JP2008016249A - Sample holder, sample inspection method, sample inspection device, and sample inspection system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample holder, a sample inspection method, a sample inspection device, and a sample inspection system allowing sample inspection to be efficiently carried out by selecting a constituent in a sample, and allowing the sample inspection to be carried out well. <P>SOLUTION: This sample holder having a sample holding space 55 arranged between an electron beam-permeable film 11 and a base 12 facing to the film, and supply passages 54 used for supplying a sample to the sample holding space 55 is provided, in at least either of the supply passages 54 and the sample holding space 55, with a filter structure separating a constituent in the sample. In addition, the sample holder having a sample holding space arranged between two electron beam-permeable films arranged oppositely to each other, and the supply passages for supplying the sample to the sample holding space is provided, in at least either of the supply passage and the sample holding space, with a filter structure separating a constituent in the sample. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides a sample holder, a sample inspection method, a sample inspection apparatus, and a sample capable of selectively inspecting a constituent of a liquid sample (a constituent of a sample containing a liquid) in the inspection of a sample using an electron beam. It relates to the inspection system.

  In a sample inspection apparatus having a configuration of a scanning electron microscope (hereinafter referred to as “SEM”), a sample to be inspected is placed in a sample chamber whose pressure is reduced by evacuation. Then, an electron beam is irradiated onto the sample placed in the sample chamber in such a reduced pressure atmosphere, and secondary signals such as reflected electrons (backscattered electrons) and secondary electrons generated from the sample by the irradiation are detected. Is done.

  Further, even in a sample inspection apparatus having a configuration of a transmission electron microscope (hereinafter referred to as “TEM”), a sample to be inspected is placed in a sample chamber whose pressure is reduced by evacuation. In the case of TEM, the sample placed in the sample chamber is irradiated with an electron beam, and a transmission electron beam transmitted through the sample is detected.

  In sample inspection using such a sample inspection apparatus, the sample is exposed to a reduced-pressure atmosphere in the sample chamber. Therefore, when a sample containing moisture is an object to be inspected, if the sample is placed as it is in a sample chamber in a reduced pressure atmosphere, moisture will evaporate from the sample. In this case, the inspection of the sample in a state where moisture is contained is hindered.

  As one example of performing sample inspection using an SEM without exposing the sample to a reduced-pressure atmosphere in this way, the sample is placed inside a sample container (sample chamber) whose opening (aperture) is sealed by a film, A method of placing this sample container in the sample chamber of the SEM in a reduced pressure atmosphere has been considered.

  Here, the inside of the sample container in which the sample is arranged is not decompressed. The film that covers the opening formed in the sample container can withstand a pressure difference between a reduced pressure atmosphere in the sample chamber of the SEM and an unreduced atmosphere (for example, an atmospheric pressure atmosphere) inside the sample container. Is transmitted (see Patent Document 1).

  When performing sample inspection, an electron beam is irradiated to the sample in the sample container from the outside of the sample container through the film of the sample container disposed in the sample chamber of the SEM in a reduced pressure atmosphere. Reflected electrons are generated from the sample irradiated with the electron beam, and the reflected electrons pass through the film of the sample container and are detected by a reflected electron detector provided in the sample chamber of the SEM. Thereby, the image (SEM image) by SEM will be acquired.

  An example of acquiring an SEM image by irradiating a sample with an electron beam through a film that can withstand a pressure difference between vacuum and atmospheric pressure and detecting reflected electrons generated from the sample is described in Non-Patent Document 1. (Chapter 1 Introduction of the document).

  In addition, a cell structure (sample holder) including a pair of films through which an electron beam is transmitted is placed in the sample chamber of the TEM, the sample is placed in the cell structure, the sample is irradiated with the electron beam, and the sample is transmitted through the sample. Examples of detecting an electron beam are described in Patent Document 2 and Patent Document 3.

Special table 2004-515049 gazette JP 47-24961 A JP-A-6-318445 `` Atmospheric scanning electron microscopy '' Green, Evan Drake Harriman, Ph.D., Stanford University, 1993

  When a constituent contained in a liquid sample containing moisture or the like is an object to be inspected, the sample often contains a plurality of types of constituents having different sizes. At this time, when inspecting a specific component having a predetermined dimension as an inspection target, it is efficient to select the component and perform the inspection.

  However, in the prior art, a component having a specific size in a liquid sample is selected and extracted from a sample container, and the component thus selected and extracted is inspected as an inspection target. The method has not been studied.

  In addition, among the constituents in the liquid sample, when a specific constituent having a property to be adsorbed by a predetermined adsorption function film or other constituents other than the specific constituent is to be inspected, these are included in the sample container. The method of selecting and extracting the test among them has not been studied.

  Further, particularly in the case of sample inspection by SEM, when a liquid sample is placed in the sample container, the constituents of the sample are precipitated in the sample container, and a film through which an electron beam is transmitted (usually on the upper part of the sample container). Are often separated from the In this case, the electron beam that has passed through the film and entered the sample is attenuated until it reaches the component (precipitate), and the reflected electrons generated from the component irradiated with the electron beam also It will decay before reaching the membrane. As a result, an appropriate SEM image cannot be acquired, and sample inspection cannot be performed satisfactorily.

  The present invention has been made in view of the above points, and it is possible to select a component in a sample and perform a sample inspection efficiently, and to perform a sample holding that can perform the sample inspection satisfactorily. An object is to provide a body, a sample inspection method, a sample inspection apparatus, and a sample inspection system.

  A first sample holder according to the present invention includes a sample holding space provided between a film through which an electron beam passes and a base facing the film, and a supply path for supplying a sample to the sample holding space. And a filter structure that separates constituents in the sample in at least one of the supply path and the sample holding space.

  A second sample holder according to the present invention includes a sample holding space provided between two opposed films through which an electron beam is transmitted, and a supply path for supplying a sample to the sample holding space. A sample holder having a filter structure for separating constituents in a sample in at least one of the supply path and the sample holding space.

  A third sample holder according to the present invention includes a sample holding space provided between a film through which an electron beam passes and a base facing the film, a flow path for supplying a sample, and a flow path from the flow path. A sample holder having a supply path for supplying a sample to the sample holding space, and comprising a filter structure for separating components in the sample in at least one of the supply path and the sample holding space. It is characterized by.

  A fourth sample holder according to the present invention includes a sample holding space provided between two opposingly arranged films through which an electron beam passes, a channel to which a sample is supplied, and the sample from the channel. A sample holder having a supply path for supplying a sample to the holding space, and comprising a filter structure for separating components in the sample in at least one of the supply path and the sample holding space. And

A fifth sample holder according to the present invention includes a sample holding space provided between a film through which an electron beam passes and a base facing the film, and a supply path for supplying a sample to the sample holding space. A sixth sample according to the present invention, wherein the width of the sample holding space and the supply path is set to 10 nm to 20 μm, or 10 nm to 5 μm. The holder is a sample holder having a sample holding space provided between two opposingly arranged films through which an electron beam passes, and a supply path for supplying a sample to the sample holding space, The width of the sample holding space and the supply path is set to 10 nm to 20 μm, or set to 10 nm to 5 μm. The first sample inspection method according to the present invention is characterized in that the sample holding body Sample held in the sample holding space A sample is obtained by irradiating an electron beam through the film and detecting a secondary signal generated from the sample by the irradiation or a transmitted electron beam transmitted through the sample.

  A second sample inspection method according to the present invention includes a supply step of supplying a sample to the supply path of the sample holder and supplying the sample to the sample holding space through the supply path, and the sample holding space. An irradiation step of irradiating the sample held on the sample with the electron beam through the film, and a detection of acquiring a sample information by detecting a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample. And a process.

  A sample inspection apparatus according to the present invention is a sample inspection apparatus including the sample holder, and an irradiation unit that irradiates a sample held in the sample holding space of the sample holder with an electron beam through the film. And detecting means for detecting a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample.

  A sample inspection system according to the present invention includes the sample inspection apparatus, and includes a sample pretreatment means, a sample supply means for supplying the pretreated sample to the flow path of the sample holder, and a sample supplied And a moving means for moving the sample holder to the sample inspection apparatus.

  In the present invention, at least one of the supply path and the sample holding space provided in the sample holder is provided with a filter structure for separating components in the sample, or the width of the supply path and the sample holding space. The function of bringing the sample close to the sample holding space by limiting the above is provided.

  Therefore, the sample structure that has passed through the filter structure is supplied to the sample holding space of the sample holder, and the sample held in the sample holding space is used as an inspection target. The sample inspection can be efficiently performed in a state in which the components are selected and extracted in the sample holder. When the film of the sample holder has an adsorption function, it is possible to selectively adsorb the components in the sample to the film and perform the sample inspection efficiently.

  On the other hand, when the filter structure includes an adsorption function film, the sample inspection can be efficiently performed in a state where the constituents in the sample that are not adsorbed by the adsorption function film are selected and extracted in the sample holder.

  Further, when the width of the sample holding space in the direction perpendicular to the surface of the film of the sample holder is specified, the constituent in the selected sample approaches the film in the sample holding space. As a result, the film and the component can be prevented from being separated from each other, and the electron beam reaching the component and the reflected electrons generated from the component can be prevented from being attenuated. A good SEM image or TEM image can be acquired and a good sample inspection can be performed.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 shows a schematic configuration of an embodiment of the present invention. This embodiment comprises an electron source 1, a condenser lens 2, an objective lens 3, an electron beam column 5 comprising a scanning unit 4, an electron beam detector 6, a vacuum chamber 7, and a sample cell (sample holder) 8. Since the sample cell 8 is in contact with the chamber 7 using the O-ring 9, the inside of the chamber 7 can be evacuated or decompressed. Note that grease may be used in place of the O-ring 9. Similarly, grease can be used in the following examples.

  The structure of the sample cell 8 is shown in FIG. The sample cell 8 includes a film 11 that can transmit the electron beam 16, a cell base 12, a cell cap 13, and an adhesive 14 for bonding them. Further, as shown in the figure, an inlet 8a, an outlet 8b, and a supply path 8c are provided in the cell base 12 portion. An opening 13a is formed in the cell cap 13, so that a part of the film 11 is exposed. Under the part of the film 11, a sample holding space 8d is located.

  Pipes are connected to both the inlet 8a and the outlet 8b of the sample cell 8, and the sample is supplied to the supply path 8c of the sample cell 8 through the pipe 22a connected to the inlet 8a. The sample is appropriately discharged from the pipe 22b connected to the outlet 8b of the supply path 8c (see FIG. 3).

  By using such a structure, the sample can be easily put into the cell 8. Further, the focused electron beam 16 can be irradiated and scanned on the sample 15 disposed in the supply path 8c inside the cell 8 through the opening 13a, and the reflected electrons generated by the electron beam 16 can be converted into an electron beam. It can be detected by the detector 6. Thereby, a scanning electron microscope image (SEM image) of the sample 15 can be obtained.

FIG. 4 shows how to make the sample cell 8. Resin raw materials are processed using nanoimprint technology. Thereby, the cell base 12 and the cell cap 13 are produced. Next, the adhesive 14 is applied to the adhesive surface between the cell base 12 and the cell cap 13 using a spray. Finally, the polyimide organic film 11 is bonded to the cell base 12 and the cell cap 13. By adopting such a structure, an inexpensive and stable cell (sample cell) 8 can be produced.
In the cell 8 having such a structure, a sample can be easily introduced into the cell 8 by connecting pipes 22a and 22b to the inlet 8a and the outlet 8b (see FIG. 3). Further, since the cell 8 is arranged in the chamber 7 via the O-ring 9, the sample can be easily exchanged. Thus, a sample in an atmospheric pressure state can be easily observed.

