JP6492602B2 - Sample storage cell - Google Patents

Description

  The present invention relates to a cell for accommodating a sample (Object) to be observed.

  In observation of a sample using an electron microscope, the sample to be observed is generally exposed to a special space such as a vacuum. On the other hand, in recent years, in a technical field called mechanobiology, there is a demand for observing living cells, living tissues and the like as they are. However, if the sample is exposed to a vacuum during observation with an electron microscope, the cells and the like are altered as the liquid components volatilize, and the measurement environment is contaminated. Various methods have been developed to prevent this.

  For example, there is a technique in which a sample is quickly frozen and confined in thin ice and observed in a frozen state. However, the technique for observing in a frozen state cannot easily prepare a sample for observation, and the sample preparation apparatus is very special and expensive.

  Furthermore, Patent Document 1 discloses a technique for directly observing a sample by observing the sample while holding it in a liquid. In this technique, a liquid containing a sample is disposed between thin films constituting an observation cell, and a space in which the sample is disposed is blocked from an external space of the cell. The electron beam passes through the thin film and reaches the sample between the thin films.

JP 2007-165271 A

  Further, among the cells disclosed in Patent Document 1, the size of the observation hole is small in a cell in which a liquid containing a sample is injected from an injection port. Therefore, it is difficult to confirm whether the sample to be observed has reached the observation hole before measurement with an electron microscope. Further, Patent Document 1 discloses a cell in which a sample is placed on a seat and covered with a lid, and the seat and the lid are joined with an adhesive. In such a cell, the user is required to have a high level of technology for joining, the joint between the seat and the lid does not work well, the internal liquid leaks out, and it is difficult to control the gap between the seat and the lid. There was a case. As a result of the occurrence of at least one of these problems, the cell containing the sample is often in a state where it cannot be used for observation with an electron microscope, which impairs user convenience.

  One of the objects of the present invention is to improve the convenience of a cell for accommodating a sample.

  According to an embodiment of the present invention, a first substrate having a first opening, and a second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening. A first thin film that closes the second substrate side of the first opening, a second thin film that closes the first substrate side of the second opening, and a beam disposed in the first thin film. And for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and an external space between the first substrate and the second substrate. And at least one of the first substrate and the second substrate is transparent to visible light, and the first thin film and the second thin film are Provided is a sample storage cell characterized by having permeability. According to this, the convenience of the cell which accommodates a sample can be improved.

  According to one embodiment of the present invention, a first substrate having a first opening, a second substrate disposed opposite to the first substrate, and having a second opening at a position facing the first opening. Two substrates, a first thin film that closes the second substrate side of the first opening, a second thin film that closes the first substrate side of the second opening, and a beam disposed in the first thin film Between the first substrate and the second substrate, the observation space between the first thin film and the second thin film in which the sample is arranged, and the observation space and the external space are connected And at least one of the first substrate and the second substrate is a silicon substrate, and the first thin film and the second thin film are transmissive to an electron beam. A sample storage cell is provided. According to this, the convenience of the cell which accommodates a sample can be improved.

  Further, a beam is also disposed in the second thin film, and a region of the first thin film where the beam is not disposed and a region of the second thin film where the beam is not disposed are at least partially opposed to each other. You may do it. According to this, it is possible to efficiently secure a region where the sample can be observed while maintaining the strength of the first thin film and the second thin film.

  The beam may be made of a material different from that of the first thin film. According to this, it is possible to easily produce a beam on a thin film.

  The beam may be an amorphous silicon film, and the first thin film may be a silicon nitride film. According to this, it is possible to easily produce a beam on a thin film.

  The beam may be a silicon nitride film, and the first thin film may be a silicon nitride film having a higher nitrogen content than the beam. According to this, it is possible to easily produce a beam on a thin film.

  The beam may be the same material as the first thin film. According to this, it is not necessary to form the film | membrane used for a beam separately from a thin film, and a manufacturing process can be simplified.

  The beam may be disposed on the first opening side of the first thin film. According to this, the surface of the thin film on which the sample is arranged can be flattened, and the sample can be easily introduced into the observation space.

  A protrusion is disposed on the surface of the first thin film on the observation space side, the region of the first thin film where the protrusion is not disposed, and the beam of the first thin film is not disposed. The region may be opposed at least in part. According to this, it is possible to efficiently secure a region where the sample can be observed while suppressing the occurrence of the sticking phenomenon between the first thin film and the second thin film.

  The protrusion is disposed on the observation space side of the first thin film and the second thin film, and the protrusion disposed on the first thin film and the protrusion disposed on the second thin film are opposed to each other. It may be. According to this, it is possible to further suppress the occurrence of the sticking phenomenon between the first thin film and the second thin film.

  The beam may be disposed on the observation space side of the first thin film. According to this, it is possible to further suppress the occurrence of the sticking phenomenon between the first thin film and the second thin film.

  The first thin film may have a portion that contacts a side surface of the beam. According to this, the manufacturing process which makes formation of a beam easy can also be employ | adopted.

  Further, a beam may be disposed on the second thin film, and the second thin film may have a portion that contacts a side surface of the beam. According to this, the manufacturing process which makes formation of a beam easy can also be employ | adopted.

  The beam may be arranged as a part of the first substrate. According to this, the manufacturing process which makes formation of a beam easy can also be employ | adopted.

  The region of the first thin film in which the beam is not disposed may be divided into a plurality of divided regions, and at least one of the divided regions may have a shape different from that of the other divided regions. According to this, when observing with an electron microscope, it is also possible to easily grasp the place being observed.

  The beam may be thicker than the first thin film. According to this, the strength of the thin film can be further improved.

  The first substrate may be a silicon substrate, and the second substrate may be a glass substrate. According to this, a cell can be manufactured easily. Further, anodic bonding can be used as a bonding method between the first substrate and the second substrate, and excellent bonding capable of vacuum sealing can be easily realized.

  The first substrate may be transmissive to visible light. This makes it easier to observe the state of the sample-containing liquid injected into the interior.

  According to the present invention, the convenience of the cell that accommodates the sample can be enhanced.

It is a figure which shows the external appearance of the sample storage cell in 1st Embodiment of this invention. It is the top view which looked at the sample storage cell in a 1st embodiment of the present invention from the 1st substrate side. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line A-A 'in FIG. 1, 2) of the sample storage cell in 1st Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line B-B 'in FIG. 1, 2) of the sample storage cell in 1st Embodiment of this invention. It is a figure explaining the method of inject | pouring a sample containing liquid into the sample storage cell in 1st Embodiment of this invention. It is a figure explaining the method to form resin for sealing the sample storage cell in 1st Embodiment of this invention. It is a figure explaining the sample injection process of the sample injection apparatus in 1st Embodiment of this invention. It is a figure explaining the cell sealing process of the sample injection apparatus in 1st Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 1st Embodiment of this invention. FIG. 10 is a diagram illustrating a manufacturing process of the first substrate following FIG. 9. It is a figure explaining the manufacturing process of the 2nd board | substrate among the sample accommodation cells in 1st Embodiment of this invention. It is a figure which shows the external appearance of the sample storage cell in 2nd Embodiment of this invention. It is the top view which looked at the sample accommodation cell in a 2nd embodiment of the present invention from the 1st substrate side. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line C-C 'in FIG. 12, 13) of the sample storage cell in 2nd Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line D-D 'in FIG. 12, 13) of the sample storage cell in 2nd Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 2nd Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate following FIG. It is a figure which shows the external appearance of the sample storage cell in 3rd Embodiment of this invention. It is the top view which looked at the sample accommodation cell in a 3rd embodiment of the present invention from the 1st substrate side. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line E-E 'in FIG. 18, 19) of the sample storage cell in 3rd Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line F-F 'in FIG. 18, 19) of the sample storage cell in 3rd Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 3rd Embodiment of this invention. It is a figure which shows the external appearance of the sample storage cell in 4th Embodiment of this invention. It is the top view which looked at the sample accommodation cell in a 4th embodiment of the present invention from the 1st substrate side. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line G-G 'in FIG. 23, 24) of the sample storage cell in 4th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure (cross-sectional structure of the cross-sectional line H-H 'in FIG. 23, 24) of the sample storage cell in 4th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 5th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 6th Embodiment of this invention. It is a schematic diagram which shows the marker provided in the sample storage cell in 7th Embodiment of this invention. It is a figure explaining the transmittance | permeability characteristic of the 2nd thin film of the sample storage cell in 8th Embodiment of this invention. It is a figure explaining the example of arrangement | positioning of the sample containing liquid inject | poured into the flow-path space of the sample storage cell in the comparative example of this invention. It is a figure explaining the shape of the flow-path space of the sample storage cell in 9th Embodiment of this invention. It is a figure explaining the shape of the reserve space provided in the flow-path space of the sample storage cell in 10th Embodiment of this invention. It is a figure explaining the shape of the reserve space provided in the flow-path space of the sample storage cell in 11th Embodiment of this invention. It is a figure explaining the surface treatment of a sample storage cell, sample injection processing, and cell sealing processing in a 12th embodiment of the present invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 13th Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 13th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 14th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 15th Embodiment of this invention. It is a schematic diagram which shows the production method of the beam in 15th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 16th Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 16th Embodiment of this invention. It is a figure explaining another manufacturing method of the sample storage cell in 16th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 17th Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 17th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 18th Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 18th Embodiment of this invention. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 19th Embodiment of this invention. It is a figure explaining the manufacturing process of the 1st board | substrate among the sample accommodation cells in 19th Embodiment of this invention. FIG. 50 A diagram for describing the manufacturing processes for the first substrate, following FIG. 49. It is a figure explaining the manufacturing process of the 2nd board | substrate among the sample accommodation cells in 19th Embodiment of this invention. It is a figure explaining the manufacturing process which bonds together the 1st substrate and the 2nd substrate in a 19th embodiment of the present invention. It is a schematic diagram which shows the example of various arrangement | positioning of a beam. It is a schematic diagram which shows the example of various shapes of a beam.

