speed of 6,000 rpm.

[0072] A semiconductor chip was covered with the polyimide thin film having a thickness of 4 μm with a slight tension being applied to the thin film and observed with a scanning electron microscope under a condition of an accelerating voltage of 15 kV, and the resulting image is shown in FIG. 7. The same semiconductor chip was covered with the polyimide thin film having a thickness of 1.5 μm in the same way and observed under the same condition, and the resulting image is shown in FIG. 8. By comparison between the two images, it is shown that the shape of the surface of the semiconductor chip is more accurately reflected in the case of the thin film having a smaller thickness.

EXAMPLE 6

[0073] In the same way as in Example 4, the heart extracted from a mouse was sealed on the sample holder 1, followed by taking a video of the state where the thin film 3 closely adhered to the heart tissue and where the heart and the thin film expanded and contracted. A still image at that moment is shown in the left side of FIG. 9. Changes of the strain over time were measured by dividing each of a relative displacement (distance variation indicated by the solid double-pointed arrow) in the horizontal direction and a relative displacement (distance variation indicated by the dashed double-pointed arrow) in the vertical direction among relative displacements between three appropriate regions on the thin film encircled by circles in the image shown in the drawing by the time-average distance. The measurement result is shown on the right side of the image in FIG. 9. The measurement result shows that the heart tissue was vibrating in all directions such as horizontal, oblique, and vertical directions because changes in the horizontal direction and the vertical direction vibrated while coinciding with each other and being independent from each other. That is, such physical properties (restoring force) and behavior of the heart tissue that the contractile system of the cardiac muscle is oriented in a twisted way and can contract and relax in a plurality of directions is reflected.

EXAMPLE 7

[0074] The heart extracted from a mouse was sealed on the sample holder 1 in the same way as in Example 4. At the time of forming the thin film 3, a step of applying on the substrate a polyimide precursor caused to contain microbubbles by stirring instead of applying the normal polyimide precursor and heating the product on a hot plate at 200° C. for 5 minutes immediately after the application was added before the heating step with an electric furnace, so that spherical markers derived from the microbubbles were obtained on the surface of the thin film 3 as shown in FIG. 12. Among the spherical markers derived from the microbubbles, movement of a marker (at the upper right position near the center of FIG. 12) having a diameter of 1.4 μm was analyzed, and the result is shown in FIG. 13. The drawing shows that the locus of the movement of the marker was able to be measured with high precision. Since relative movement between the marker and the thin film 3 or between the thin film 3 and the heart does not occur, the movement of the marker corresponds to that of the heart directly under the marker.

[0075] The marker may be produced by mixing minute particles with the raw material of the thin film to disperse the particles at the time of forming the film or by printing the markers on the film after formation of the film without mixing minute particles with the raw material of the thin film.

EXAMPLE 8

[0076] A product obtained by adding 10% of newborn calf serum, 100 unit/ml of penicillin, and 100 μg/ml of streptomycin to a 1:1 mixed culture medium of Dulbecco's Modified Eagle's Medium and Ham's F12 Nutrient Mixture was used as the fluid material 9 and sealed on the sample holder 1. A calcium phosphate crystal separated from the fluid material 9 near the thin film 3 was used as the sample 8, and the behavior of the crystal was imaged. The result is shown in FIG. 14. In the drawing, the upper right and lower right images show the same portions as the upper left and lower left images, respectively, after the lapse of time.

[0077] As shown in the drawing, it is possible to observe a crystal floating and moving in the fluid material 9 directly under the thin film 3. That is, it is possible to confirm that the crystal indicated by the white arrows in the drawing moved from the position shown in the upper left image to the position shown in the upper right image. As the separation of a crystal progresses, the thin film 3 is deformed to follow the shape of the crystal, and the shape of the crystal can be three-dimensionally observed. The shape of the crystal can be observed in the lower left portions of the upper left image and the upper right image and in the right portions of the lower left image and the lower right image. In addition, since the crystal in the portion indicated by the white arrow in the lower left image is not observed in the lower right image, it is possible to confirm that the crystal that had been separated near the thin film 3 and deformed the thin film 3 moved away from the thin film 3 and sank into the fluid material 9.

REFERENCE SIGNS LIST

[0078] 1, 14 sample holder [0079] 2 sample stage [0080] 3 thin film [0081] 4 sealing member [0082] 5 spacer [0083] 6 pressing member [0084] 7 bolt [0085] 8 sample [0086] 9 fluid material [0087] 10 radiation source [0088] 11 electron beam (incident electron) [0089] 12 detector [0090] 13 secondary electron [0091] 15 adhesive