  In the present embodiment, the thickness of the film 11 is set between 10 nm and 1000 nm. If a thin film is used, the scattering of the electron beam 16 can be reduced, so that the resolution of SEM observation can be improved. However, the strength of the film 11 decreases. In contrast, when a thick film is used, the resolution is degraded, but the strength is improved. They were used as needed. More desirably, the film 11 has a thickness of 20 nm to 200 nm.

  In this embodiment, an example in which polyimide is used for the film 11 is shown, but it goes without saying that organic films such as polypropylene and polyethylene can be used. Needless to say, carbon is used.

  In the cell 8 having such a structure, by setting the widths of the sample holding space 8d and the supply path 8c directly below the membrane 11 to 1.1 to 1.2 times the size of the component including the liquid sample, The composition can be brought into close proximity to the membrane. By performing SEM observation in this state, the attenuation of the electron beam in the gap between the film 11 and the component could be suppressed as much as possible, and a high-resolution image could be obtained. A more specific range of width depends on the size of the structure, but is generally 10 nm to 20 μm in consideration of the size of a sample that is generally an observation target in SEM. 10 nm to 5 μm is suitable.

  The present embodiment basically has the same configuration as that of the first embodiment. However, the point which arrange | positioned the X-ray detector 22 differs (refer FIG. 5).

  By arranging the X-ray detector 22, it is possible to detect characteristic X-rays generated from the sample 15 as a result of irradiating the sample 15 with the electron beam 16. Since the energy of the characteristic X-ray is unique to the element, the composition of the region of the sample 15 irradiated with the electron beam 16 is known.

  The present embodiment is basically the same as the first embodiment. However, in order to adhere | attach the film | membrane 11 and a cell main body (cell base 12 and cell cap 13), the point which used the fusion | melting instead of the adhesive agent differs (refer FIG. 6). Since it was set as such a structure, the adhesion process at the time of cell preparation could be simplified.

  The present embodiment is basically the same as the first embodiment. However, the difference is that a conductive organic film 11a is further added to the film 11 (see FIG. 7). The positional relationship between the film 11 and the organic film 11a may be reversed, or the film 11 itself may have conductivity.

  With such a structure, the film 11 can be prevented from being charged. Thereby, more stable observation than Example 1 can be performed.

 The present embodiment is basically the same as the first embodiment. However, the difference is that a mesh 21b is added to the membrane 11 portion of the sample cell 8 (see FIG. 8).

  The method for creating this cell is shown in FIG. A mesh 21b and an opening 21c are formed on the resin by using a nanoimprint technique to form a cell cap 13. Next, the cell base 12 is similarly formed using the nanoimprint technique. Adhesive 20 is applied to the adhesive surface between the cell cap 13 and the cell base 12 using a spray. Finally, the cell cap 13, the membrane 11, and the cell base 12 are bonded via the adhesive 20.

  When the inside of the chamber 7 is evacuated or decompressed, a pressure difference is generated on both sides of the film 11. This pressure difference may cause the membrane 11 to break. On the other hand, by supporting the film 11 with the mesh 21b, the film 11 is hardly broken.

  In the present embodiment, the above mesh structure is adopted, but it goes without saying that a line and space type structure or a honeycomb structure is used. In the present embodiment, an example in which polyimide is used for the film 11 is shown, but it goes without saying that an organic film such as polypropylene or polyethylene can be used. Needless to say, carbon is also used. Furthermore, it goes without saying that adhesion can be performed by fusion as in Example 3, or a conductive film can be added as in Example 4.

The present embodiment is basically the same as the first embodiment. However, the difference is that the sample cell 8 is formed of another material using a process similar to that of a semiconductor device (see FIG. 10).
FIG. 11 shows how to make the sample cell. An oxide film 31 is formed on the Si substrate 30 by thermal oxidation. The thickness of the oxide film 31 is controlled to a thickness that allows transmission of electron beams. Next, a resist pattern (not shown) is formed using a photoresist and lithography. Using this resist pattern as a mask, Si is selectively etched using deep RIE (reactive ion etching). Finally, ashing is performed to remove the resist pattern. The cell cap 13 was formed by these steps.

  Next, the cell base 12 is processed. A thermal oxide film 33 is formed on another Si substrate 32. Similar to the cell cap, the thermal oxide film (silicon oxide) 33 and the Si substrate 32 are etched using a resist pattern (not shown) as a mask. The Si substrate 32 has an appropriate depth so as not to penetrate through the etching. Thereby, the cell base 12 was able to be formed. Finally, the cell cap 13 and the cell base 12 are bonded by glass fusion. Through these steps, the cell 8 was formed.

  In this example, since silicon oxide was used for the film 11, the durability of the film 11 could be improved. Although silicon oxide is used for the film 11 in this embodiment, it goes without saying that other inorganic films such as silicon nitride and boron nitride can be used. It goes without saying that various films can be formed by using chemical or physical film forming methods such as CVD and sputtering.

  The present embodiment is basically the same as the sixth embodiment. However, the difference is that a mesh is added to the film 11 portion of the sample cell 8.

  The method for creating this cell is shown in FIG. A silicon oxide 35 is formed on the surface of the SOI wafer 34 (the surface of the Si layer 34c) including the Si layer 34a, the silicon oxide layer 34b, and the Si layer 34c. Next, a resist pattern 36 is formed on the opposite surface of the SOI wafer 34 (the surface of the Si layer 34a) using a photoresist and lithography. Using this resist pattern 36 as a mask, Si is selectively etched using deep RIE. Thereby, an observation region was formed. Ashing is performed to remove the resist pattern 36.

  Next, a mesh is formed for the processed product. A resist 37 is applied by spraying on the side where the observation part is formed. After the pattern 37a was formed by lithography, a mesh 38 was formed by successively performing oxide film etching and Si selective RIE to form the cell cap 13.

  Further, the supply path 8 c was formed on another Si substrate 39 by RIE using the resist pattern 40 as a mask, and the cell base 12 was obtained. Finally, the cell cap 13 and the cell base 12 were fused. Through these steps, a cell with a mesh could be formed.

  When the inside of the chamber 7 is evacuated or decompressed, a pressure difference is generated on both sides of the film 11. This pressure difference may cause the membrane 11 to break. On the other hand, by supporting the film 11 with this mesh, the film 11 is hardly broken.

  In the present embodiment, the mesh structure is used, but it goes without saying that a line-and-space structure or a honeycomb structure is used. In this embodiment, silicon oxide is used for the film 12, but it goes without saying that other inorganic films such as silicon nitride and boron nitride can be used. Furthermore, it goes without saying that various films can be formed using chemical or physical film forming methods such as CVD and sputtering.

  The present embodiment is basically the same as the first embodiment. However, the difference is that a plurality of cells are formed on the same resin (see FIG. 13). FIG. 14 shows an outline of connecting a pipe 141 for introducing a sample into the cell. With such a configuration, a plurality of samples can be efficiently observed.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. .

  The cell used in this example is the same as in Example 8. When inserting a sample into the cell, extreme caution is required so that the membrane does not break. In particular, it is necessary to adjust the pressure difference between the inside and outside of the cell to be small. For this reason, in this embodiment, the pressure adjustment mechanism 42 was created (see FIG. 15).

  The outline is shown in FIG. The pressure adjustment mechanism 42 includes a plate 43, an O-ring 44, and a pipe 45 connected to a pressure adjustment device (not shown). The pressure adjusting mechanism 42 is connected to the cell 8 as shown in FIG. When the sample is inserted into the cell 8, the pressure adjustment mechanism 42 is used to adjust so that the pressure difference between both sides of the membrane 11 is minimized. By using such a configuration, it was possible to prevent the film 11 from being broken.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided.

  The present embodiment is the same as the ninth embodiment. However, the difference is that a pressure difference is applied to both sides of the membrane 11 using the pressure adjusting mechanism 42 before putting the sample into the cell 8 to check whether the membrane 11 of the cell 8 is damaged.

  Since the ultrathin film 11 is used for the cell 8, the influence of the accuracy of creation and processing of the film 11 is increased. In the worst case, the film 11 of the cell 8 may be destroyed during observation due to these fluctuations. At this time, the sample may be contaminated by scattering inside the lens barrel or the chamber.

  On the other hand, by introducing this procedure, it is possible to examine in advance whether the film 11 is broken due to the fabrication or processing fluctuation of the film 11. Thereby, it was possible to prevent contamination of the lens barrel and the chamber due to the film 11 being damaged inside the chamber after the liquid-containing sample was introduced into the cell 8.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided.

  This example is the same as Example 1 or Example 8. However, it has a filter structure.

  FIG. 17 shows the configuration of the cell 8. 17A is a plan view (including a cross section), FIG. 17B is a cross-sectional view taken along line AA in FIG. 17A, and FIG. 17C is a cross-sectional view taken along line BB in FIG. is there. An inlet (supply port) 51, an outlet (discharge port) 52, a channel 53, a plurality of branch channels (supply channels) 54 branched from the channel 53, and an observation unit (sample holding space) 55 communicating with the branch channel 54. Is done. As shown in FIG. 17, it is characterized by having a structure (filter structure) having a different cross-sectional area (opening cross-sectional area) from the flow path 53 to the observation unit 55. As a means for changing the cross-sectional area, in this embodiment, the width in the direction along the surface of the film 11 of the branch path 54 is changed. Instead of the branch path 54 or in addition to the branch path 54, the opening cross-sectional area of the observation unit 55 may be changed. In each of the following embodiments, the structure in which the opening cross-sectional area of the branch path and / or the observation part is changed corresponds to the filter structure in the present invention.

  With such a configuration, the structure (component) in the sample was separated by the filter structure, and structures having different sizes could be guided to the observation unit 55. As a result, it was possible to selectively observe structures having different sizes.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 The cell using an inorganic film such as silicon oxide, silicon nitride, or boron nitride described in Example 6 or the cell that supports the film with the structure described in Example 5 or 7 can be used. Needless to say. In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided.

  In Example 11, the depth (width in the direction perpendicular to the film 11) of the flow path 53, the branch path 54a, and the branch path 54b was the same. In contrast, in this embodiment, the depths of the branch path 54 a and the branch path 54 b connected to the observation unit 55 are shallower than the depth of the flow path 53. That is, the width in the direction perpendicular to the film 11 in each branch path 54 was reduced.

  A schematic configuration is shown in FIG. 18A corresponds to the AA cross-sectional view of FIG. 17 of Example 11, and FIG. 18B corresponds to the BB cross-sectional view of FIG. 18A. As described above, even when the depth of each branch path 54 is reduced (the width in the direction perpendicular to the film is reduced), the sample enters the observation section 55 by capillary action through each branch path 54.

  With such a configuration, a small structure (a component in the sample) could be selectively guided to the observation unit 55.

  Next, when the thickness of the observation portion 55 immediately below the film 11, that is, the width in the direction perpendicular to the film 11 is 10 nm to 20 μm (1.1 to 1.2 times the size of the component included in the sample) Especially good). As a result, when a sample containing a liquid component is introduced into the cell 8, the structure to be inspected comes close to and close to the film 11, so that it is interposed between the film 11 and the structure. The degree to which the electron beam is scattered by the liquid component or the like can be reduced. As a result, a good scanning electron microscope image could be obtained throughout the observation region. The same applies when the sample contains air.

  Next, the depth of the observation part 55 immediately below the film 11, that is, the branch path width in the direction perpendicular to the film 11 was set to 10 nm to 5 μm. Thus, when a sample containing a liquid component is introduced into the cell 8, the electron beam path in the cell 8 is shortened, so that scattering of the electron beam by the liquid or gas can be reduced. As a result, a good scanning electron microscope image could be obtained throughout the observation region.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided.