<First Embodiment>
Hereinafter, a sample storage cell according to an embodiment of the present invention will be described in detail with reference to the drawings. In addition, embodiment shown below is an example of embodiment of this invention, This invention is limited to these embodiment, and is not interpreted. Note that in the drawings referred to in the present embodiment, the same portion or a portion having a similar function is denoted by the same reference symbol or a similar reference symbol (a reference symbol simply including A, B, etc. after a number) and repeated. The description of may be omitted. In addition, the dimensional ratios of the drawings (the ratios between the components, the ratios in the vertical and horizontal height directions, etc.) may be different from the actual ratios for convenience of explanation, or some of the configurations may be omitted from the drawings.

[Configuration of sample storage cell]
FIG. 1 is a diagram showing an appearance of a sample storage cell in the first embodiment of the present invention. The sample storage cell 1 is manufactured by bonding the first substrate 10 and the second substrate 20. The first substrate 10 is, for example, a silicon substrate. The second substrate is a substrate that is transparent to visible light, and is, for example, a glass substrate. Note that the first substrate may also be a substrate that is transparent to visible light, like the second substrate. A space is disposed between the first substrate 10 and the second substrate 20. The sample storage cell 1 is a cell that stores a sample to be observed by an electron microscope in this internal space in a state where the sample is included in a liquid.

  Hereinafter, a liquid containing a sample may be expressed as a sample-containing liquid. The size of the sample storage cell 1 is a square having a side of about 2.5 mm to 3 mm, and in this example is 2.6 mm. The thickness of the sample storage cell 1 is about 0.3 to 1.2 mm including the first substrate 10 and the second substrate 20.

  Openings are formed in the first substrate 10 and the second substrate 20, respectively. In this example, openings 110, 120, and 130 are arranged on the first substrate 10. An opening 210 (see FIGS. 3 and 4) is arranged on the second substrate. Next, a detailed structure including the internal structure of the sample storage cell 1 will be described with reference to FIGS. 2, 3, and 4.

  FIG. 2 is a plan view of the sample storage cell in the first embodiment of the present invention as viewed from the first substrate side. FIG. 3 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along the cross-sectional line A-A ′ in FIGS. 1 and 2) of the sample storage cell in the first embodiment of the present invention. FIG. 4 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along a cross-sectional line B-B ′ in FIGS. 1 and 2) of the sample storage cell in the first embodiment of the present invention.

  The second substrate 20 side of the opening 110 is closed by the first thin film 150. The first substrate 10 side of the opening 210 is closed by the second thin film 250. The first thin film 150 and the second thin film 250 are disposed to face each other. The first thin film 150 and the second thin film 250 are films that are permeable to electron beams.

  The first thin film 150 and the second thin film 250 are made of, for example, silicon nitride. The film thicknesses of the first thin film 150 and the second thin film 250 are 10 nm or more and 200 nm or less, preferably 15 nm or more and 50 nm or less, and in this example, 20 nm. The first thin film 150 and the second thin film 250 may have the same film thickness or different film thicknesses. If the first thin film 150 and the second thin film 250 are thinner than 10 nm, the strength is lost and the first thin film 250 may be damaged. On the other hand, when it becomes thicker than 200 nm, an electron beam will not permeate | transmit. Therefore, it is desirable to make the first thin film 150 and the second thin film 250 as thin as possible while obtaining the strength of the film not to break. However, as shown below, since the thin film has a beam, the thin film can be made thinner and the area of the thin film can be expanded compared to the case where no beam is present.

  A beam 150R is disposed on the second thin film 250 (observation space MS) side of the first thin film 150. A beam 250R is disposed on the second thin film 250 on the first thin film 150 (observation space MS) side. In this example, the beams 150R and 250R are amorphous silicon films. The beams 150R and 250R may be silicon nitride films. In this case, the silicon nitride film that forms the first thin film 150 and the second thin film 250 preferably has a higher nitrogen content than the silicon nitride film that forms the beams 150R and 250R. The beams 150R and 250R formed of such a material also exist between the substrate (the first substrate 10 and the second substrate 20) and the thin film (the first thin film 150 and the second thin film 250). The thin film of the beam 150R also formed between the first substrate 10 and the first thin film 150 has higher adhesion to the first substrate 10 than when the first substrate 10 and the first thin film 150 are in contact with each other. Therefore, the bonding strength between the first substrate 10 and the first thin film 150 can be increased. The same applies to the relationship between the second substrate 20 and the second thin film 250.

  The beam 150R and the beam 250R are arranged on the thin film in the same lattice pattern. It is desirable that the end of the pattern of the beam 150R extends to the outside of the opening 110. That is, a thin film extending from the end of the beam 150 </ b> R to the outside of the opening 110 sandwiches the first thin film 150 with the first substrate 10. The width of the beam 150R is 2 μm or more and 100 μm or less, desirably 5 μm or more and 30 μm or less, and in this example, 10 μm. The width of the beam 150R and the width of the beam 250R may be the same or different. If this width is narrow, pattern formation (processing) becomes difficult, and if it is wide, the region that can be observed becomes narrow.

  The thickness of the beam 150R is 20 nm or more and 10 μm or less, preferably 30 nm or more and 1 μm or less. In this example, the thickness is 50 nm thicker than the first thin film 150. The beam 150R is desirably thicker than the first thin film 150, but may be the same thickness or may be thin. The same applies to the beam 250R.

  The beam 150R and the beam 250R may have the same thickness or different thicknesses. One of the beam 150R and the beam 250R may not exist. By disposing the beam 150R on the first thin film 150, the overall strength of the first thin film 150 can be increased. By disposing the beam 250R on the second thin film 250, the overall strength of the second thin film 250 can be increased.

  In the portion where the beams 150R and 250R are disposed, the electron beam may not be transmitted or may be transmitted. The electron beam only needs to be transmitted through a region of the first thin film 150 where the beam 150R is not disposed and a region of the second thin film 250 where the beam 250R is not disposed. In addition, the first thin film 150 has at least a region where the beam 150R is not disposed and the region of the second thin film 250 where the beam 250R is not disposed (region through which the electron beam is transmitted). doing.

  The distance between the first thin film 150 and the second thin film 250 is 10 nm or more and 400 nm or less, preferably 50 nm or more and 300 nm or less, and in this example, 200 nm. In addition, since the 1st thin film 150 and the 2nd thin film 250 are each 20 nm, the distance of the 1st board | substrate 10 and the 2nd board | substrate 20 in this part will be 240 nm. Since the distance between the first thin film 150 and the second thin film 250 depends on the size of the sample, that is, the observation object (for example, a cell), it needs to be at least larger than the observation object. On the other hand, if the distance between the first thin film 150 and the second thin film 250 is too large with respect to the observation object, the observation objects overlap and it is difficult to obtain the observation results of the individual observation objects.

  If the observation object is too large, the electron beam cannot pass through the observation object itself, so that observation with a transmission electron microscope is not possible. That is, the distance between the first thin film 150 and the second thin film 250 does not have to be greater than the distance that the electron beam cannot pass. On the other hand, even if the object to be observed is small, if the distance between the first thin film 150 and the second thin film 250 is too small, a sticking phenomenon described later tends to occur.

  The opening 110 and the opening 210 are arranged to face each other, and are designed to open with substantially the same size when viewed from the first substrate 10 side. In this example, the shape of the opening of the opening 110 and the opening 210 is a square of 60 μm × 60 μm. The shape of the opening may be a rectangle such as 5 μm × 20 μm. As described above, the width and thickness of the beam can be adjusted to improve the strength of the thin film, and as a result, a larger opening shape can be set.

  In this example, the beams 150R are arranged in a lattice pattern, and the first thin film 150 in the opening is divided into 3 × 4. Therefore, the first thin film 150 is divided into regions divided by 20 μm × 15 μm by the beam 150R. Similarly, the second thin film 250 is divided into regions divided by 20 μm × 15 μm by the beam 250R. Considering the widths of the beams 150R and 250R, the region where the thin film surface is exposed is smaller than the size of each partitioned region by this width.

  The shape of the openings 120 and 130 is larger than that of the opening 110, and in this example, it is a rectangle of 1.0 mm × 1.5 mm. The shape of the opening may be a square such as 1.0 mm × 1.0 mm. As described above, the opening 110 is very small compared to the openings 120 and 130. However, in the drawings used for the description, these sizes are adjusted and shown for easy understanding of the structure. Yes.

  In addition, the shape of the opening of these openings 110, 120, and 130 may be a polygon other than a rectangle, a circle, an ellipse, or the like surrounded by a curve, or a straight line. It may be a shape surrounded by a curve. Further, the shape of the opening 120 and the shape of the opening 130 may be different.

  The inner walls of the openings 110 and 210 are formed with an inclination (tapered shape) with respect to a plane (substrate surface) on which the first thin film 150 and the second thin film 250 expand. The inner walls of the openings 120 and 130 are also inclined with respect to the substrate surface. The degree of inclination may not be constant in the opening but may vary. That is, the openings 110, 120, and 130 have an opening area on the first thin film 150 side that is smaller than an opening area on the external space 1000 side. The opening 210 has an opening area on the second thin film 250 side that is smaller than an opening area on the external space 1000 side. If the inner walls of the openings 110 and 210 are tapered, a margin for the incident angle of the electron beam can be secured.

  The first substrate 10 and the second substrate 20 are joined in a region CA along the outer periphery. A space is formed between the first substrate 10 and the second substrate in a region surrounded by the region CA. This space includes an observation space MS and a flow path space FP. The observation space MS is a space formed in a region between the opening 110 and the opening 210, and is a space through which an electron beam used for an electron microscope passes.

  As described above, at least a region of the first thin film 150 where the beam 150R is not disposed and a region of the second thin film 250 where the beam 250R is not disposed are opposed to each other. The observation space MS is a space sandwiched between the opposing regions. In order to simplify the notation, the observation space MS is represented as one space in each figure. However, strictly speaking, the observation space MS includes a plurality of observation spaces MS in a space sandwiched between a region where the beam 150R is arranged in the first thin film 150 and a region where the beam 250R is arranged in the second thin film 250. Have been separated. In other words, the observation space MS corresponds to a region between the first thin film 150 and the second thin film 250 that is sandwiched between regions where the surfaces of both thin films are exposed (regions where no beams are arranged). .

  The channel space FP is a space for connecting the observation space MS and the external space 1000. In this example, the flow path space FP is connected to the external space 1000 through at least two openings, and in this example, is connected to the external space 1000 through the openings 120 and 130.