  This example is the same as Example 19. However, the difference is that the depth of the branch path 54 communicating with the observation unit 55, that is, the branch path width in the direction perpendicular to the film 11 is set to a plurality of different values.

  In order to show a schematic configuration, FIG. 19 shows a cross-sectional view corresponding to FIG. The depth was changed between the branch path 54c and the branch path 54d. Further, the depth is changed between the branch path 54e and the branch path 54f. By adopting such a structure, it was possible to selectively lead structures having different sizes to the observation unit 55. As a result, it was possible to selectively observe structures having different sizes.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. .

  Modification: This embodiment is the same as the twelfth embodiment. However, the difference is that the cross-sectional area of the branch path 54 changes continuously (see FIG. 20).

  With such a structure, structures having different sizes can be selectively classified. In particular, since the cross-sectional area continuously changes, the structure does not get caught in the branch path 54 as compared with a case where the cross-sectional area changes discretely (discontinuously). For this reason, structures with different sizes can be classified smoothly.

  Further, the depth of the observation section 55, that is, the flow path width in the direction perpendicular to the film 11 can be continuously changed directly below the film 11 (see FIG. 21). In this way, the large structure stays in the deep part, but the small structure moves to the shallow part. Therefore, structures having different sizes can be separated and observed in the observation unit 55 according to the depth of the flow path.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided.

  This example is the same as Example 11. However, the difference is that a plurality of protrusions are arranged as a filter structure in a part of the branch path 54.

  A schematic configuration of the present embodiment is shown in FIG. The cylinder shown in FIG. 23 was provided as a protrusion on each of the branch path 54g and the branch path 54h connected to the observation unit 55. The size and interval of the protrusions were different values.

  By adopting such a configuration, it was possible to guide structures having different sizes to the observation unit 55. As a result, it was possible to selectively observe structures having different sizes. One protrusion may be provided. Moreover, as a protrusion, you may make it arrange | position a protrusion (convex part) other than a cylindrical thing. Further, a structure in which balls (spheres) are arranged may be used.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided.

  This example is the same as Example 11. However, as shown in FIG. 24, the difference is that the inlet 51 and the outlet 52 of the sample are provided on the same surface of the cell 8. Further, after the sample was supplied into the cell 8, the sample was sealed in the cell 8 using an adhesive (not shown).

  By doing in this way, after supplying a sample without connecting a pipe to the cell 8, the cell 8 could be put in the chamber. For this reason, the configuration could be simplified.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided. Needless to say, the cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, or the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment.

  This example is the same as Example 11. However, the difference is that, in addition to the inlet (supply port) 51 and the outlet (discharge port) 52 communicating with the flow path 53, a pulling port 52a is provided.

  A schematic configuration of this embodiment is shown in FIG. A branch path 54 i and a branch path 54 j having a small cross-sectional area are connected to a flow path 53 extending from the inlet 51 to the outlet 52. These branch paths 54 are connected to a lead-out path 61 connected to the pulling port 52a through the observation unit 55 and the communication path 60. By introducing the sample from the inlet 51 toward the outlet 52, the sample enters the flow path 53. A sample usually enters the branch path 54 by capillary action. However, if the cross-sectional area of the branch path 54 is extremely small, a sufficient amount of sample may not be introduced into the observation unit 55. On the other hand, in the present embodiment, a sufficient amount of sample can be put into the observation unit 55 via the branch path 54 by lowering the pressure of the drawing port 52a with respect to the inlet 51 and the outlet 52.

  By adopting such a configuration, the sample can be introduced into the observation unit 55 via the branch path 54 having a small cross-sectional area, so that a small structure can be observed.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided. The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. -Needless to say, the outlet is provided above the cell (on the same surface).

  This example is the same as Example 16. However, the difference is that, as shown in FIG. 26, filters 62a, 62b, 62c, and 62d are provided in the branch passage 54 having a small cross-sectional area connected to the flow path 53, respectively. These filters consisted of protrusions shown in FIG. As the filters 62a, 62b, 62c, and 62d, filters having different protrusion intervals were employed.

  By adopting such a configuration, it was possible to guide structures having different sizes to the observation unit 55. As a result, it was possible to selectively observe structures having different sizes.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided. The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, 51 and the outlet 52 can be provided on the upper side of the cell.

  This example is the same as Example 17. However, the difference is that other filters 63a, 63b, 63c, 63d are installed in the communication path 60 located between the observation unit 55 and the extraction path 61 (see FIG. 27). These filters were composed of protrusions as in Example 17. However, the other filter 63 is characterized in that it is configured with protrusions whose intervals are narrower than those of the filter 62.

  The operation of the present embodiment is as follows. First, similarly to Example 17, a sample is introduced from the inlet 51 toward the outlet 52, and the channel 53 is filled with the sample. Next, the inlet 52 a is decompressed, and the sample is filled in the branch path 54 communicating with the observation unit 55. At this time, structures having different sizes enter under the respective observation units 55 by the filter 62.

  Next, the pressure at the inlet 51, outlet 52, and outlet 52 a is made substantially equal, and the sample is discharged from the outlet 52. At this time, the sample remains in the observation unit 55 and the sample is removed from the flow path 53. By reducing the pressure of the drawing port 52a in this state, only the liquid component can be discharged from the drawing port 52a through the communication path 60 and the drawing path 61 through the filter 63. Thereby, only the structure without a liquid component can be selectively left in the observation unit 55.

  The electron beam 16 incident on the film 11 is scattered by the liquid when there is a liquid inside the cell 8. On the other hand, in this embodiment, since the liquid was selectively removed and only the structure could be arranged in the observation unit 55, scattering by the liquid could be eliminated. For this reason, since the scattering of the electron beam 16 is reduced, the resolution during observation can be improved.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided. The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, the outlet 51 is attached to the upper side of the cell, or the outlet 52a can be attached as in the sixteenth embodiment.

  This example is the same as Example 18. However, it differs in that saturated water vapor is introduced into the cell 8 after the liquid is removed.

  After removing the liquid component in the same manner as in Example 18, saturated water vapor was introduced into the flow path 53 from the inlet 51, and the pressure at the outlet 52a was adjusted so that saturated water vapor also entered the observation unit 55. By adopting such a procedure, it was possible to prevent the sample from drying.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . In this embodiment, a plurality of observation units (sample holding spaces) are provided, but one observation unit may be provided. The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, the outlet 51 is attached to the upper side of the cell, or the outlet 52a can be attached as in the sixteenth embodiment.

  In this embodiment, the sample 15 is arranged on the upper side of the film 11 of the cell 8 and the electron beam 16 can be irradiated from the lower side of the film 11 of the cell 8. FIG. 28 shows a schematic configuration of the present embodiment. The cell 8 was placed on the chamber 7 so that the portion of the film 11 through which the electron beam 16 passes was on the lower side (the side pulled by gravity). A sample is disposed in the flow path of the cell 8 and inside the observation section. The electron beam column 5 was arranged so that the electron beam 16 could be irradiated from below to above so that the sample 15 could be irradiated with the electron beam 16 through the film 11. By using this structure, the sample 15 inside the cell 8 can be irradiated and scanned with the electron beam 16 focused from below, and the reflected electrons generated by the electron beam 16 are detected by the electron beam detector 6. It can be detected. Thereby, a scanning electron microscope image of the lower part of the sample can be obtained.

  In the cell 8 having such a structure, the sedimentary structure inside the sample 15 contacts the film 11 that transmits the electron beam 16 by precipitation. By irradiating / scanning the focused electron beam 16 from the lower part of the film 11, the focused electron beam 16 can be irradiated / scanned on the sedimentary structure. By forming an image using the reflected electrons generated thereby, the structure precipitated in the sample 15 can be observed with a scanning electron microscope while maintaining atmospheric pressure.

  In this example, the thickness of the film was between 10 nm and 1000 nm. When a thin film is used, the electron beam scattering can be reduced, so that the resolution of SEM observation can be improved. However, the strength of the film 11 decreases. In contrast, when a thick film is used, the resolution is degraded, but the strength is improved. They were used as needed. More desirably, the thickness of the film 11 is 20 to 200 nm.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . Needless to say, a plurality of cells can be integrated as in the eighth embodiment. The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, the outlet 51 is attached to the upper side of the cell, or the outlet 52a can be attached as in the sixteenth embodiment. It goes without saying that the liquid can be removed as in Example 18 or saturated water vapor can be introduced as in Example 19.

  In this example, the sample cell 8 was placed inside the vacuum chamber 7 (see FIG. 29). A stage 9b for moving the cell 8 was installed. Furthermore, the cell 8 described in Example 15 was adopted, and the inlet 51 and the outlet 52 were sealed before entering the vacuum chamber 7.

  With such a structure, the stage 9b for moving the sample 15 could be easily created.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . Needless to say, a plurality of cells can be integrated as in the eighth embodiment. The cross-sectional area of the branch path 14 may be changed as in the eleventh embodiment or the twelfth embodiment, the protrusion may be disposed on a part of the branch path 14 as in the fourteenth embodiment, or the inlet may be used as in the nineteenth embodiment. Needless to say, the outlet 51 is attached to the upper side of the cell, or the outlet 52a can be attached as in the sixteenth embodiment. Needless to say, liquid can be removed as in Example 18, saturated water vapor can be applied as in Example 19, or electron beam can be irradiated and scanned from below as in Example 20.

  This example is the same as Example 21. However, the depth of the observation part 55 of the cell 8, that is, the width in the direction perpendicular to the film 11 is 10 nm to 20 μm (more desirably 10 nm to 5 μm), and the stage 9 b that can tilt the sample is adopted. Are different (see FIG. 30).

  By reducing the depth of the observation unit 55, the structure in the sample 15 is difficult to move inside the observation unit 55 even if the sample 15 in the observation unit 55 is tilted. As a result, tilt observation was easy. As a result, a three-dimensional structure of the structure could be obtained. In particular, it is possible to construct a three-dimensional stereoscopic image (computer topography) by reconstructing the contour of the structure in the sample from a plurality of tilt images using a computer.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . Needless to say, a plurality of cells can be integrated as in the eighth embodiment. The cross-sectional area of the branch path 14 may be changed as in the eleventh embodiment or the twelfth embodiment, the protrusion may be disposed on a part of the branch path 14 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, the outlet 51 is attached to the upper side of the cell, or the outlet 52a can be attached as in the sixteenth embodiment.

  This example is the same as Example 11. However, the difference is that an index pattern indicating a dimension reference as a dimension index is formed on a part of the cell 8.

The configuration of the cell 8 according to this embodiment is shown in FIG. A standard pattern (index pattern) 200 of length was formed on the surface of the cell 8 and in the vicinity of each observation portion 55. An example of a standard length pattern is shown in FIG. It consists of lines and spaces with different intervals and periods.
When observing the sample, an image of such a length standard is acquired, and the exact dimensions of the structure in the sample can be obtained by comparing and referring to the image of the structure in the sample.

  In this embodiment, all processes including observation and inspection described in Embodiments 1 to 23 can be automatically performed.

  An outline of an apparatus for automatic observation is shown in FIG. The flow of automatic observation is shown in FIG. First, automatic sample pretreatment is performed. After the sample is automatically put into the first pretreatment unit 71 using the autopipette 70, the fixing solution is put into the first pretreatment unit 71 using the autopipette. In this state, while stirring as necessary, wait until the fixing process is completed. The temperature during processing was maintained at 37 ° C. Next, the sample after the fixing process is transferred to the second pretreatment unit 72 using an autopipette. Then, after discarding unnecessary supernatant, the sample is washed.

  Thereafter, the conductive staining solution is put into the second pretreatment unit 72 using an autopipette. In this state, it waits for the dyeing process to be completed while stirring as necessary. Discard unnecessary supernatant and wash the sample. The above is the pretreatment process.