  One opening is an opening for injecting the sample-containing liquid, and the other functions as an exhaust port for pushing the air in the flow path space FP to the external space 1000. In addition, one opening part (for example, opening part 130) may be formed in the other part of the 1st board | substrate 10, may be formed in the 2nd board | substrate 20, and embodiment mentioned later is used. As illustrated, it may be formed between the first substrate 10 and the second substrate 20.

  The above is the description of the configuration of the sample storage cell 1. Then, the process (observation cell preparation process) for arrange | positioning a sample containing liquid to the sample storage cell 1 and making it the state which can be observed with an electron microscope is demonstrated.

[Observation cell fabrication process]
FIG. 5 is a diagram for explaining a method of injecting a sample-containing liquid into the sample storage cell according to the first embodiment of the present invention. When the sample-containing liquid 700 is injected into the space between the first substrate 10 and the second substrate 20 from the opening 120, the sample-containing liquid 700 moves in the space to reach the observation space MS, and further to the opening 130. To reach. Note that the sample-containing liquid 700 may be injected into the opening 130 instead of the opening 120, but in the following description, it is assumed that the sample-containing liquid 700 is injected into the opening 120.

  The openings 110 and 210 corresponding to the observation space MS are very small compared to the overall size of the sample storage cell 1. Therefore, it is difficult to determine whether the sample-containing liquid 700 has reached the observation space MS simply by visually recognizing the openings 110 and 210. In the sample storage cell 1, since the second substrate 20 is formed of a material that transmits visible light, the position of the sample-containing liquid 700 can be confirmed from the second substrate 20 side.

  Therefore, by controlling the injection amount, injection pressure, and the like from the opening 120 while confirming the position of the sample-containing liquid 700 or the sample itself in the liquid from the second substrate 20 side, the target range (for example, flow The sample-containing liquid 700 can be spread in the path space FP, the observation space MS, and the opening 130). At this time, it is desirable that the sample-containing liquid 700 is injected so as to fill the entire flow path space FP. If a portion not filled with the sample-containing liquid 700 exists as a bubble in the sealed space including the flow path space FP after being sealed with a sealing material as described later, that is until the observation with an electron microscope is performed. This is because bubbles may move to the observation space MS.

  FIG. 6 is a diagram for explaining a method of forming a resin for sealing the sample storage cell in the first embodiment of the present invention. After the sample-containing liquid 700 is filled in the flow path space FP and the observation space MS, the openings 120 and 130 are closed with the sealing materials 320 and 330, so that the flow path space FP and the observation space MS are separated from the external space 1000. To be separated. The sealing materials 320 and 330 are, for example, a resin such as an epoxy resin, and may be a UV curable resin, or a two-component mixed curable resin (for example, a two-component room temperature curing type or a one-component low-temperature curing type) ). In the case of the UV curing type, a resin before curing is formed so as to block the openings 120 and 130, and cured by UV irradiation to form the sealing materials 320 and 330. The internal space separated from the external space 1000 by the sealing materials 320 and 330 may not contain bubbles, or at least the resin so that the resin before curing and the sample-containing liquid 700 are not mixed. Until they are cured, they may be separated from each other (bubbles are present between the sample-containing liquid and the sealing material).

  Since the sample-containing liquid 700 arranged in the observation space MS is separated from the external space 1000, even when the sample storage cell 1 is exposed to a vacuum environment when observation is performed with an electron microscope, the sample-containing liquid 700 is used. Can be prevented from volatilizing and the liquid state can be maintained. In addition, the observation space MS is surrounded by a first thin film 150 and a second thin film 250 of about several tens of nanometers that are transparent to an electron beam.

  Further, since the beam 150R is arranged on the first thin film 150 and the beam 250R is arranged on the second thin film, the overall strength of the thin film can be improved, and the beams 150R and 250R are not arranged. Compared to the case, the area of the observation space MS can be enlarged or the thin film can be made thin. Furthermore, since the external space 1000 is in a reduced pressure state compared to the observation space MS, there is a possibility that the observation space MS is expanded by applying a force so that the first thin film 150 and the second thin film 250 expand to the external space 1000 side. The presence of 150R and 250R can prevent the observation space MS from expanding. The height of the observation space MS (distance between the first thin film 150 and the second thin film 250), that is, the thickness of the sample-containing liquid 700 is large enough to be transmissive to the electron beam.

  Therefore, the electron beam used in the electron microscope (electron beam EB in FIG. 6) passes through the opening 110, passes through the first thin film 150, the sample-containing liquid 700, and the second thin film 250, and further passes through the opening 210. Thus, the entire sample storage cell 1 can be passed. The direction of the electron beam EB may be opposite to the direction shown in FIG.

  The sample injection process in FIG. 5 and the cell sealing process in FIG. 6 may be manual processes or automatic processes. In the case of manual processing, the positional relationship between the tip of the glass capillary attached to the micromanipulator and the openings 120 and 130 is confirmed using a stereomicroscope, and the sample-containing liquid 700 is detected using an injector connected to the glass capillary. Or the sealing materials 320 and 330 may be injected.

  In the case of automatic processing, an apparatus that automatically executes sample injection processing and cell sealing processing to create an observation cell may be used.

[Observation cell manufacturing equipment]
FIG. 7 is a view for explaining sample injection processing of the observation cell manufacturing apparatus according to the first embodiment of the present invention. The observation cell manufacturing apparatus 800 includes a sample injector 810, a UV irradiator 860, and a stage 888. A chip base 825, cup bases 835 and 845, and a sample base 850 are attached to the stage 888. The chip base 825 accommodates the chip 820. The tip 820 is a pipette tip having a nozzle for sucking out the sample-containing liquid 700. The cup base 835 accommodates the sample cup 830 that holds the sample-containing liquid 700. The cup base 845 accommodates a sample cup 840 that holds the pre-curing resin 300 that serves as a sealing material. The sample stage 850 is provided with the sample storage cell 1. The stage 888 is provided with a disposal port 870 for discarding the chip 820.

  With respect to the sample injector 810, the stage 888 can move in the horizontal direction (the left-right direction in FIG. 7, hereinafter referred to as the X direction). In addition, the sample injector 810 has a horizontal direction that is perpendicular to the moving direction of the stage 888 (the depth direction in FIG. 7, hereinafter referred to as the Y direction) and the vertical direction (the vertical direction in FIG. 7, hereinafter referred to as Z). Direction). Therefore, the sample injector 810 and the stage 888 are relatively movable in the X, Y, and Z directions. Note that the sample stage 850 on the stage 888 may be separately movable in the Y direction.

  The sample injector 810 includes a chip attachment part 811, a support part 813, a control part 815 and a chip removal part 817. The tip attachment portion 811 is a portion to which the tip 820 is inserted and attached at the tip. The support portion 813 supports the sample injector 810 so as to move in the Y direction and the Z direction with respect to the apparatus ceiling. The control unit 815 performs control for sucking and holding the liquid in the sample cup in the chip 820 attached to the chip mounting unit 811 and discharging the liquid held in the chip 820. The chip removal unit 817 moves downward to push the chip 820 downward and remove it from the chip attachment unit 811.

  The UV irradiator 860 is a device that irradiates UV light for curing the pre-curing resin 300. The irradiation range of the UV light may include the entire sample storage cell 1 installed on the sample stage 850 or may irradiate portions corresponding to the openings 120 and 130 with spots. If the spots corresponding to the openings 120 and 130 are irradiated with spots, the influence of UV light on the sample can be suppressed.

  FIG. 7A shows a state in which the sample storage cell 1 is transported from a cell storage or the like and installed on the sample stage 850 of the observation cell manufacturing apparatus 800. Subsequently, the stage 888 and the sample injector 810 are moved, and processing is performed in the order shown below. First, the chip 820 is mounted on the chip mounting portion 811 (FIG. 7B). Thereafter, the sample injector 810 sucks up the sample-containing liquid 700 in the sample cup 830 and holds it in the tip 820 (FIG. 7C). The chip 820 holding the sample-containing liquid 700 is moved onto the opening 120 of the sample storage cell 1, and the sample-containing liquid 700 in the chip 820 is discharged and injected into the sample storage cell 1 from the opening 120 (FIG. 7 (d)).

  When the chip 820 is moved onto the opening 120 of the sample storage cell 1, for example, the sample injector 810 recognizes the shape of the sample storage cell 1 using an imaging unit such as a camera, and further, The position of the opening 120 is recognized, and the chip 820 is moved to the position of the opening 120. In addition, by using an imaging unit that images the internal state of the sample storage cell 1 from the second substrate 20 side on the sample stage 850, the degree of injection of the sample-containing liquid 700 is detected, and the injection amount according to the degree, The injection pressure may be adjusted. Subsequently, a sealing process of the observation cell manufacturing apparatus 800 will be described.

  FIG. 8 is a diagram for explaining cell sealing processing of the observation cell manufacturing apparatus according to the first embodiment of the present invention. The sample injector 810 sucks up the pre-curing resin 300 in the sample cup 840 and holds it in the chip 820 (FIG. 8A). The chip 820 holding the pre-curing resin 300 is moved onto the opening 120 of the sample storage cell 1, the pre-curing resin 300 in the chip 820 is discharged and dropped into the opening 120 (FIG. 8D). Then, it is dripped at the opening part 130 (FIG.8 (e)). Note that the resin 300 before curing may be sucked into the chip 820 again before dropping into the opening 130. The same is true even if the pre-curing resin 300 is not the UV curable type but the above-described two-component room-temperature curing type or one-component low-temperature curing type. In this case, irradiation with UV light described later is unnecessary. .

  Subsequently, the sample stage 850 is moved below the UV irradiator 860, and the sample storage cell 1 is irradiated with UV light from the UV irradiator 860. By this irradiation, the pre-curing resin 300 dropped into the openings 120 and 130 of the sample storage cell 1 is cured. As a result, the sample-containing liquid 700 is stored in the internal space of the sample storage cell 1 in a state of being separated from the external space 1000. Further, the sample injector 810 removes the tip 820 from the tip attachment portion 811 by the tip removal portion 817 and discards it in the disposal port 870. The chip 820 may be discarded during the irradiation with UV light.

  Thereafter, the sample storage cell 1 containing the sample-containing liquid 700 is recovered, and a new sample storage cell 1 is installed on the sample stage 850 (FIG. 7A). In addition, although the process of replacing | exchanging the chip | tip 820 for every sample accommodation cell 1 was demonstrated, you may replace | exchange the chip | tip 820 before sucking up the resin 300 before dripping at the opening part 120. FIG.

  The above is the description of the sample injection process and the cell sealing process performed by the observation cell manufacturing apparatus 800. Then, the manufacturing method of the sample storage cell 1 is demonstrated using FIGS.

[Manufacturing method of sample storage cell 1]
The sample storage cell 1 is formed by bonding a first substrate 10 and a second substrate 20. Each of the first substrate 10 and the second substrate 20 is bonded after undergoing a predetermined manufacturing process described below.

[Manufacturing Process for First Substrate 10]
FIG. 9 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the first embodiment of the present invention. FIG. 10 is a diagram illustrating the manufacturing process of the first substrate following FIG. Each figure shows a cross-sectional structure corresponding to FIG. First, the first substrate 10 shown in FIG. 9A is prepared. As described above, the first substrate 10 is a silicon substrate, and in this example, has a thickness of 750 μm. In addition, since the thinning process of FIG.10 (c) mentioned later is unnecessary by using a silicon substrate about 300 micrometers, it is not necessary to use the support substrate 16 shown in FIG.10 (b).

  A thermal oxide film 12 is formed on the first substrate 10 (FIG. 9B), and the thermal oxide film 12 in a region corresponding to the flow path space FP and the observation space MS is removed using a photolithography technique (FIG. 9). (C)). When the thermal oxide film 12 is formed on both surfaces of the first substrate 10, all of the thermal oxide film 12 on one surface (the lower surface in FIG. 9) is removed. Note that both dry etching and wet etching can be applied to remove the film, and the same applies to the following description unless otherwise specified. Further, the thermal oxide film 12 may be left at this time and removed in a later process. Instead of a thermal oxide film, a film formed by a vapor deposition process such as CVD (Chemical Vapor Deposition) or PVD (Physical Vapor Deposition) may be used. Various films formed below may be formed by vapor deposition, plating, or the like.

  Subsequently, the first substrate 10 is etched using the thermal oxide film 12 as a mask to form a cavity 101 (FIG. 9D). In this example, the depth of the cavity 101 is 240 nm. A film 1501 to be the first thin film 150 and a film 1502 to be the beam 150R are formed so as to cover the cavity 101 (FIG. 9E). Then, using the photolithography technique, a part of the films 1501 and 1502 is etched to form the first thin film 150 and the beam 150R in the cavity 101 (FIG. 9F). In this example, the film 1501 is a silicon nitride film and has a thickness of 20 nm. The film 1502 is an amorphous silicon film and has a thickness of 50 nm.

  A stopper film 14 serving as an etching stopper performed in FIG. 10D is formed so as to cover the cavity 101, the first thin film 150, and the beam 150R (FIG. 10A). In this example, the stopper film 14 is an aluminum film and has a thickness of 1 μm. A support substrate 16 including an adhesive layer is attached to the stopper substrate 14 side of the first substrate 10 (FIG. 10B). The pressure-sensitive adhesive layer of the support substrate 16 is provided on the bonding surface, and the pressure-sensitive adhesive force is reduced by applying a stimulus such as heat or light. In this example, an adhesive layer whose adhesive strength is reduced by irradiation with UV light is used. In addition, since the depth of the cavity 101 is very small compared with the stopper film | membrane 14, in FIG. ing. Further, by attaching the support substrate 16, the level difference of the depth of the cavity 101 is embedded by the adhesive layer or the like, but there may be a portion that is not embedded.

  The first substrate 10 supported by the support substrate 16 is thinned (FIG. 10C). For the thinning of the first substrate 10, for example, CMP (Chemical Mechanical Polishing) or wet etching is used. When wet etching is used, for example, a mixed liquid of hydrofluoric acid and ammonium fluoride is used as a liquid that can etch the first substrate 10. The first substrate 10 has a thickness of 750 μm, but is thinned to about 250 μm by this thinning process. Since the first substrate 10 is supported by the support substrate 16, even if the first substrate 10 is thinned, the warp of the first substrate 10 is suppressed, and the strength during the manufacturing process can be maintained.

  Subsequently, openings 110, 120, and 130 are formed in the first substrate 10 using a photolithography technique (FIG. 10D). In this etching process, D-RIE (Deep Reactive Ion Etching) is used. During the etching, the inner walls of the openings 110, 120, and 130 may be processed so as to be inclined with respect to the surface of the first substrate 10. In this etching process, the stopper film 14 and the first thin film 150 serve as an etching stopper.

  By using the stopper film 14, it is possible to prevent the adhesive layer of the support substrate 16 from being exposed when the openings 120 and 130 are opened. When the adhesive layer is exposed, gas may be generated from the organic adhesive layer, but this can be prevented. As described above, if a thin silicon substrate is used as the first substrate 10 in advance, the support substrate 16 may not be used. In this etching step, a space for circulating He gas, which is a refrigerant, is provided between the stage and the stage in order to cool the substrate. If the support substrate 16 is not used and the stopper film 14 does not exist, the He gas flows into the etching chamber when the openings 120 and 130 are opened, causing a problem. Under such conditions, the stopper film 14 needs to be present, and it may be desirable that the stopper film 14 exist even under other conditions.

  After the openings 110, 120, and 130 are formed, UV light is irradiated to reduce the adhesive strength of the adhesive layer and the support substrate 16 is peeled off (FIG. 10E), and the stopper film 14 is etched away (FIG. 10). 10 (f)). Through these steps, the manufacturing process on the first substrate 10 side is completed. Note that the lower surface of the portion constituting the wall of the cavity 101, that is, the area CA (the lower surface in the drawing) of the first substrate 10 is an area to be bonded to the second substrate 20.

[Manufacturing Process for Second Substrate 20]
FIG. 11 is a diagram illustrating a manufacturing process of the second substrate in the sample storage cell according to the first embodiment of the present invention. This figure shows a cross-sectional structure corresponding to FIG. First, the second substrate 20 shown in FIG. As described above, the second substrate 20 is a substrate having transparency to visible light, and is a glass substrate in this example. In this example, the second substrate 20 has a thickness of 700 μm. In addition, since the thinning process of FIG.11 (e) mentioned later becomes unnecessary by using a glass substrate about 300 micrometers, it is not necessary to use the support substrate 26 shown in FIG.11 (d). When the support substrate 26 is not used, it is desirable to use a configuration corresponding to the stopper film as described above instead of the support substrate 26.

  A film 2501 to be the second thin film 250 and a film 2502 to be the beam 250R are formed on the second substrate 20 (FIG. 11B). Then, the second thin film 250 and the beam 250R are formed by using a photolithography technique (FIG. 11C). In this example, the film 2501 is a silicon nitride film like the first thin film 150 and has a thickness of 20 nm. The film 2502 is an amorphous silicon film and has a thickness of 50 nm.

  Subsequently, as in the manufacturing process of the first substrate 10, the support substrate 26 including the adhesive layer is attached so as to cover the second thin film 250 and the beam 250R of the second substrate 20 (FIG. 11D). The support substrate 26 has the same configuration as the support substrate 16. The second substrate 20 supported by the support substrate 26 is thinned (FIG. 11E). Thinning of the second substrate 20 uses, for example, CMP or wet etching. The second substrate 20 has a thickness of 700 μm, but is thinned to about 250 μm by this thinning process. Since the second substrate 20 is supported by the support substrate 26, even if the second substrate 20 is thinned, the warp of the second substrate 20 is suppressed, and the strength during the manufacturing process can be maintained.

  Subsequently, an opening 210 is formed in the second substrate 20 by using a photolithography technique (FIG. 11F). During the etching, the inner wall of the opening 210 may be inclined with respect to the surface of the second substrate 20. Note that before the opening 210 is formed, the portion corresponding to the opening 210 may be irradiated with laser to increase the etching rate of the second substrate 20. In this case, the opening 210 may be formed by opening the laser irradiation portion by wet etching without using a resist mask or the like.

  After forming the opening 210, the support substrate 26 is peeled off by irradiating UV light to reduce the adhesive strength of the adhesive layer (FIG. 11 (g)). Through these steps, the manufacturing process on the second substrate 20 side is completed. The surface around the second thin film 250, that is, the region CA (surface below the drawing) of the second substrate 20 is a region bonded to the first substrate 10.

[Bonding of the first substrate 10 and the second substrate 20]
The first substrate 10 and the second substrate 20 formed in this way are joined in the region CA so that the first thin film 150 and the second thin film 250 face each other. In this example, since the first substrate 10 is silicon and the second substrate 20 is glass, anodic bonding is used for this bonding. By this bonding, the first substrate 10 and the second substrate 20 are firmly bonded, and a space including the flow path space FP and the observation space MS is formed therebetween.

  The sample storage cell 1 described above has been described as one cell in each figure, but in the actual manufacturing process, a plurality of sample storage cells 1 are formed on one substrate at the same time. Therefore, dicing is performed to divide each sample storage cell 1 into individual pieces. In addition, about various conditions, such as the material of each structure in the manufacturing method mentioned above, and the etching method, it is an example, Comprising: It can set to various conditions. The above is the description of the method for manufacturing the sample storage cell 1.

  In the sample storage cell 1 according to the above-described embodiment, since the first substrate 10 and the second substrate 20 are firmly bonded in advance, the sample-containing liquid 700 injected therein hardly leaks, Further, since the second substrate 20 has transparency to visible light, the sample-containing liquid 700 is injected into the internal space between the first substrate 10 and the second substrate 20 while confirming the injection state. be able to. The first thin film 150 is provided with a beam 150R, and the second thin film 250 is provided with a beam 250R. Therefore, since the overall strength of the thin film can be improved as compared with the case where no beam is present, the first thin film 150 and the second thin film 250 can be thinned, or the observation space MS can be expanded. Thus, the sample storage cell 1 with high convenience can be provided.