  Here, glutaraldehyde, formaldehyde, formalin, or the like can be used as the fixing solution, and an osmium acid aqueous solution, tannic acid aqueous solution, phosphotungstic acid aqueous solution, or the like can be used as the conductive staining solution, and these are properly used depending on the purpose. Both are chemical solutions for fixation and conductive staining developed for SEM and TEM for vacuum observation.

  Next, the sample is automatically put into the cell 8 by using an autopipette (sample setting to the sample cell). By connecting an autopipette to a pipe connected to the inlet of the cell 8, a sample can be put into the flow path of the cell 8. A pipe for flowing the overflowed sample to the waste liquid tank 73 is connected to the outlet of the cell 8 (see FIG. 33).

  Then, after the cell 8 is moved directly below the O-ring 9 using the transfer unit 74 (automatic loading of the sample cell), the inside of the chamber 7 is evacuated. The cell 8 is moved using the sample moving mechanism 9d, and the sample in the cell 8 is moved to the observation position (automatic movement to the observation position). After the electron beam 16 is automatically adjusted in focus and stigma (automatic SEM adjustment), an SEM image is acquired (automatic SEM observation). The acquired image is automatically stored in the image server 75 for each observation sample and pre-processing (image transfer to the server). The image transfer from the sample transfer to the server is repeated as many times as necessary. In the figure, 75a is a computer and 75b is a main body controller.

  By using such a method, it was possible to automate all observation steps including sample pretreatment. As a result, human labor has been saved.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . Needless to say, a plurality of cells can be integrated as in the eighth embodiment. The cross-sectional area of the branch passage 14 is changed as in the eleventh embodiment or the twelfth embodiment, or the protrusion 51 is arranged on a part of the branch passage 14 as in the fourteenth embodiment. Needless to say, the outlet 52 can be mounted on the upper side of the cell, the pulling port 52a can be mounted as in the sixteenth embodiment, or the tilt mechanism can be mounted as in the twenty-second embodiment.

  This example is the same as Example 24. However, the difference is that the pressure control described in the ninth embodiment can be automatically performed.

  After pre-processing in the same manner as in Example 24, the sample is supplied to the cell 8 using an autopipette and a pipe connected to the inlet of the cell 8. At this time, the pressure adjusting mechanism described in Example 9 is used. 42 was automatically connected to the cell and automatically adjusted so that the pressure difference between the two sides of the membrane 11 when the sample was put into the cell 8 did not increase. After putting the sample into the cell 8, automatic conveyance and automatic observation were performed in the same manner as in Example 24.

  By performing this automation, it was possible to prevent the film 11 of the cell 8 from being broken and to save human labor.

  This example is similar to Example 24. However, the difference is that the fluorescent marker is added to the sample at the beginning of the pretreatment, and the fluorescence detector is added to the detector (see FIG. 35). Specifically, an antibody to which a fluorescent marker was added was added to the sample by using an antigen-antibody reaction.

  When the sample is irradiated with a focused electron beam, the fluorescent marker causes cathodoluminescence. The light generated thereby is detected by a fluorescence detector. In this embodiment, at the time of observation, first, reflected electrons are detected and the focus and stigma of the electron beam 15 are adjusted. Then, a fluorescent location is specified using a fluorescence detector, and finally reflected again. A backscattered electron image was acquired using an electron detector. By adopting such a process, it can be understood which part is fluorescent. As a result, the site where the fluorescent marker reacts could be identified. Moreover, the vicinity of the part to which the fluorescent marker was added could be observed in detail with a backscattered electron image.

  This example is similar to Example 24. However, the difference is that a metal marker is added to the sample at the beginning of the pretreatment (for example, using a protein in which protein A is adsorbed to gold particles). Specifically, the antibody to which the gold marker was added was added to the sample by using an antigen-antibody reaction. At this time, gold particles having a diameter of 10 nm to 50 nm were used as markers.

  When the sample is irradiated with a focused electron beam, the metal marker has a larger atomic weight than that of the living body, and therefore can be clearly observed as a reflected electron image or a transmitted electron image.

  In this embodiment, automatic image processing and image classification are added to Embodiment 24 (see FIG. 36).

  After the sample was automatically preprocessed and automatically observed in the same manner as in Example 24, the acquired images were classified by the following method. First, feature amounts are extracted from an image (image processing). In this example, specific structures such as the structure size, eccentricity, edge roughness, density, surface morphology, and protrusions (protrusions on the structure surface) were extracted. Samples were automatically classified according to the combination of these feature quantities. Repeat as many times as necessary from sample movement to classification.

  As for the relationship between the feature quantity and the classification method, the classification accuracy was improved by learning a large number of samples in advance using the result of manual classification. This automation has saved human effort.

This embodiment is basically the same as the embodiment 24. However, not only the observation from one direction but also the point that the observation is automatically performed from a plurality of directions using an apparatus in which the lens barrel is inclined is different. Using these images, the three-dimensional shape of the structure in the sample can be calculated.
Since it is necessary to put the cell 8 in the vacuum chamber 7, as shown in FIG. 37, after supplying the sample to the cell 8, the inlet and outlet were sealed with adhesives 78 and 79. The cell 8 can be introduced into the vacuum chamber 7 using a transfer robot similar to that in Example 24. By automating observations from multiple directions, human labor was saved.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . Needless to say, a plurality of cells can be integrated as in the eighth embodiment. The cross-sectional area of the branch path is changed as in Example 11 and Example 12, or the protrusions are arranged on a part of the branch path as in Example 14, and the inlet / outlet is a cell as in Example 19. Needless to say, it is possible to attach to the upper side of the head, or to attach a pulling mechanism as in the sixteenth embodiment, or to attach a tilting mechanism as in the twenty-second embodiment.

  This embodiment is basically the same as the embodiment 29. However, the difference is that the three-dimensional shape construction process described in the embodiment 29 can be automatically performed (see FIG. 38). This automation has saved human effort.

  In this embodiment, automatic image recognition and image classification are added to the embodiment 30.

  Similar to Example 30, a three-dimensional shape was automatically obtained after automatic pretreatment and automatic observation of the sample. A feature amount is extracted from the three-dimensional shape. In this example, specific structures such as the size of the structure, three-dimensional eccentricity, edge roughness, density, surface form, and protrusions (projections on the structure surface) were extracted. Samples were automatically classified according to the combination of these feature quantities.

  As for the relationship between the feature quantity and the classification method, the classification accuracy was improved by learning a large number of samples in advance using the result of manual classification. This automation has saved human effort.

  This example is the same as Example 31. However, the difference is that the etiology can be automatically determined using the classification result by particularly observing blood cells and using the relationship between the classification result and the etiology of the three-dimensional shape. This automation has saved human effort. Other than blood cells, the same effect could be obtained using living cells.

  This example is the same as Example 31. However, by observing the powder and using the relationship between the result of classifying the three-dimensional shape and the past measurement result, it is possible to automatically determine whether the product is good or defective using the classification result. The difference is that. In this example, the toner for copying, cosmetics, pigments, and graphite were inspected as powders. This automation has saved human effort.

  FIG. 39 shows the structure of the cell used in this example. A sample inlet 81, an outlet 82, a channel 83, a branch channel (supply channel) 84, and an observation unit (sample holding space) 85 were provided. Sample inlet and outlet pipes can be connected to these inlet 81 and outlet 82 as in the first embodiment. A channel 83 is provided between the inlet 81 and the outlet 82. The sample is injected from the inlet 81 and extracted from the outlet 82 through the flow path.

  A plurality of branch paths 84 branching from the flow path 83 are provided, and each branch path 84 communicates with the observation unit 85. Each observation unit 85 is constituted by a gap between two films arranged opposite to each other that transmit an electron beam. Thereby, the sample introduced into each observation part 85 is irradiated with an electron beam through one film | membrane. The transmitted electron beam that has passed through the sample reaches the outside of the cell through the other film. Using this observation unit 55, scanning transmission electron microscope observation or transmission electron microscope observation of the sample disposed therein can be performed.

  A method of creating this cell will be described (see FIG. 40 (a)). The resin 91 and the resin 92 were each provided with a flow path, a branch path, and an observation unit using nanoimprint technology. Adhesives 93 and 94 were applied to the bonding surfaces of the resin 91 and the resin 92 by spraying, and the films 95 and 96 were bonded to the respective resins 91 and 92.

  An adhesive 97 is applied to one film 96 so that the two films 95 and 96 can be bonded with a thin gap, and then both the films 95 and 96 are bonded. Only a necessary part was applied as an adhesive by using a metal mask 98. The gap between the films can be controlled by controlling the amount of adhesive, viscosity, and pressure at the time of bonding. Alternatively, a gap can be controlled by inserting a spacer between the two films 95 and 96. After these adhesions, the excess film was cut.

  Further, the film near the center of the flow path is removed using another metal mask 99 and the ion beam 100. After forming the portion 101 that becomes the cap of the flow path by nanoimprinting, the adhesive 102 was applied by spraying and then adhered to the resin 91. A cell was formed by these steps.

  An overall configuration for observing a scanning transmission electron image is shown in FIG. An electron beam column 5 comprising an electron source 1, a condenser lens 2, an objective lens 3, a scanning unit 4, a vacuum chamber 7, an upper electron beam detector 103, an on-axis electron beam detector 104, an off-axis electron beam detector 106, It consists of a sample cell 108 and a stage 107.

  By using this structure, it is possible to irradiate and scan the electron beam 16 focused on the sample inside the observation unit 85 of the cell 108 and to detect the reflected electrons generated by the electron beam 16 with the upper electron beam detector 103. it can. Thereby, a scanning electron microscope image of the sample can be obtained. Simultaneously, by using the on-axis electron beam detector 104 and the off-axis electron beam detector 105, a scanning transmission electron beam microscope image can be obtained.

  In this embodiment, the thickness of the films 95 and 96 is set between 10 nm and 1000 nm. If a thin film is used, the scattering of the electron beam 16 can be reduced, so that the resolution of SEM observation can be improved. However, the strength of the film is reduced. In contrast, when a thick film is used, the resolution is degraded, but the strength is improved. They were used as needed. More desirably, the thickness of the films 95 and 96 is 20 nm to 200 nm.

  In the present example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3 or the conductive film described in Example 4 was used. Needless to say, you can use the cells you had. Needless to say, the inlet / outlet can be mounted on the upper side of the cell as in the fifteenth embodiment.

  The same effect could be obtained even with a sample holder having a simpler structure than the above example. FIG. 40B shows the structure. There is one sample holding space 85, and a supply path 84 is connected to the sample holding space 85. In such a sample holder, by setting the widths of the sample holding space 85 and the supply path 84 immediately below the membrane 95 to 1.1 to 1.2 times the size of the constituent contained in the liquid sample, The composition can be brought into close proximity to the membrane. TEM observation was performed in this state, and a high resolution image could be obtained. A more specific range of the width depends on the size of the component, but is generally 10 nm to 20 μm in consideration of the size of a sample to be generally observed with a TEM or SEM. For this, 10 nm to 5 μm is suitable. The method for making the sample holder is the same as in FIG.

  This embodiment is basically the same as the embodiment 34. However, the difference is that the X-ray detector 22 is arranged (see FIG. 42).

  By arranging the X-ray detector 22, it is possible to detect characteristic X-rays generated from the sample as a result of irradiating the sample with the electron beam 16. Since the energy of the characteristic X-ray is unique to the element, the composition of the region irradiated with the electron beam 16 is known.

  When inserting a sample into the cell, extreme caution is required so that the membrane does not break. In particular, it is necessary to adjust the pressure difference between the inside and outside of the cell to be small. For this purpose, a pressure adjustment mechanism 142 was created. The basic configuration is the same as in Example 9, except that the adjustment mechanism is arranged on both sides of the cell because the membrane is on both sides of the sample.