  Further, the presence of the beams 150R and 250R suppresses direct contact between the first thin film 150 and the second thin film 250. As a result, the occurrence of a phenomenon (sticking phenomenon) in which the first thin film 150 and the second thin film 250 are brought into contact with each other can be suppressed. In addition, the beams 150R and 250R may have a function of fixing the sample depending on the relationship between the shape of the thickness, the arrangement interval, and the like and the size of the sample contained in the liquid.

Second Embodiment
In the second embodiment, a sample storage cell 1A in which a gap film disposed between a first substrate and a second substrate is formed on the first substrate to form a space therein will be described.

  FIG. 12 is a diagram showing the appearance of the sample storage cell in the second embodiment of the present invention. In the first embodiment, the sample storage cell 1 is formed by bonding the first substrate 10 and the second substrate 20, but in the second embodiment, between the first substrate 10 </ b> A and the second substrate 20. The gap film 30 is disposed on the two layers and joined to each other. The height of the gap film 30 determines the distance between the first thin film 150 and the second thin film 250.

  FIG. 13 is a plan view of the sample-receiving cell in the second embodiment of the present invention as viewed from the first substrate side. FIG. 14 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along a cross-sectional line C-C ′ in FIGS. 12 and 13) of the sample storage cell according to the second embodiment of the present invention. FIG. 15 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along the cross-sectional line D-D ′ in FIGS. 12 and 13) of the sample storage cell according to the second embodiment of the present invention.

  The gap film 30 is made of, for example, silicon oxide and has a thickness of 200 nm. The gap film 30 is disposed along the outer periphery between the first substrate 10 </ b> A and the second substrate 20. Therefore, the channel space FP and the observation space MS are formed as a space surrounded by the first substrate 10 </ b> A, the second substrate 20, and the gap film 30. As in the first embodiment, the gap film 30 is determined such that the distance between the first thin film 150 and the second thin film 250 is 10 nm or more and 400 nm or less, and preferably 50 nm or more and 300 nm or less.

  Although not shown in FIGS. 12 and 13, as shown in FIGS. 14 and 15, the same laminated film as the first thin film 150 and the beam 150R is interposed between the gap film 30 and the first substrate 10A. A peripheral thin film 151 is disposed. In this example, the peripheral thin film 151 is formed so as to surround the first thin film 150 and the beam 150R. The first thin film 150 and the peripheral thin film 151 may be connected to form an integral film. In this case, the integral film of the first thin film 150 and the peripheral thin film 151 may be provided with an opening partly on the second substrate 20 side of the openings 120 and 130 so as not to block the opening. . Further, the same film as the second thin film may exist between the second substrate 20 and the gap film 30. This film may be an integral film extending from the second thin film 250 in contact with the observation space MS, or may be a film separated from the second thin film 250. When the integrated film is used, the process for forming the second thin film 250 shown in FIG. 11C is unnecessary in the method for manufacturing the second substrate 20, and the film 2501 may be used as it is as the second thin film 250.

  Subsequently, a manufacturing process on the first substrate 10A side of the sample storage cell 1A will be described. Note that the second substrate 20 is the same as that of the first embodiment, and a description thereof will be omitted. Moreover, in the following description, a different part from 1st Embodiment is demonstrated in detail, and a part other than that may be set as simple description, and the description may be abbreviate | omitted.

[Manufacturing Process for First Substrate 10A]
FIG. 16 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the second embodiment of the present invention. FIG. 17 is a diagram illustrating the manufacturing process of the first substrate following FIG. Each figure shows a cross-sectional structure corresponding to FIG. First, the first substrate 10A shown in FIG. The first substrate 10A is the same as the first substrate 10 in the first embodiment.

  A film 1501 to be the first thin film 150, a film 1502 to be the beam 150R, and a film 3001 to be the gap film 30 are formed on the first substrate 10A (FIG. 16B). In this example, the film 1501 is a silicon nitride film and has a thickness of 20 nm. The film 1502 is an amorphous silicon film and has a thickness of 50 nm. The film 3001 is a silicon oxide film and has a thickness of 200 nm.

  Subsequently, by using a photolithography technique, the gap film 30 is formed by removing the film 3001 in the region corresponding to the channel space FP and the observation space MS (FIG. 16C), and the films 1501 and 1502 are further formed. A part is etched and the 1st thin film 150, the beam 150R, and the peripheral thin film 151 are formed (FIG.16 (d)). The peripheral thin film 151 may be etched using the gap film 30 as a mask.

  A support substrate 16 including an adhesive layer is attached so as to cover the laminated film of the first thin film 150 and the film 1501R of the first substrate 10A (FIG. 16E). Before the support substrate 16 is attached, the stopper film 14 in the first embodiment may be formed. The first substrate 10A supported by the support substrate 16 is thinned (FIG. 17A), and openings 110, 120, and 130 are formed in the first substrate 10A using a photolithography technique (FIG. 17B). ).

  After the openings 110, 120, and 130 are formed, the support substrate 16 is peeled off by irradiating UV light to reduce the adhesive strength of the adhesive layer (FIG. 17C). Through these steps, the manufacturing process on the first substrate 10A side is completed. Note that the lower surface of the gap film 30 (the lower surface in the drawing), that is, the region CA is a region bonded to the second substrate 20.

<Third Embodiment>
In the third embodiment, a sample storage cell 1B in which an internal space and an external space formed between a first substrate and a second substrate are connected on a side surface will be described.

  FIG. 18 is a diagram showing the appearance of the sample storage cell in the third embodiment of the present invention. In the first embodiment, the sample storage cell 1 has the openings 120 and 130 formed in the first substrate 10, but in the third embodiment, the sample storage cell 1 is provided between the first substrate 10B and the second substrate 20 (sample storage). A configuration corresponding to the openings 120 and 130, that is, a configuration in which the internal space and the external space are connected is disposed on the side surface portion of the cell 1B.

  FIG. 19 is a plan view of the sample-receiving cell in the third embodiment of the present invention as viewed from the first substrate side. FIG. 20 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along a cross-sectional line E-E ′ in FIGS. 18 and 19) of the sample storage cell according to the third embodiment of the present invention. FIG. 21 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along the cross-sectional line F-F ′ in FIGS. 18 and 19) of the sample storage cell in the third embodiment of the present invention.

  There are no openings 120 and 130 in the first substrate 10B. On the other hand, as shown in FIGS. 19 and 20, the flow path space FP is connected to the external space 1000 through openings 125 and 135 opened on the side surface of the sample storage cell 1B. The openings 125 and 135 may be the same as the width of the opening 110 (the distance in the vertical direction in FIG. 19), may be narrow, or may be wide.

  Then, the manufacturing process by the side of the 1st board | substrate 10B of the sample storage cell 1B is demonstrated. Note that the second substrate 20 is the same as that of the first embodiment, and a description thereof will be omitted. Moreover, in the following description, a different part from 1st Embodiment is demonstrated in detail, and a part other than that may be set as simple description, and the description may be abbreviate | omitted.

[Manufacturing Process for First Substrate 10B]
FIG. 22 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the third embodiment of the present invention. This figure shows a cross-sectional structure corresponding to FIG. Since the manufacturing process up to FIG. 9F described in the first embodiment is the same in the third embodiment, the description thereof is omitted. The diagram shown in FIG. 22A is a diagram corresponding to FIG. 9F in the first embodiment although the orientation of the cross section shown in the drawing is different. 9F has a shape in which four sides are surrounded by the first substrate 10, the cavity 101B in FIG. 22A has a wall surface (Fig. 22 (a) corresponding to the left and right walls), and the other two sides have no wall surface. That is, the cavity 101B has a groove shape.

  A support substrate 16 including an adhesive layer is attached so as to cover the first thin film 150 and the beam 150R of the first substrate 10B (FIG. 22B). The first substrate 10B supported by the support substrate 16 is thinned (FIG. 22C), and an opening 110 is formed in the first substrate 10B using a photolithography technique (FIG. 22D).

  After forming the opening 110, the support substrate 16 is peeled off by irradiating UV light to reduce the adhesive strength of the adhesive layer (FIG. 22E). Through these steps, the manufacturing process on the first substrate 10B side is completed. Note that the lower surface of the portion constituting the wall of the cavity 101B, that is, the area CA (the lower surface in the figure) of the first substrate 10 is an area where the second substrate 20 is bonded.

<Fourth embodiment>
In the fourth embodiment, a gap film formed on the first substrate is formed between the first substrate and the second substrate so as to form a space inside as in the second embodiment, and A sample storage cell 1C in which the internal space and the external space are connected at the side surfaces as in the third embodiment will be described.

  FIG. 23 is a diagram showing an appearance of a sample storage cell in the fourth embodiment of the present invention. In the second embodiment, the gap film 30 is disposed between the first substrate 10A and the second substrate 20 and bonded to each other. However, in the fourth embodiment, an opening is formed in the side surface portion of the sample storage cell 1C. A gap film 30C having a structure corresponding to the portions 120 and 130 is provided.

  FIG. 24 is a plan view of the sample-receiving cell in the fourth embodiment of the present invention as viewed from the first substrate side. FIG. 25 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along the cross-sectional line G-G ′ in FIGS. 23 and 24) of the sample storage cell in the fourth embodiment of the present invention. FIG. 26 is a schematic diagram showing a cross-sectional configuration (a cross-sectional structure taken along a cross-sectional line H-H ′ in FIGS. 23 and 24) of the sample storage cell according to the fourth embodiment of the present invention.

  The gap film 30C is made of, for example, silicon oxide and has a thickness of 200 nm. The gap film 30 </ b> C is disposed along the two outer sides between the first substrate 10 </ b> C and the second substrate 20. Therefore, the flow path space FP and the observation space MS are formed as a space surrounded by the first substrate 10C, the second substrate 20, and the gap film 30C, and openings 125 and 135 are formed at the ends thereof.

  Although not shown in FIGS. 23 and 24, as shown in FIGS. 25 and 26, between the gap film 30C and the first substrate 10C, the same laminated film as the first thin film 150 and the beam 150R is used. A peripheral thin film 151 is disposed. In this example, the peripheral thin film 151 is formed so as to surround the first thin film 150 and the beam 150R. The first thin film 150 and the peripheral thin film 151 may be connected to form an integral film.

  The manufacturing process on the first substrate 10C side of the sample storage cell 1C is almost the same as that of the second embodiment. The difference is that the arrangement pattern when forming the gap film 30 in FIG. 16C and the openings 120 and 130 in FIG. 17B are not formed. The second substrate 20 is the same as that in the first embodiment. Therefore, the description about the manufacturing method of the sample storage cell 1C is omitted.

<Fifth Embodiment>
In the fifth embodiment, a sample storage cell 1D in which the position of the beam with respect to the thin film is different from that of the first embodiment will be described.

  FIG. 27 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in the fifth embodiment of the present invention. As shown in FIG. 27, the beam 150R is arranged on the outer space 1000 side (opening 110 side) of the first thin film 150. The beam 250R is disposed on the outer space 1000 side (opening 210 side) of the second thin film 250. According to this structure, when the 1st thin film 150 and the 2nd thin film 250 approached, it may contact in a large area. In this case, a phenomenon of sticking due to electrostatic attraction (sticking phenomenon) may occur. At the time of sample observation, since the sample storage cell is exposed to a vacuum environment, a force acts in a direction away from the first thin film 150 and the second thin film 250. However, if the sticking phenomenon is strong and contacts once, even if it is exposed to a vacuum environment, it may not be separated.

  Therefore, in this example, the protrusion 155 is disposed on the surface of the first thin film 150 on the second thin film 250 side, and the protrusion 255 is disposed on the surface of the second thin film 250 on the first thin film 150 side. And the protrusion part 155 and the protrusion part 255 are arrange | positioned facing each other.

  On the other hand, in the case where the projecting portions 155 and 255 are present as in the sample storage cell 1D, even if the first thin film 150 and the second thin film 250 approach each other, the presence of the projecting portion prevents the contact in a wide area. The occurrence of the sticking phenomenon can be suppressed, and even if the sticking phenomenon occurs, the force can be weakened and the first thin film 150 and the second thin film 250 can be easily separated.

  In this example, the protrusion amount of the protrusions 155 and 255 is 40 nm. It is desirable that the protruding amount is determined so that the distance between the protruding portions 155 and 255 is 50% or more of the distance between the first thin film 150 and the second thin film 250. In some cases, only one of the protruding portion 155 and the protruding portion 255 exists.

  It is desirable that the protruding portion 155 be disposed so as to sandwich the first thin film 150 between a part of the region where the beam 150R exists. It is desirable that the protruding portion 255 is disposed so as to sandwich the second thin film 250 between a part of the region where the beam 250R exists. In other words, at least a part of the region of the first thin film 150 where the protruding portion 155 is not disposed and the region of the first thin film 150 where the beam 150R is not disposed are opposed. By arranging in this way, the influence on the region through which the electron beam is transmitted, that is, the observation space MS can be reduced.

  Only one of the beam 150R and the beam 250R may be arranged on the external space 1000 (opening 110) side. In many cases, the first substrate 10 side in which an opening for injecting the sample-containing liquid 700 is provided is installed upward. Therefore, in consideration of the case where bubbles tend to accumulate in the observation space MS, only the beam 150R is arranged on the external space 1000 side for the first thin film 150 arranged on the first substrate 10 in which the opening is formed. Also good. Of course, depending on the application, only the beam 250R may be disposed on the external space 1000 side.

<Sixth Embodiment>
In the sixth embodiment, a sample storage cell 1E in which the configuration corresponding to the openings 120 and 130 in the first embodiment is formed on the second substrate side will be described.

  FIG. 28 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in the sixth embodiment of the present invention. The first substrate 10E is configured such that the openings 120 and 130 are not formed in the first substrate 10 of the first embodiment. Therefore, the manufacturing process on the first substrate 10E side is almost the same as that of the first embodiment, and the openings 120 and 130 are not formed in the process of forming the opening 110.

  In the second substrate 20E, openings 220 and 230 are formed in a configuration corresponding to the openings 120 and 130 in addition to the opening 210. Therefore, the manufacturing process on the second substrate 20E side is substantially the same as the manufacturing process on the second substrate 20 side in the first embodiment, and the openings 220 and 230 are formed in the step of forming the opening 210.

  Thus, an opening for injecting the sample-containing liquid 700 may be formed on the side of the substrate having transparency to visible light.

  Further, instead of forming a cavity on the first substrate side to form an internal space such as the flow path space FP, a cavity may be formed on the second substrate side, or a cavity is formed on both substrates. May be.

<Seventh embodiment>
In the seventh embodiment, a case where a marker is formed on the surface of the sample storage cell 1 will be described. This marker is used for confirming the position of the observation space MS or the position of the sample arranged in the observation space MS during observation with an electron microscope.

  FIG. 29 is a schematic diagram showing markers provided in the sample storage cell according to the seventh embodiment of the present invention. In this example, markers M1, M2, M3, and M4 are formed on the surface of the first substrate 10 (the surface opposite to the surface on which the first thin film 150 is formed). Although the shape of the marker can be various, in this example, a graphic is drawn such that an arrow points to the center of gravity of the observation space MS. The marker may be formed on the second substrate 20 side. Moreover, like the marker M5, a part of the beam 150R may have a shape different from the surroundings (for example, a shape corresponding to the markers M1, M2, M3, and M4).

  The markers M1, M2, M3, M4, and M5 are of a size that can be observed with an electron microscope, and preferably have a size that can be observed even with an optical microscope. The position of the sample with reference to the marker can be confirmed in advance with an optical microscope, and the observation position can be adjusted with the electron microscope as a reference. Various examples in which the marker function is given depending on the shape of the beam will be described later.

<Eighth Embodiment>
In the above-described manufacturing process of the second substrate 20, when performing a process of increasing the etching rate of the second substrate 20 by irradiating the portion corresponding to the opening 210 with a laser before forming the opening 210, It is desirable to form the second thin film 250 (film 2501) with a film that is difficult to absorb laser. In addition, since the wavelength of the laser used in this process is often 300 nm or more and 400 nm or less, it is desirable that the transmittance of the second thin film 250 be high in this wavelength range.

  FIG. 30 is a diagram illustrating the transmittance characteristics of the second thin film of the sample storage cell in the eighth embodiment of the present invention. In FIG. 30, in the silicon nitride film applied to the second thin film 250, a film having a relatively large silicon composition ratio (SiN—Si rich) and a film having a relatively large nitrogen composition ratio (SiN—N). rich). When expressed as SixNy, in this example, SiN-Si rich is x = 5 and y = 1, and SiN-N rich is x = 2 and y = 1. The film thickness is 20 nm.

  Under the above conditions, SiN-N rich has a transmittance of 90% or more at a wavelength of 300 nm to 400 nm. Since the second thin film 250 has such a transmittance, the influence on the second thin film 250 by the laser irradiation can be suppressed. On the other hand, as described above, the film 2502 to be the beam 250R may be a SiN-Si rich silicon nitride film or an amorphous silicon film, and as a result, may be affected by laser irradiation.

  The first thin film 150 may be a silicon nitride film of SiN—Si rich. This silicon nitride film (SiN-Si rich) has a transmittance of 15% or less at a wavelength of 300 nm to 400 nm. In this way, when a sample that is weak with respect to a wavelength of 300 nm or more and 400 nm or less, for example, an organic substance such as a cell is arranged in the observation space MS, the deterioration of the sample can be suppressed. Note that a silicon nitride film having a transmittance of 10% or less with respect to a wavelength of 350 nm or less may be used. Since the first thin film 150 on the side where the openings 120 and 130 are provided is easily affected by UV light or the like when the sealing material is cured, such a low transmittance is desirable. The thin film 250 may also be a SiN-Si rich silicon nitride film.

  As described above, when a UV curable resin is used as the sealing materials 320 and 330 for sealing the openings 120 and 130, the UV light irradiated at the time of curing is prevented from reaching the observation space MS. You can also. Similarly, the second thin film 250 may be a SiN-Si rich silicon nitride film.

<Ninth Embodiment to Eleventh Embodiment>
In the embodiment described above, the openings (openings 120, 125, 130, 135) that connect the flow path space FP and the external space 1000 are disposed at both ends of the flow path space FP. When the end portion of the flow path space FP is located outside the opening portion, the sample-containing liquid 700 does not fully spread at the end portion, and bubbles are easily generated. This situation will be described with reference to FIG.

  FIG. 31 is a diagram for explaining an arrangement example of the sample-containing liquid injected into the channel space of the sample storage cell in the comparative example of the present invention. The sample storage cell 1Z in this example has a flow path space FP formed by a cavity formed in the first substrate 10Z. The end of this flow path space FP exists outside the openings 120 and 130. Such an end portion generates a portion BS in which the sample-containing liquid 700 injected from the opening 120 cannot be spread. The part BS becomes a bubble.

  Such bubbles do not affect the observation in the electron microscope unless their positions are changed. However, the bubbles may move due to the movement until observation. If bubbles move to the observation space MS, observation with an electron microscope becomes impossible. Therefore, it is desirable to prevent bubbles from entering the flow path space FP (and the observation space MS, including the opening). In addition, it is desirable to prevent the bubbles from moving into the observation space MS even if bubbles enter. Therefore, the shape of the flow path space FP is the same as described in the above embodiment. Further, sample storage cells in which measures against bubbles are taken by another method will be described as ninth to eleventh embodiments.

  FIG. 32 is a diagram illustrating the shape of the flow path space of the sample storage cell according to the ninth embodiment of the present invention. The sample storage cell 1 </ b> F has a channel space FP having a shape that once expands from the range of the opening 120 and then narrows to the range of the opening 130 between the opening 120 and the opening 130. The sample-containing liquid 700 injected from the opening 120 is more easily injected into the space when the flow path space FP is expanded. Then, toward the opening 130, the flow path space FP is narrowed, so that bubbles are pushed out from the opening 130. Therefore, it is difficult for bubbles to remain in the flow path space FP.

  In the tenth and eleventh embodiments, sample storage cells 1G and 1H having a configuration that prevents the movement of bubbles when bubbles remain in the flow path space FP will be described with reference to FIGS. The sample storage cells 1G and 1H have a spare space for fixing the position of the remaining bubbles. The spare space is a space narrower than the flow path space FP.