  The outline is shown in FIG. The pressure adjustment mechanism 142 includes a plate 143, an O-ring 144, and a pipe 145 connected to a pressure adjustment device (not shown). The pressure adjusting mechanism 142 is connected to the cell 108 as shown in FIG. When the sample is inserted into the cell 108, adjustment is performed using the pressure adjusting mechanism 142 so that the pressure difference between both sides of the membrane is minimized. By using such a configuration, it was possible to prevent the film from being broken.

  In the present example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3 or the conductive film described in Example 4 was used. Needless to say, you can use the cells you had. Needless to say, the inlet 51 and the outlet 52 can be mounted on the upper side of the cell as in the fifteenth embodiment.

  This example is the same as Example 34. However, as in Example 12, the depth of the branch path 84 connected to the observation unit 55 was shallower than the depth of the flow path 83. That is, the difference is that the width of the branch path 84 in the direction perpendicular to the film has a plurality of different values.

  FIG. 44 shows the configuration of the cell. 44A is a plan view (including a cross section), and FIG. 44B is a cross-sectional view taken along line BB in FIG. 44A.

  The depth of the branch path 84a and the branch path 84b, that is, the width in the direction perpendicular to the film was changed. Further, the depth, that is, the width in the direction perpendicular to the film was changed between the branch path 84c and the branch path 84d. By adopting such a structure, it was possible to selectively lead structures having different sizes to the observation part. As a result, it was possible to selectively observe structures having different sizes.

  In the present example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3 or the conductive film described in Example 4 was used. Needless to say, you can use the cells you had. Needless to say, the inlet 51 and the outlet 52 can be mounted on the upper side of the cell as in the fifteenth embodiment.

  This example is the same as Example 34. However, the difference is that a plurality of protrusions are arranged on a part of the branch path in the same manner as in Example 14.

  A schematic configuration of the present embodiment is shown in FIG. A column shown in FIG. 46 is provided as a projection on each of the branch path 84e and the branch path 84f connected to the observation unit 55. By adopting such a configuration, it was possible to guide structures having different sizes to the observation part. As a result, it was possible to selectively observe structures having different sizes.

  In the present example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3 or the conductive film described in Example 4 was used. Needless to say, you can use the cells you had. Needless to say, the inlet 51 and the outlet 52 can be mounted on the upper side of the cell as in the fifteenth embodiment, or the sectional area of the branch path can be changed as in the eleventh and twelfth embodiments.

  This example is the same as Example 34. However, in the same manner as in the sixteenth embodiment, in addition to the inlet 51 and the outlet 52 communicating with the flow path, the difference is that a drawing port 52a is added.

  A schematic configuration of the present embodiment is shown in FIG. A branch path 54 i and a branch path 54 j having a small cross-sectional area are connected to a flow path 53 extending from the inlet 51 to the outlet 52. These branch paths 54 are connected to a lead-out path 61 connected to the pulling port 52a through the observation unit 55 and the communication path 60. By introducing the sample from the inlet 51 toward the outlet 52, the sample enters the flow path 53. A sample usually enters the branch path 54 by capillary action. However, if the cross-sectional area of the branch path 54 is extremely small, a sufficient amount of sample may not be introduced into the observation unit 55. On the other hand, in the present embodiment, a sufficient amount of sample can be put into the observation unit 55 via the branch path 54 by lowering the pressure of the drawing port 52a with respect to the inlet 51 and the outlet 52.

  By adopting such a configuration, the sample can be introduced into the observation unit 55 via the branch path 54 having a small cross-sectional area, so that a small structure can be observed.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, 51 and the outlet 52 are provided on the upper side (on the same surface) of the cell.

  This example is the same as Example 39. However, the difference is that filters 62a, 62b, 62c, and 62d are provided in the branch passage 54 having a small cross-sectional area connected to the flow path 53, as shown in FIG. These filters consisted of protrusions shown in FIG. As the filters 62a, 62b, 62c, and 62d, filters having different protrusion intervals were employed.

  By adopting such a configuration, it was possible to guide structures having different sizes to the observation unit 55. As a result, it was possible to selectively observe structures having different sizes.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, the outlet 51 is provided on the upper side of the cell, or the outlet 52a is provided as in the sixteenth embodiment.

  This example is the same as Example 40. However, the difference is that other filters 63a, 63b, 63c, 63d are installed in the communication path 60 located between the observation unit 55 and the extraction path 61 (see FIG. 49). These filters were formed of protrusions as in Example 40. However, the other filter 63 is characterized in that it is configured with protrusions whose intervals are narrower than those of the filter 62.

  The operation of the present embodiment is as follows. First, as in Example 40, a sample is introduced from the inlet 51 toward the outlet 52, and the flow path 53 is filled with the sample. Next, the inlet 52 a is decompressed, and the sample is filled in the branch path 54 communicating with the observation unit 55. At this time, structures having different sizes enter under the respective observation units 55 by the filter 62.

  Next, the pressure at the inlet 51, outlet 52, and outlet 52 a is made substantially equal, and the sample is discharged from the outlet 52. At this time, the sample remains in the observation unit 55 and the sample is removed from the flow path 53. By reducing the pressure of the drawing port 52a in this state, only the liquid component can be discharged from the drawing port 52a through the communication path 60 and the drawing path 61 through the filter 63. Thereby, only the structure without a liquid component can be selectively left in the observation unit 55.

  The electron beam 16 incident on the film 11 is scattered by the liquid when there is a liquid inside the cell 8. On the other hand, in this embodiment, since the liquid was selectively removed and only the structure could be arranged in the observation unit 55, scattering by the liquid could be eliminated. For this reason, since the scattering of the electron beam 16 is reduced, the resolution during observation can be improved.

  In this example, an example using a cell containing a resin as in Example 1 was shown. However, a cell adhered by fusion as in Example 3, a cell using the conductive film described in Example 4 Needless to say, a cell using an inorganic film such as silicon oxide or silicon nitride described in Example 6 or a cell supporting a film with a structure described in Example 5 or 7 can be used. . The cross-sectional area of the branch path 54 may be changed as in the eleventh and twelfth embodiments, the protrusions may be disposed on a part of the branch path 54 as in the fourteenth embodiment, or the inlet may be used as in the fifteenth embodiment. Needless to say, the outlet 51 is attached to the upper side of the cell, or the outlet 52a can be attached as in the sixteenth embodiment.

  This example is the same as Example 41. However, it differs in that saturated water vapor is introduced into the cell 8 after the liquid is removed.

  After removing the liquid component in the same manner as in Example 41, saturated water vapor was introduced into the flow path 53 from the inlet 51, and the pressure at the outlet 52a was adjusted so that saturated water vapor also entered the observation unit 55. By adopting such a procedure, it was possible to prevent the sample from drying.

  This embodiment is basically the same as the embodiment 34. However, the difference is that a mesh is added to the film portion of the sample cell. The manufacturing method is the same as in Example 5. Also, the same creation method as that in the seventh embodiment may be used.

  FIG. 50A shows a cross-sectional structure of a sample cell to which a mesh 501 is added. Similarly to Example 34, the resin 91 and the resin 92 were each provided with a flow path, a supply path, and an observation unit using nanoimprint technology. At this time, a mesh 501 was also formed. The subsequent steps are the same as in Example 34, and the membranes 95, 96 are fixed to the resins 91, 92 using adhesives 93, 94, 97.

  When the inside of the chamber is evacuated or decompressed, a pressure difference is generated on both sides of the membrane. This pressure difference may break the membrane. On the other hand, by supporting the membrane with the present mesh, the membrane is difficult to break.

  In the present embodiment, the above mesh structure is adopted, but it goes without saying that a line and space type structure or a honeycomb structure is used. In this embodiment, an example in which polyimide is used for the film is shown, but it goes without saying that organic films such as polypropylene and polyethylene are used. Needless to say, carbon is also used. Furthermore, it goes without saying that adhesion can be performed by fusion as in Example 3, or a conductive film can be added as in Example 4.

  Needless to say, the embodiments of the above-described embodiments can be applied.

  This embodiment is basically the same as the embodiment 34. However, the difference is that the sample cell is formed of another material using a process similar to that of a semiconductor device.

FIG. 51 shows how to make the sample cell. A portion 511 of the Si substrate is processed by dry etching using a resist pattern formed by photolithography as a mask. Next, an oxide film 512 is formed on the processed surface by thermal oxidation. Again, using a resist pattern formed by photolithography as a mask, processing is performed using dry etching. Thereby, the flow path 513 and the membrane 514 are formed.
An observation region was formed on the opposite side of the processed surface by the same method. At this time, Si was selectively etched using RIE. Two sets of these were bonded using glass attraction so that the films were close to each other. A cell was formed by these steps.
In this example, since silicon oxide was used for the film, the durability of the film could be improved. In this embodiment, silicon oxide is used for the film, but it goes without saying that other inorganic films such as silicon nitride and boron nitride can be used. It goes without saying that various films can be formed using a chemical or physical film forming method such as CVD or sputtering.

  Needless to say, the embodiments described above can also be applied to this embodiment.

  Also in this embodiment, it is needless to say that a mesh structure similar to that in Embodiment 43 can be formed as shown in FIG. In this case, the film is unlikely to be broken as in Example 43.

  This example is the same as Example 34. However, the difference is that the depth of the observation part of the cell, that is, the width in the direction perpendicular to the film is set to 10 nm to 20 μm (more desirably 10 nm to 5 μm), and a stage that can tilt the sample is adopted (FIG. 52). reference).

  By reducing the depth of the observation unit 85, the structure in the sample 15 is difficult to move inside the observation unit 55 even if the sample 15 in the observation unit 85 is tilted. As a result, tilt observation was easy. As a result, a three-dimensional structure of the structure could be obtained. In particular, it is possible to construct a three-dimensional stereoscopic image (computer topography) by reconstructing the contour of the structure in the sample from a plurality of tilt images using a computer.

  Needless to say, the embodiments described above can also be applied to this embodiment.

  This embodiment is the same as the embodiment 45. However, the difference is that the depth of the observation part of the cell, that is, the width in the direction perpendicular to the film is set to 5 μm or less, and a stage capable of tilting the sample is employed.

  By reducing the depth of the observation unit 55, the structure in the sample 15 is difficult to move inside the observation unit 55 even if the sample 15 in the observation unit 55 is tilted. As a result, tilt observation was easy. As a result, a three-dimensional structure of the structure could be obtained. In particular, it is possible to construct a three-dimensional stereoscopic image (computer topography) by reconstructing the contour of the structure in the sample from a plurality of tilt images using a computer.

  Needless to say, the embodiments described above can also be applied to this embodiment.

  In this embodiment, all processes including the observation and inspection can be automatically performed.

  An outline of an apparatus for automatic observation is shown in FIG.

  First, automatic sample pretreatment is performed. After the sample is automatically put into the first pretreatment unit 71 using the autopipette 70, the fixing solution is put into the first pretreatment unit 71 using the autopipette. In this state, while stirring as necessary, wait until the fixing process is completed. The temperature during processing was maintained at 37 ° C. Next, the sample after the fixing process is transferred to the second pretreatment unit 72 using an autopipette. Then, after discarding unnecessary supernatant, the sample is washed.

  Thereafter, the conductive staining solution is put into the second pretreatment unit 72 using an autopipette. In this state, it waits for the dyeing process to be completed while stirring as necessary. Discard unnecessary supernatant and wash the sample. The above is the pretreatment process.

  Here, glutaraldehyde, formaldehyde, formalin, or the like can be used as the fixing solution, and an osmium acid aqueous solution, tannic acid aqueous solution, phosphotungstic acid aqueous solution, or the like can be used as the conductive staining solution, and these are properly used depending on the purpose. Both are chemical solutions for fixation and conductive staining developed for SEM and TEM for vacuum observation.

  Next, the sample is automatically placed in the cell 8 using an autopipette. By connecting an autopipette to a pipe connected to the inlet of the cell 8, a sample can be put into the flow path of the cell 8. A pipe for flowing the overflowed sample to the waste liquid tank 73 is connected to the outlet of the cell 8 (see FIG. 33).

  And after arrange | positioning the cell 8 in the chamber 7 using the conveyance unit 74, the inside of the chamber 7 is evacuated. Using the sample moving mechanism 9d, the cell 8 is moved, and the sample in the cell 8 is moved to the observation position. After automatically adjusting the focus of the electron beam 16 and the stigma, an SEM image or a TEM image is acquired. The acquired image is automatically stored in the image server 75 for each observation sample and preprocessing. The image transfer from the sample transfer to the server is repeated as many times as necessary.

  By using such a method, it was possible to automate all observation steps including sample pretreatment. As a result, human labor has been saved.

  This example is the same as Example 47. However, the difference is that the pressure control can be automatically performed.

  After pre-processing in the same manner as in Example 47, the sample is supplied to the cell 8 using an autopipette and a pipe connected to the inlet of the cell 8. At this time, the pressure adjustment mechanism described in Example 9 is used. 42 was automatically connected to the cell and automatically adjusted so that the pressure difference between the two sides of the membrane 11 when the sample was put into the cell 8 did not increase. After putting the sample in the cell 8, automatic conveyance and automatic observation were performed.

  By performing this automation, it was possible to prevent the film 11 of the cell 8 from being broken and to save human labor.

  The present embodiment is different in that a fluorescent marker is added to the sample at the beginning of the pretreatment and a fluorescent detector is added to the detector. Specifically, an antibody to which a fluorescent marker was added was added to the sample by using an antigen-antibody reaction.

  When the sample is irradiated with a focused electron beam, the fluorescent marker causes cathodoluminescence. The light generated thereby is detected by a fluorescence detector. In this embodiment, at the time of observation, first, reflected electrons are detected and the focus and stigma of the electron beam 16 are adjusted. Then, a fluorescent spot is specified by using a fluorescence detector, and finally reflected again. A backscattered electron image was acquired using an electron detector. By adopting such a process, it can be understood which part is fluorescent. As a result, the site where the fluorescent marker reacts could be identified. In addition, the part where the fluorescent marker is added

  This embodiment is different in that a metal marker addition process is performed on the sample at the beginning of the pretreatment. Specifically, the antibody to which the gold marker was added was added to the sample by using an antigen-antibody reaction. At this time, gold particles having a diameter of 10 nm to 50 nm were used as markers.

  When the sample is irradiated with a focused electron beam, the metal marker has a larger atomic weight than that of the living body, and therefore can be clearly observed as a reflected electron image or a transmitted electron image.

  In this embodiment, automatic image processing and image classification are added.

  After the sample was automatically preprocessed and automatically observed in the same manner as in Example 24, the acquired images were classified by the following method. First, feature amounts are extracted from an image (image processing). In this example, specific structures such as the structure size, eccentricity, edge roughness, density, surface morphology, and protrusions (protrusions on the structure surface) were extracted. Samples were automatically classified according to the combination of these feature quantities. Repeat as many times as necessary from sample movement to classification.

  As for the relationship between the feature quantity and the classification method, the classification accuracy was improved by learning a large number of samples in advance using the result of manual classification. This automation has saved human effort.

Modification: The present embodiment is different from the first embodiment in that the observation is automatically performed from a plurality of directions using an apparatus in which the lens barrel is inclined. Using these images, the three-dimensional shape of the structure in the sample can be calculated.
Since it is necessary to put the cell 8 in the vacuum chamber 7, after supplying the sample to the cell, the inlet and outlet were sealed with an adhesive. A cell can be introduced into the vacuum chamber 7 using a transfer robot. By automating observations from multiple directions, human labor was saved.

  This embodiment is different in that the three-dimensional shape construction process can be automatically performed. This automation has saved human effort.

  In this embodiment, automatic image recognition and image classification are added.

  After automatic pretreatment and observation of the sample, a three-dimensional shape was automatically obtained. A feature amount is extracted from the three-dimensional shape. In this example, specific structures such as the size of the structure, three-dimensional eccentricity, edge roughness, density, surface form, and protrusions (projections on the structure surface) were extracted. Samples were automatically classified according to the combination of these feature quantities.

  As for the relationship between the feature quantity and the classification method, the classification accuracy was improved by learning a large number of samples in advance using the result of manual classification. This automation has saved human effort.

  The present embodiment is different from the first embodiment in that blood cells are observed and the relationship between the result of classification of the three-dimensional shape and the etiology is used to automatically determine the cause using the classification result. This automation has saved human effort.

  In this example, powder is observed, and the result of classification of the three-dimensional shape and the relationship between past measurement results are used, and the result of classification is automatically used to determine whether the product is good or defective. The difference is that the judgment can be made. In this example, the toner for copying, cosmetics, pigments, and graphite were inspected as powders. This automation has saved human effort.

  This embodiment is different in that the protein is observed, and the three-dimensional shape can be automatically reconstructed. This automation has saved human effort.

  The present embodiment is different in that the objective lens is composed of two stages of an upper magnetic pole 3a and a lower magnetic pole 3b (see FIG. 54).

  With such a configuration, the aberration of the objective lens can be reduced, so that the electron beam can be easily focused. Therefore, observation with higher resolution was possible.

  This embodiment is different in that a scattered electron angle selection lens 3c is added to select a detected scattering angle when detecting forward scattered electrons (see FIG. 55). Thereby, when an electron is scattered by the sample, an electron scattering angle can be selected. Therefore, various contrasts could be selected. Thereby, a clearer image could be obtained.

  The present embodiment is different in that a lens 3d for imaging electrons transmitted through the sample is added and that the scanning unit 4 is used for alignment (see FIG. 56).

  With this configuration, a normal transmission electron microscope image could be obtained. For this reason, it is easy to compare with an image acquired with a normal transmission electron microscope. In addition, 3e in a figure is a highly sensitive electron beam camera.

  This example is similar to Example 59. However, the difference is that a phase plate 3f is added to obtain a phase difference image of electrons transmitted through the sample (see FIG. 57).

  With such a configuration, a phase difference image of the sample can be obtained. As a result, it was possible to clearly observe a sample with less material contrast. In addition, clear observation was possible even when no staining or the like was performed.

  The present embodiment is different in that a phase plate is added in order to obtain a differential interference image of electrons transmitted through the sample.

  With such a configuration, a differential interference image of the sample can be obtained. As a result, it was possible to clearly observe a sample with less material contrast. In addition, clear observation was possible even when no staining or the like was performed.

  In this embodiment, a plurality of inlets 51 communicating with the same channel 53 are provided so that the sample and the drug can be mixed in the cell, and the sample and the chemical solution can be mixed in each channel 53. This is different (see FIG. 58).

  By adopting such a configuration, the sample and the chemical could be mixed on the cell.

  The substance generated by this mixing is introduced into the sample holding space 55 through the supply path 54. The structure around the sample holding space 55 is as shown in FIG. The cell is placed in a sample chamber of a scanning electron microscope, a transmission electron microscope, or a scanning transmission electron microscope. The substance existing in the sample holding space of the cell was irradiated with an electron beam, and the secondary signal generated by this was detected, or the transmitted electron was detected, and the sample information could be acquired.

Here, when the sample is a living cell, the drug solution includes a substance that fixes or stains the living cell. Specifically, glutaraldehyde, formaldehyde, formalin or the like can be used as the fixing solution, and an osmium acid aqueous solution, a tannic acid aqueous solution, or a phosphotungstic acid aqueous solution can be used as the conductive staining solution. Both are chemical solutions for fixation and conductive staining developed for SEM and TEM for vacuum observation.
In the present embodiment, the sample and one medicine are mixed, but it goes without saying that the sample and a plurality of medicines can be mixed. Needless to say, the mixing timing of the chemical solution can be controlled by arranging a delay or the like in the flow path.

  As described above, in the embodiment, before or after the sample is put into the sample holding space 55, it is adhered to the sample holding space 55 when it is washed with distilled water, ethanol or the like from the supply path 54 connected to the sample holding space 55. Foreign matter can be removed. Thereby, the captured image became clearer.

  The embodiments of the present invention have been described above.

  The sample holder of the present invention is a sample having a sample holding space provided between a film through which an electron beam passes and a base facing the film, and a supply path for supplying the sample to the sample holding space. A holding body comprising a filter structure for separating components in a sample in at least one of the supply path and the sample holding space. That is, the filter structure can be provided in the supply path of the sample holder or / and the sample holding space.

  Further, the sample holder of the present invention is a sample having a sample holding space provided between two opposingly arranged films through which an electron beam is transmitted, and a supply path for supplying a sample to the sample holding space. It is a holding | maintenance body, Comprising: At least one of this supply path and this sample holding space is provided with the filter structure which fractionates the structure in a sample, It is characterized by the above-mentioned. Similarly to the above, the filter structure can be provided in the supply path of the sample holder or / and the sample holding space.

  Furthermore, the sample holder of the present invention includes a sample holding space provided between a membrane through which an electron beam permeates and a base facing the membrane, a channel to which a sample is supplied, and the sample from the channel. A sample holder having a supply path for supplying a sample to the holding space, the filter holding structure for separating components in the sample in at least one of the supply path and the sample holding space. And Similarly to the above, the filter structure can be provided in the supply path of the sample holder or / and the sample holding space.

  The sample holder of the present invention includes a sample holding space provided between two opposed films through which an electron beam is transmitted, a flow path to which a sample is supplied, and the sample holding space from the flow path. A sample holding body having a supply path for supplying a sample to the apparatus, wherein at least one of the supply path and the sample holding space is provided with a filter structure for separating components in the sample. . Similarly to the above, the filter structure can be provided in the supply path of the sample holder or / and the sample holding space.

  The sample holder of the present invention is also characterized in that the filter structure has a function of preventing a constituent in the sample exceeding the set first dimension from passing.

  Furthermore, the sample holder of the present invention specifies the width of a part of the supply path or a part of the sample holding space in a direction along the surface of the membrane or / and a direction perpendicular to the surface. It is also characterized by the construction of a filter structure. In each of the above examples, such a configuration can be provided.

  The sample holder of the present invention is also characterized in that the width is set to 10 nm to 20 μm, or 10 nm to 5 μm.

  Further, the sample holder of the present invention is characterized in that a difference portion or an inclined portion is formed in the supply path or the sample holding space, and the filter structure is configured by any one of these portions. .

  Furthermore, the sample holder of the present invention is characterized in that at least one protrusion or column is provided in the supply path or the sample holding space, thereby forming the filter structure. That is, a protrusion or a column can be provided as a filter structure in the supply path or the sample holding space.

  The sample holder of the present invention is characterized in that the filter structure includes an adsorption function film that adsorbs a specific component in the sample. That is, an adsorption function film can be provided as a filter structure in the supply path or the sample holding space.

  The sample holder of the present invention is also characterized in that the adsorption functional membrane is an extracellular matrix component. That is, an adsorption function film made of an extracellular matrix component can be provided as a filter structure in the supply path or the sample holding space.

  Furthermore, the sample holder of the present invention is characterized in that the adsorption functional membrane is composed of at least one of poly L lysine, poly D lysine, collagen, matrigel, fibronectin, laminin, and cell tack. That is, an adsorption function film made of at least one of poly L lysine, poly D lysine, collagen, matrigel, fibronectin, laminin, and cell tack can be provided in the supply channel or the sample holding space.