  FIG. 33 is a diagram for explaining the shape of the reserve space provided in the flow path space of the sample storage cell in the tenth embodiment of the present invention. In this example, the spare space SS1 is arranged at the end of the flow path space FP. In this example, the spare space SS1 is arranged on the outer periphery side of the cell with respect to the opening 130. The spare space SS1 may be arranged at a different position as long as it is connected to the end of the flow path space FP.

  The flow path space FP and the spare space SS1 are connected by a connection space CS1. The connection space CS1 connects the flow path space FP and the spare space SS1 in a narrow space, and is the area that is the narrowest in the flow path when going from the flow path space FP to the spare space SS1. That is, the cross-sectional area (passage area) having the direction from the flow path space FP toward the spare space SS1 as a normal vector is the smallest in the connection space CS1.

  If there are bubbles remaining in the flow path space FP, the sample storage cell 1G is tilted or shaken to move the bubbles to the spare space SS1. Due to the shape of the connection space CS1, the bubbles that have entered the spare space SS1 do not move to the flow path space FP unless a large force is received. Therefore, it is possible to reduce the possibility that bubbles are unexpectedly moved to the observation space MS before observation with an electron microscope.

  FIG. 34 is a diagram for explaining the shape of the reserve space provided in the flow path space of the sample storage cell in the eleventh embodiment of the present invention. In this example, the spare space SS2 is arranged at the end of the flow path space FP. In this example, the spare space SS2 is disposed on the first substrate 10H side of the flow path space FP. Note that the spare space SS2 may be disposed on the second substrate 20 side of the flow path space FP.

  The flow path space FP and the spare space SS2 are connected by a connection space CS2. The connection space CS2 connects the flow path space FP and the spare space SS2 in a narrow space, and is the area that is the narrowest in the flow path when going from the flow path space FP to the spare space SS2. That is, the area (passage area) of the cross section having the direction from the flow path space FP toward the spare space SS2 as a normal vector has a minimum value in the connection space CS2.

  If there are bubbles remaining in the flow path space FP, the sample storage cell 1H is tilted or shaken to move the bubbles to the spare space SS2. Due to the shape of the connection space CS2, the bubbles that have entered the spare space SS2 do not move to the flow path space FP unless a large force is received. Therefore, it is possible to reduce the possibility that bubbles are unexpectedly moved to the observation space MS before observation with an electron microscope. Until the observation by the electron microscope is completed, it is desirable to hold the sample storage cell 1H so that the spare space SS2 is located above the flow path space FP. In this way, it is possible to prevent bubbles from coming out of the spare space SS2.

  Note that the connection space CS2 does not have to be narrower than the spare space SS2. In this case, the force to keep the bubbles in the spare space SS2 is weakened, but by keeping the spare space SS2 positioned above the flow path space FP, the bubbles enter the spare space SS2 in the horizontal direction. The movement to can be suppressed.

<Twelfth embodiment>
For the opening and the inner wall portion of the space between the first substrate and the second substrate (flow path space FP and observation space MS), a hydrophilic treatment that imparts hydrophilicity or a lipophilic property that imparts lipophilicity (hydrophobicity) (Hydrophobic) treatment may be applied. For example, as long as it is a hydrophilic treatment, it may be a treatment that covers the surface of the silicon substrate with an oxide film, may be formed by plasma treatment with fluorine and oxygen, or may be formed by heat treatment in an oxygen-containing atmosphere. Further, a natural oxide film formed on the silicon substrate surface may be a hydrophilic surface. On the other hand, in the case of lipophilic (hydrophobic) treatment, fluoride may be formed on the silicon substrate surface by plasma treatment with fluorine and nitrogen, or methyl groups are formed on the silicon substrate surface by HMDS (hexamethyldisiloxane) treatment. May be.

  Although the silicon substrate has been described as an example, even for a glass substrate, the hydrophilic treatment is a treatment for forming a hydrophilic group such as a hydroxyl group, a carboxyl group, or an amino group on the substrate surface, and the lipophilic treatment is a substrate surface. In this case, a hydrocarbon group such as a methyl group, a vinyl group, or an alkyl group may be formed.

  If a sample storage cell that has been processed according to the characteristics of the sample-containing liquid 700 to be injected is used, the sample-containing liquid 700 can be easily injected. For example, when the moisture-based sample-containing liquid 700 is used, the inner wall portion may be subjected to a hydrophilic treatment. At this time, the outer surface of the sample storage cell may be similarly subjected to a hydrophilic treatment.

  On the other hand, the outer surface of the sample storage cell may be subjected to a process opposite to the process performed on the inner wall portion. For example, when the hydrophilic treatment is performed on the inner wall portion of the opening, the outer surface of the sample storage cell may be subjected to a lipophilic (hydrophobic) treatment. In this way, it becomes easy to remove the sample-containing liquid 700 spilled outside during the injection from the cell surface. Moreover, if the sealing material is alcoholic (oil-based), it becomes difficult to enter the opening, and contact with the sample-containing liquid can be avoided to prevent mixing. In addition, surface treatment, such as hydrophilic treatment, may not be performed on part of the inner wall of the opening, and surface treatment may be performed on part of the inner wall.

  FIG. 35 is a diagram for explaining the surface treatment, sample injection treatment, and cell sealing treatment of the sample storage cell in the twelfth embodiment of the present invention. In this example, the sample storage cell 1J has a region SS on the entire inner wall of the opening 120 that has been subjected to a surface treatment that is not compatible with the pre-curing resin 300 used as the sealing material. For example, if the sample-containing liquid 700 is a water system and the sealing material is an alcohol system (oil system), the region SS is subjected to a hydrophilic treatment so as to have hydrophilicity. At this time, the hydrophilic treatment may be applied to the inner wall of the flow path space FP. Note that the opening 130 may be subjected to the same surface treatment as the opening 120. Further, the outer surface of the sample storage cell 1J may be subjected to the same surface treatment as the opening 120 (in this example, hydrophilic treatment).

  If it does in this way, as shown in FIG.35 (c), (d), it will become difficult for the resin 300 before hardening dripped from the chip | tip 820 to enter the inside of the opening part 120 by the influence of area | region SS. Therefore, the sample-containing liquid 700 and the pre-curing resin 300 are hardly brought into contact with each other, and can be made difficult to mix with each other. As shown in the figure, the sample-containing liquid 700 may be injected in such an amount that it does not reach the region SS (so that it does not enter the opening) or slightly overlaps (so that it partially covers the opening). desirable.

  In this configuration, for example, if the sample-containing liquid 700 is alcohol-based (oil-based) like the pre-curing resin 300, it may be difficult to enter the interior due to the influence of the region SS. Even in this case, as shown in FIGS. 35A and 35B, by bringing the chip 820 into contact with the opening 120, the opening 120 is almost closed with the chip 820, and the sample-containing liquid 700 is injected. In this case, it can be easily injected into the flow path space FP. At this time, the inner wall of the flow path space FP may be subjected to a surface treatment having characteristics opposite to those performed on the inner wall of the opening 120, in this example, a lipophilic treatment.

  Note that, regardless of whether the sample-containing liquid 700 is water-based or alcohol-based (oil-based), the sample-containing liquid 700 is removed from a position as close as possible to the flow path space FP by substantially closing the opening 120 with the chip 820. It can also be injected under pressure. At this time, even if the chip 820 and the opening 120 are not completely in contact with each other due to the presence of another opening 130 (exhaust port) connected to the flow path space FP, a certain degree of contact is possible. , Pressurized injection is possible.

  Specifically, as shown in FIGS. 35A and 35B, by bringing the chip 820 into contact with the opening 120, the opening 120 is substantially closed with the chip 820, and the sample-containing liquid 700 is injected. desirable. When the opening 120 has a tapered shape, it is easy to bring the tip of the nozzle of the chip 820 into contact with the opening 120. That is, the opening area on the external space 1000 side (upper opening) of the opening 120 is large, and the opening area on the flow path space FP side (lower opening) is small. At this time, a tip 820 in which the size of the tip of the nozzle of the tip 820 is smaller than the upper opening and larger than the lower opening is used for the injection of the sample-containing liquid 700.

  In this manner, when the tip of the nozzle of the chip 820 is pushed into the opening 120, the tip of the nozzle can be brought into contact with the inner wall of the opening 120 and stopped. Since the nozzle tip can be pushed into the opening 120 and the opening 120 can be closed with the nozzle tip, the sample-containing liquid 700 can be easily injected under pressure. In addition, since the sample-containing liquid 700 can be directly injected from a portion of the opening 120 close to the lower opening side, the sample-containing liquid 700 can be made difficult to remain particularly in the upper opening side of the region SS. As described above, the sealing material is less likely to enter the opening due to the influence of the region SS, and as a result, contact with the sample-containing liquid 700 can be prevented.

<13th Embodiment>
In the thirteenth embodiment, a sample storage cell 1K in the case where the gap film in the second embodiment is in direct contact with the first substrate, for example, a silicon thermal oxide film will be described.

  FIG. 36 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in a thirteenth embodiment of the present invention. In this example, the first substrate 10K is a silicon substrate. The gap film 30K is formed as a thermal oxide film on the surface of the silicon substrate of the first substrate 10K. On the surface of the gap film 30K, a peripheral thin film 151K having the same laminated film as the first thin film 150 and the beam 150R is disposed. The peripheral thin film 151K is in contact with the second thin film 250K and the peripheral thin film 251K of the same laminated film as the beam 250R.

  In this way, the first substrate 10K and the second substrate 20K are connected via the gap film 30K, the peripheral thin film 151K, and the peripheral thin film 251K. The distance between the first substrate 10K and the second substrate 20K is determined by the total film thickness of the gap film 30K, the peripheral thin film 151K, and the peripheral thin film 251K, and the distance between the first thin film 150 and the second thin film 250K is the first thin film 150. And the thickness of the second thin film 250K are determined by the thickness of the gap film 30K. When the gap film 30K is formed of the thermal oxide film of the first substrate 10K (silicon substrate), the distance between the first thin film 150 and the second thin film 250K is precisely controlled due to the good film thickness controllability of the thermal oxide film. can do. The gap film may be a film formed by vapor deposition such as CVD (Chemical Vapor Deposition) or PVD (Physical Vapor Deposition) instead of a thermal oxide film.