  The sample holder of the present invention includes a sample holding space provided between a film through which an electron beam is transmitted and a base facing the film, and a supply path for supplying the sample to the sample holding space. It is also characterized in that the width of the sample holding space and the supply path is set to 10 nm to 20 μm, or the width is set to 10 nm to 5 μm.

  Further, the sample holder of the present invention is a sample having a sample holding space provided between two opposingly arranged films through which an electron beam is transmitted, and a supply path for supplying a sample to the sample holding space. It is a holding | maintenance body, Comprising: The width | variety of this sample holding space and this supply path is also set to 10 nm-20 micrometers, or it is characterized by being set to 10 nm-5 micrometers.

  The sample holder of the present invention is characterized in that the sample holding space communicates with a drawing path for drawing a part of the sample.

  Further, in the sample holder of the present invention, the sample holding space is connected to the extraction path via a communication path, and a component in the sample exceeding the second dimension passes through the communication path. It is also characterized in that another filter structure having the function of preventing is provided. In each of the above examples, such a configuration can be adopted.

  Furthermore, the sample holder of the present invention is characterized in that the film contains at least one of silicon oxide, silicon nitride, carbon, polyimide, polypropylene, polyethylene, and boron nitride.

  The sample holder of the present invention is also characterized in that the thickness of the film is 10 nm to 1000 nm.

  The sample holder of the present invention is also characterized in that a conductive layer is provided on the film. In each of the above examples, such a configuration can be adopted.

  Furthermore, the sample holder of the present invention is also characterized by including a grid or mesh that supports the membrane. That is, a grid or a mesh can be arranged on the film provided in the sample holder.

  The sample holder of the present invention is characterized in that a plurality of the sample holding spaces are provided, and supply paths are provided individually corresponding to the respective sample holding spaces. In each of the above examples, such a configuration can be adopted.

  Further, the sample holder of the present invention is provided with a plurality of the sample holding spaces, and individually provided supply paths corresponding to the respective sample holding spaces, and a filter structure provided in these supply paths or the sample holding spaces. 2 is characterized in that two or more dimensions are set as the first dimension. In each of the above examples, such a configuration can be adopted.

  Furthermore, the sample holder of the present invention is characterized in that the supply port and the discharge port communicating with the flow path are provided on the same surface of the sample holder.

  The sample holder of the present invention is also characterized in that a plurality of supply ports communicating with the flow path are provided.

  The sample holder of the present invention is also characterized in that an index pattern serving as a dimension index is formed on the surface.

  Further, in the sample inspection method of the present invention, the sample held in the sample holding space of the sample holder is irradiated with an electron beam through the film, and a secondary signal or sample generated from the sample by the irradiation is obtained. The transmitted electron beam is detected to acquire sample information.

  In the sample inspection method of the present invention, after the sample is supplied to the sample holding space of the sample holder, the liquid component is removed from the sample in the sample holding space, and the configuration of the sample remaining in the sample holding space The object is also characterized by irradiating an object with an electron beam through the film and detecting a secondary signal generated from the sample by the irradiation or a transmitted electron beam transmitted through the sample to acquire information on the sample.

  In the sample inspection method of the present invention, after removing a liquid component from the sample in the sample holding space, water vapor is supplied to the sample holding space through the supply path, and then information on the sample is acquired. Features.

  Furthermore, in the sample inspection method of the present invention, among the plurality of supply ports provided in the sample holder, the sample is supplied to the flow path via the first supply port, and via the second supply port. A chemical solution is supplied to the flow channel, and a sample that has reacted with the chemical solution in the flow channel is supplied to the sample holding space via the supply channel, and an electron is supplied to the sample held in the sample holding space via the film. It is also characterized in that information on the sample is acquired by detecting a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample.

  The sample inspection method of the present invention is characterized in that the sample includes a biological component, and the chemical solution includes a substance that fixes or stains the biological component.

  In the sample inspection method of the present invention, the sample held in the sample holding space of the sample holder is irradiated with an electron beam through the film, and a secondary signal generated from the sample by the irradiation or the sample is transmitted. Is a sample inspection method for detecting the transmitted electron beam and acquiring sample information, and acquiring pattern information obtained when the index pattern formed on the surface of the sample holder is irradiated with the electron beam. Further, it is also characterized by referring to the obtained sample information and the pattern information.

  Furthermore, the sample inspection method of the present invention includes a supplying step of supplying a sample to the supply path of the sample holder and supplying the sample to the sample holding space through the supply path, and holding the sample in the sample holding space. An irradiation step of irradiating the irradiated sample with an electron beam through the film, and a detection step of acquiring a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample to obtain information on the sample; It is characterized by having.

  The sample inspection method of the present invention is also characterized in that, in the supplying step, the pressure on at least one surface side of the film is adjusted so that the difference in pressure on both surfaces of the film becomes small.

  Further, in the sample inspection method of the present invention, in the supplying step, before supplying the sample to the supply path, a predetermined pressure difference is generated on both sides of the film, thereby determining whether the film is broken or not. It is also characterized by confirming the strength of the film.

  Furthermore, the sample inspection method of the present invention is further characterized by further comprising a sample pretreatment step performed before the supply step.

  The sample inspection method of the present invention is characterized in that in the pretreatment step, at least one of a sample fixing process, a staining process, a fluorescent marker addition process, and a metal marker addition process is performed.

  Further, the sample inspection method of the present invention is characterized in that the secondary signal is at least one of reflected electrons, secondary electrons, X-rays, and fluorescence.

  Furthermore, in the sample inspection method of the present invention, fluorescence is detected as the secondary signal to search for a fluorescent region of the sample, and then reflected electrons or secondary electrons are detected as a secondary signal from the fluorescent region. To obtain the structure of the fluorescent region.

  The sample inspection method of the present invention is characterized by detecting reflected electrons as the secondary signal and simultaneously detecting a transmitted electron beam.

  Further, the sample inspection method of the present invention is characterized in that image processing is performed on the acquired sample information data, and image classification is performed based on the image processing result.

  Furthermore, the sample inspection method of the present invention is characterized in that pathogenesis determination or pass / fail determination is performed based on the image classification result.

  The sample inspection method of the present invention further includes a cleaning step of cleaning at least the supply path and the sample holding space of the sample holder before the supply step.

  In addition, the sample inspection method of the present invention further includes a sealing step of sealing a supply port and / or a discharge port communicating with the supply path of the sample holder after the supply step. .

  Each of these sample inspection methods can be implemented using the sample holder.

  Furthermore, the sample inspection apparatus according to the present invention is a sample inspection apparatus including the sample holder, and the irradiation unit irradiates the sample held in the sample holding space of the sample holder with an electron beam through the film. And a detection means for detecting a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample.

  The sample inspection apparatus of the present invention includes a vacuum chamber that supports the irradiation unit and includes the detection unit therein, and the sample holder is installed in the vacuum chamber via an O-ring or grease. It is also characterized by being.

  In addition, the sample inspection apparatus of the present invention is characterized by having a tilting means for tilting the sample holder.

  Furthermore, the sample inspection apparatus of the present invention is characterized by further comprising selection means capable of selecting the detected scattering angle of the transmitted electrons.

  The sample inspection apparatus of the present invention is also characterized by comprising an imaging means for imaging the phase difference or differential interference component of the transmitted electrons.

  The sample inspection apparatus of the present invention is also characterized by comprising a reflected electron detector that detects reflected electrons as the secondary signal and a transmitted electron detector that detects the transmitted electrons.

  Furthermore, the sample inspection apparatus of the present invention is also characterized in that image processing is performed on the acquired sample information data and image classification is performed based on the image processing result.

  The sample inspection apparatus of the present invention is also characterized in that the etiology determination or the pass / fail determination is performed based on the classification result.

  Each of these sample inspection apparatuses can be configured using the sample holder.

  The sample inspection system of the present invention includes the sample inspection apparatus, and includes a sample pretreatment means, a sample supply means for supplying the pretreated sample to the flow path of the sample holder, and a sample. And a moving means for moving the sample holder to the sample inspection apparatus.

  Furthermore, the sample inspection system of the present invention is characterized in that the pretreatment means performs at least one of a sample fixing process, a staining process, a fluorescent marker adding process, and a metal marker adding process.

  The sample inspection system of the present invention is characterized in that the sample supply means can adjust the pressure on at least one surface side of the film of the sample holder.

  Thus, in the present invention, at least one of the supply path and the sample holding space provided in the sample holder is provided with a filter structure that separates the components in the sample.

  Therefore, the sample structure that has passed through the filter structure is supplied to the sample holding space of the sample holder, and the sample held in the sample holding space is used as an inspection target. The sample inspection can be efficiently performed in a state in which the components are selected and extracted in the sample holder.

  Further, when the filter structure includes an adsorption function film, the sample inspection can be efficiently performed in a state where the constituents in the sample that are not adsorbed by the adsorption function film are selected and extracted in the sample holder.

  Further, when the width of the sample holding space in the direction perpendicular to the surface of the film of the sample holder is specified, the constituent in the selected sample approaches the film in the sample holding space. As a result, the film and the component can be prevented from being separated from each other, and the electron beam reaching the component and the reflected electrons generated from the component can be prevented from being attenuated. A good SEM image can be acquired and a good sample inspection can be performed.

1 is a diagram illustrating a device configuration of Example 1. FIG. FIG. 3 is a diagram showing a configuration of a sample cell of Example 1. It is a figure which shows the state which connects a pipe to a sample cell. It is a figure which shows the preparation method of a sample cell. FIG. 6 is a diagram illustrating Example 2. FIG. 6 is a diagram for explaining a third embodiment. FIG. 10 is a diagram for explaining a fourth embodiment. FIG. 10 is a diagram for explaining a fifth embodiment. FIG. 10 is a diagram for explaining a fifth embodiment. FIG. 10 is a diagram illustrating Example 6. FIG. 10 is a diagram illustrating Example 6. FIG. 10 is a diagram illustrating Example 7. FIG. 10 is a diagram illustrating an eighth embodiment. FIG. 10 is a diagram illustrating an eighth embodiment. FIG. 10 is a diagram illustrating Example 9. FIG. 10 is a diagram illustrating Example 9. FIG. 10 is a diagram for explaining Example 11. FIG. 10 is a diagram for explaining Example 12; FIG. 20 is a diagram for explaining Example 13; FIG. 20 is a diagram for explaining a modified example of the thirteenth embodiment. FIG. 20 is a diagram for explaining a modified example of the thirteenth embodiment. It is a figure explaining Example 14. FIG. It is a figure explaining Example 14. FIG. FIG. 20 is a diagram for explaining Example 15; It is a figure explaining Example 16. FIG. FIG. 17 is a diagram for explaining an example 17; FIG. 20 is a diagram for explaining an example 18; FIG. 20 is a diagram for explaining Example 20; FIG. 20 is a diagram illustrating Example 21. FIG. 22 is a diagram illustrating Example 22. FIG. 20 is a diagram illustrating Example 23. FIG. 20 is a diagram illustrating Example 23. FIG. 20 is a diagram illustrating Example 24. FIG. 20 is a diagram illustrating Example 24. FIG. 26 is a diagram illustrating Example 26. FIG. 40 is a diagram for explaining Example 28; 42 is a diagram for explaining Example 29. FIG. FIG. 10 is a diagram illustrating Example 30. FIG. 20 is a diagram illustrating Example 34. FIG. 20 is a diagram illustrating Example 34. FIG. 20 is a diagram illustrating Example 34. FIG. 40 is a diagram illustrating Example 35. FIG. 40 is a diagram for explaining Example 36; FIG. 45 is a diagram illustrating Example 37. 40 is a diagram for explaining Example 38. FIG. 40 is a diagram for explaining Example 38. FIG. 40 is a diagram for explaining Example 39. FIG. It is a figure explaining Example 40. FIG. 42 is a diagram illustrating Example 41. FIG. 42 is a diagram illustrating Example 43. FIG. 42 is a diagram for explaining Example 44. FIG. It is a figure explaining Example 45. FIG. FIG. 45 is a diagram illustrating Example 47. FIG. 57 is a diagram illustrating Example 57. FIG. 66 is a diagram illustrating Example 58. FIG. 50 is a diagram illustrating Example 59. FIG. 10 is a diagram for explaining an example 60; 42 is a diagram illustrating Example 62. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Electron source, 2 ... Condenser lens, 3 ... Objective lens, 4 ... Scanning unit, 5 ... Electron beam column, 6 ... Electron beam detector, 7 ... Vacuum chamber, 8 ... Sample cell (sample holder), 9 ... O-ring, 11 ... organic film (film), 12 ... cell base (base), 13 ... cell cap, 14 ... adhesive, 15 ... sample, 53 ... flow path, 54 ... branch path (supply path), 55 ... Observation part (sample holding space), 16 ... electron beam, 62a, 62b, 62c, 62d ... filter

Claims (57)

A sample holder having a sample holding space provided between a membrane through which an electron beam passes and a base opposite to the membrane, and a supply path for supplying a sample to the sample holding space, the supply A sample holder, comprising a filter structure for separating components in the sample in at least one of the path and the sample holding space. A sample holder having a sample holding space provided between two opposingly arranged films through which an electron beam passes, and a supply path for supplying a sample to the sample holding space, the supply path and A sample holder, comprising a filter structure that separates components in a sample in at least one of the sample holding spaces. A sample holding space provided between the membrane through which the electron beam passes and the base opposite to the membrane, a flow path for supplying the sample, and a supply for supplying the sample from the flow path to the sample holding space A sample holder having a channel, wherein the sample holder has a filter structure for separating components in the sample in at least one of the supply path and the sample holding space. A sample holding space provided between two opposingly arranged films through which an electron beam passes, a flow path for supplying a sample, and a supply path for supplying a sample from the flow path to the sample holding space; A sample holder having a filter structure for separating components in the sample in at least one of the supply path and the sample holding space. The sample holder according to any one of claims 1 to 4, wherein the filter structure has a function of preventing a constituent in the sample exceeding a set first dimension from passing therethrough. The filter structure is configured by specifying a width of a part of the supply path or a part of the sample holding space in a direction along the surface of the film or / and a direction perpendicular to the surface. The sample holder according to any one of claims 1 to 5. The sample holder according to claim 6, wherein the width is set to 10 nm to 20 μm. The sample holder according to claim 6, wherein the width is set to 10 nm to 5 μm. 9. The step according to claim 1, wherein a stepped portion or an inclined portion is formed in the supply path or the sample holding space, and the filter structure is configured by any of these portions. Sample holder. 7. The sample holder according to claim 1, wherein at least one protrusion or column is provided in the supply path or the sample holding space, thereby forming the filter structure. . The sample holder according to any one of claims 1 to 4, wherein the filter structure includes an adsorption function film that adsorbs a specific component in the sample. 12. The sample holder according to claim 11, wherein the adsorption functional film is an extracellular matrix component. 12. The sample holder according to claim 11, wherein the adsorption functional film is made of at least one of poly L lysine, poly D lysine, collagen, matrigel, fibronectin, laminin, and cell tack. A sample holder having a sample holding space provided between a membrane through which an electron beam is transmitted and a base facing the membrane, and a supply path for supplying a sample to the sample holding space, A sample holder, wherein the width of the holding space and the supply path is set to 10 nm to 20 μm. A sample holder having a sample holding space provided between a membrane through which an electron beam is transmitted and a base facing the membrane, and a supply path for supplying a sample to the sample holding space, A sample holder, wherein the width of the holding space and the supply path is set to 10 nm to 5 μm. A sample holder having a sample holding space provided between two opposingly arranged films through which an electron beam is transmitted and a supply path for supplying a sample to the sample holding space, the sample holding space And a width of the supply path is set to 10 nm to 20 μm. A sample holder having a sample holding space provided between two opposingly arranged films through which an electron beam is transmitted and a supply path for supplying a sample to the sample holding space, the sample holding space And a width of the supply path is set to 10 nm to 5 μm. The sample holder according to any one of claims 1 to 17, wherein the sample holding space communicates with a drawing path for drawing a part of the sample. The sample holding space is connected to the lead-out path through a communication path, and another filter structure having a function of preventing a component in the sample exceeding the second dimension from passing through the communication path. The sample holder according to claim 18, wherein the sample holder is provided. The sample holder according to any one of claims 1 to 19, wherein the film includes at least one of silicon oxide, silicon nitride, carbon, polyimide, polypropylene, polyethylene, and boron nitride. 21. The sample holder according to claim 1, wherein the thickness of the film is 10 nm to 1000 nm. The sample holder according to any one of claims 1 to 21, wherein a conductive layer is provided on the film. The sample holder according to any one of claims 1 to 22, further comprising a grid or a mesh that supports the film. The sample holding body according to any one of claims 1 to 23, wherein a plurality of the sample holding spaces are provided, and supply paths are provided individually corresponding to the respective sample holding spaces. A plurality of the sample holding spaces are provided, and supply paths are individually provided corresponding to the respective sample holding spaces. In the filter structure provided in these supply paths or the sample holding spaces, there are two types as the first dimension. 6. The sample holder according to claim 5, wherein the above dimensions are set. 26. The sample holder according to any one of claims 1 to 25, wherein a supply port and a discharge port communicating with the flow path or the supply channel are provided on the same surface of the sample holder. The sample holder according to any one of claims 1 to 26, wherein a plurality of supply ports communicating with the flow path or the supply path are provided. The sample holder according to any one of claims 1 to 27, wherein an index pattern serving as a dimension index is formed on a surface. The sample held in the sample holding space of the sample holder according to any one of claims 1 to 28 is irradiated with an electron beam through the film, and a secondary signal generated from the sample or the sample is transmitted by the irradiation. A sample inspection method for obtaining information on a sample by detecting a transmission electron beam. 16. After supplying a sample to the sample holding space of the sample holding body according to claim 15, the liquid component is removed from the sample in the sample holding space, and the constituent of the sample remaining in the sample holding space is interposed through the membrane. A sample inspection method comprising: irradiating an electron beam and detecting a secondary signal generated from the sample by the irradiation or a transmitted electron beam transmitted through the sample to acquire information on the sample. The sample inspection according to claim 30, wherein after removing a liquid component from the sample in the sample holding space, water vapor is supplied to the sample holding space through the supply path, and then information on the sample is acquired. Method. A sample is supplied to the channel through a first supply port among the plurality of supply ports provided in the sample holder according to claim 27, and a chemical solution is supplied to the channel through a second supply port. The sample that has reacted with the chemical solution in the flow path is supplied to the sample holding space through the supply path, and the sample held in the sample holding space is irradiated with an electron beam through the film, A sample inspection method characterized in that a secondary signal generated from a sample by the irradiation or a transmission electron beam transmitted through the sample is detected to acquire information on the sample. The sample inspection method according to claim 32, wherein the sample includes a biological component, and the chemical solution includes a substance that fixes or stains the biological component. The sample held in the sample holding space of the sample holder according to claim 28 is irradiated with an electron beam through the film, and a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample is detected. A sample inspection method for acquiring sample information, wherein pattern information obtained when an electron beam is irradiated to the index pattern formed on the surface of the sample holder is obtained, and the obtained sample information And a sample inspection method characterized by referring to the pattern information. A supply step of supplying a sample to the supply path of the sample holder according to any one of claims 1 to 24 and supplying the sample to the sample holding space through the supply path, and held in the sample holding space An irradiation step of irradiating the sample with an electron beam through the film; and a detection step of acquiring a secondary signal generated from the sample by the irradiation or a transmission electron beam transmitted through the sample to acquire information on the sample Sample inspection method. 36. The sample inspection method according to claim 35, wherein, in the supplying step, the pressure on at least one surface side of the film is adjusted so that the difference in pressure between both surfaces of the film becomes small. In the supplying step, before supplying a sample to the supply path, a predetermined pressure difference is generated on both sides of the film, thereby confirming whether the film is broken or strength of the film. The sample inspection method according to claim 35 or 36. The sample inspection method according to any one of claims 35 to 37, further comprising a sample pretreatment step performed before the supply step. 39. The sample inspection method according to claim 38, wherein at least one of a sample fixing process, a staining process, a fluorescent marker addition process, and a metal marker addition process is performed in the pretreatment step. 40. The sample inspection method according to claim 35, wherein the secondary signal is at least one of reflected electrons, secondary electrons, X-rays, and fluorescence. The fluorescence is detected as the secondary signal to search the fluorescent region of the sample, and then the reflected electron or secondary electron is detected as the secondary signal from the fluorescent region, thereby obtaining the structure of the fluorescent region. 41. The sample inspection method according to claim 40. 41. The sample inspection method according to claim 35, wherein a reflected electron is detected as the secondary signal and a transmission electron beam is detected at the same time. 43. The sample inspection method according to claim 35, wherein image processing is performed on the acquired information data of the sample, and image classification is performed based on the image processing result. 44. The sample inspection method according to claim 43, wherein etiology determination or pass / fail determination is performed based on the image classification result. 45. The sample inspection method according to claim 35, further comprising a cleaning step of cleaning at least the supply path and the sample holding space of the sample holder before the supply step. The sample according to any one of claims 35 to 45, further comprising a sealing step of sealing a supply port and / or a discharge port communicating with the supply path of the sample holder after the supply step. Inspection method. A sample inspection apparatus comprising the sample holder according to any one of claims 1 to 28, wherein the sample held in the sample holding space of the sample holder is irradiated with an electron beam through the film, A sample inspection apparatus having a secondary signal generated from the sample by the irradiation or a detection means for detecting a transmission electron beam transmitted through the sample. 48. The vacuum chamber according to claim 47, further comprising a vacuum chamber that supports the irradiation unit and includes the detection unit therein, and the sample holder is installed in the vacuum chamber via an O-ring or grease. Sample inspection equipment. 49. The sample inspection apparatus according to claim 47 or 48, further comprising tilting means for tilting the sample holder. 48. The sample inspection apparatus according to claim 47, further comprising selection means capable of selecting a detected scattering angle of the transmitted electrons. 48. The sample inspection apparatus according to claim 47, further comprising imaging means for imaging the phase difference or differential interference component of the transmitted electrons. 48. The sample inspection apparatus according to claim 47, further comprising: a reflected electron detector that detects reflected electrons as the secondary signal; and a transmitted electron detector that detects the transmitted electrons. 53. The sample inspection apparatus according to claim 47, wherein the obtained sample information data is subjected to image processing, and image classification is performed based on the image processing result. 54. The sample inspection apparatus according to claim 53, wherein pathogenesis determination or pass / fail determination is performed based on the classification result. 55. A sample inspection apparatus according to any one of claims 47 to 54, comprising a sample pretreatment means, a sample supply means for supplying the pretreated sample to the flow path of the sample holder, and a sample supplied with the sample A sample inspection system comprising a moving means for moving the holder to the sample inspection apparatus. 56. The sample inspection system according to claim 55, wherein the preprocessing means performs at least one of a sample fixing process, a staining process, a fluorescent marker adding process, and a metal marker adding process. 57. The sample inspection system according to claim 55 or 56, wherein the sample supply means can adjust a pressure on at least one surface side of the film of the sample holder.

Images