  FIG. 37 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the thirteenth embodiment of the present invention. First, as shown in FIG. 37A, the first substrate 10K, which is a silicon substrate, is thermally oxidized to form a thermal oxide film 3001K on the surface. Subsequently, the thermal oxide film 3001K in the region corresponding to the flow path space FP and the observation space MS is removed by using a photolithography technique to form the gap film 30K (FIG. 37B).

  Films 1501K and 1502K are formed so as to cover the first substrate 10K and the gap film 30K (FIG. 37C), and a part of the films 1501K and 1502K is etched to etch the first thin film 150, the beam 150R, and the peripheral thin film 151K. Is formed (FIG. 37 (d)). The subsequent steps are the same as the manufacturing steps in the second embodiment shown in FIGS. The peripheral thin film 151K on the gap film 30K may not exist.

  The second substrate 20K is the same as the manufacturing method in the second embodiment, but is formed so that the film used for the second thin film and the beam, that is, the peripheral thin film 251K also remains in the peripheral portion. When connecting the first substrate 10K and the second substrate 20K, the peripheral thin film 151K and the peripheral thin film 251K are brought into contact with each other.

<Fourteenth embodiment>
In the sample storage cell in the fourteenth embodiment, a spacer is disposed between the first substrate and the second substrate in order to form the flow path space FP and the observation space MS between the first substrate and the second substrate. The case will be described.

  FIG. 38 is a schematic diagram showing a cross-sectional configuration of the sample storage cell in the fourteenth embodiment of the present invention. In this example, an adhesive 40 including a glass filler 41 having a diameter of 200 nm is applied so as to surround the periphery between the first substrate 10L and the second substrate 20L (FIG. 38A). Then, the sample substrate cell 1L is manufactured by bringing the first substrate 10L and the second substrate 20L close to each other and fixing with the adhesive 40 (FIG. 38B). Since the adhesive 40 includes the glass filler 41, the distance between the first substrate 10L and the second substrate 20L is maintained according to the diameter of the glass filler 41.

  The spacer between the first substrate and the second substrate is not limited to the case where an adhesive containing a filler is used as described above, but a material used as a metal sealing material, such as a metal that is easily deformed (for example, indium). You may use, and you may use an adhesive agent together further.

<Fifteenth embodiment>
In the fifteenth embodiment, a sample storage cell 1M in which a beam is formed of the same material as a thin film will be described.

  FIG. 39 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in the fifteenth embodiment of the present invention. In the first thin film 150, a beam 150RM formed integrally with the first thin film 150 is disposed on the observation space MS side. On the second thin film 250, a beam 250RM formed integrally with the second thin film 250 is disposed on the observation space MS side.

  FIG. 40 is a schematic diagram showing a beam manufacturing method in the fifteenth embodiment of the present invention. 40 (e) and (f) correspond to FIGS. 9 (e) and (f) described in the first embodiment. In the first embodiment, the first thin film 150 and the beam 150R are formed of a laminated film of the films 1501 and 1502, but in the fifteenth embodiment, they are collectively formed as one film 1503. The film 1503 is, for example, the same silicon nitride film as the first thin film 150 in the first embodiment.

  After the film 1503 is formed as shown in FIG. 40E, a beam 150R and the first thin film 150 are formed by further etching a part of the film 1503 using a photolithography technique (FIG. 40F). ). This etching is performed so as to leave a necessary film thickness on the first thin film 150. Therefore, the beam 150 </ b> R has a structure that protrudes continuously from the first thin film 150.

<Sixteenth Embodiment>
In the sixteenth embodiment, a sample storage cell 1N in which a thin film is also disposed on the side surface of a beam will be described.

  FIG. 41 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in the sixteenth embodiment of the present invention. In the lower part of FIG. 41, an enlarged view of the vicinity of the broken line is shown. In this example, the beam 150R exists closer to the opening 110 than the first thin film 150N. On the other hand, the protruding direction as a beam (the direction in which the first thin film 150N protrudes) is on the side opposite to the opening 110, that is, the second thin film 250N side. Further, the beam 250R is present closer to the opening 210 than the second thin film 250N. On the other hand, the protruding direction as a beam (the direction in which the second thin film 250N protrudes) is on the side opposite to the opening 210, that is, the first thin film 150N side.

  Even in such a case, the functions of the protrusions 155 and 255 described in the fifth embodiment can be provided. In other words, the presence of the beams 150R and 250R can reduce the contact area between the first thin film 150N and the second thin film 250N, thereby suppressing the occurrence of sticking.

  The surface on the opening 110 side of the beam 150R is exposed, and the side surface and the surface on the second thin film 250N side are covered with the first thin film 150N. The first thin film 150N only needs to be in contact with at least a part of the side surface of the beam 150R.

  FIG. 42 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the sixteenth embodiment of the present invention. A film 1501 is formed on the first substrate 10N (FIG. 42A). A portion of the film 1501 is etched to form a beam 150R (FIG. 42B). Since the surface exposed after etching the film 1501 is the first substrate 10N, the etching can be performed more easily than when the thin film is exposed. Note that the film 1501 is also removed from portions where the openings 120 and 130 are disposed.

  A film 1502 and a film 3001 are formed so as to cover the beam 150R (FIG. 42C). A part of the film 3001 is etched to form the gap film 30N. Further, a part of the film 1502 (the part where the openings 120 and 130 are disposed) is etched to form the first thin film 150N that covers the beam 150R (FIG. 42D). The subsequent process proceeds in the same manner as in the first embodiment, and openings 110, 120, and 130 are formed in the first substrate 10N (FIG. 42E). The same applies to the second substrate 20N.

  Further, after the process up to the step shown in FIG. 42D is completed, the first substrate 10N and the second substrate 20N may be bonded before the openings 110, 120, 130, and 210 are formed.

  FIG. 43 is a diagram illustrating another method for manufacturing the sample storage cell according to the sixteenth embodiment of the present invention. In this manner, the openings 110, 120, 130, and 210 may be formed after the first substrate 10N and the second substrate 20N are bonded. If necessary, the first substrate 10N and the second substrate 20N are thinned before the opening is formed. Note that this manufacturing method is not limited to the case where the thin film protrudes toward the observation space MS by the beam as in this example, but can be applied even when the beam is formed on the observation space side of the thin film.

<Seventeenth Embodiment>
In the seventeenth embodiment, a sample storage cell 1P in which a beam is formed by a part of a first substrate and a second substrate will be described.

  FIG. 44 is a schematic diagram showing a cross-sectional configuration of a sample storage cell according to a seventeenth embodiment of the present invention. In this example, the beam 150RP is formed using a part of the first substrate 10P remaining at the bottom of the opening 110. The beam 250RP is formed using a part of the second substrate 20P remaining at the bottom of the opening 210. In this configuration, since the observation space MS does not change even if the beam is thickened, it is effective for strengthening the beam. For example, the thickness of the beam can be 1 μm or more.

  FIG. 45 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the seventeenth embodiment of the present invention. FIG. 45A is a diagram corresponding to FIG. 16D, and is different in that a film that becomes the beam 150R is not stacked on the first thin film 150P. Thereafter, a part of the first substrate 10P is etched to form the recesses 1201P, 1301P, and 1501P (FIG. 45B). The recess 1201P is formed at a position corresponding to the opening 120. The recess 1301P is formed at a position corresponding to the recess 130. The concave portion 1501P is formed in order to form a convex portion 1501RP that finally becomes the beam 150RP.

  Then, the portions corresponding to the openings 110, 120, and 130 in the first substrate 10P are etched (FIG. 45C). At this time, the shapes of the recesses 1201P, 1301P, and 1501P (projections 1501RP) formed in FIG. 45B remain as they are. In particular, a beam 150RP is formed at the bottom of the opening 110 due to the shape of the recess 1501P (projection 1501RP). The same applies to the second substrate 20P. Thus, since it is not necessary to separately form a film for forming the beam, the beam can be easily manufactured. In the steps in FIGS. 45B and 45C, it is desirable to attach the support substrate 16 as in the first embodiment, but the illustration is omitted in the drawings.

<Eighteenth embodiment>
In the eighteenth embodiment, a description will be given of a sample storage cell 1Q in which a beam is formed by a part of a first substrate and a second substrate, and a thin film is also disposed on a side surface of the beam.

  FIG. 46 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in an eighteenth embodiment of the present invention. In this example, similarly to the seventeenth embodiment, the beam 150RQ is formed as a part of the first substrate 10Q, and the beam 250RQ is formed as a part of the second substrate 20Q. As in the sixteenth embodiment, the first thin film 150Q is also disposed on the side surface of the beam 150RQ, and the second thin film 250Q is disposed on the side surface of the beam 250RQ. As described above, the eighteenth embodiment has a structure in which the configuration of the sixteenth embodiment is applied to the configuration of the seventeenth embodiment.

  More specifically, in this example, the beam 150RQ is present closer to the opening 110 than the first thin film 150Q. On the other hand, the protruding direction as a beam (the direction in which the first thin film 150Q protrudes) is on the side opposite to the opening 110, that is, the second thin film 250Q side. Further, the beam 250RQ exists on the opening 210 side with respect to the second thin film 250Q. On the other hand, the protruding direction as a beam (the direction in which the second thin film 250Q protrudes) is on the side opposite to the opening 210, that is, the first thin film 150Q side.

  Even in such a case, the functions of the protrusions 155 and 255 described in the fifth embodiment can be provided. That is, the presence of the beams 150RQ and 250RQ can reduce the contact area between the first thin film 150Q and the second thin film 250Q, thereby suppressing the occurrence of sticking.

  The surface on the opening 110 side of the beam 150RQ is exposed, and the side surface and the surface on the second thin film 250Q side are covered with the first thin film 150Q. The first thin film 150Q may be in contact with at least a part of the side surface of the beam 150RQ.

  FIG. 47 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the eighteenth embodiment of the present invention. A part of the first substrate 10Q is etched to form the recesses 1201Q, 1301Q, and 1501Q (FIG. 47A). The recess 1201Q is formed at a position corresponding to the opening 120. The recess 1301Q is formed at a position corresponding to the opening 130. The concave portion 1501Q is formed to form a convex portion 1501RQ that finally becomes the beam 150RQ.

  A film 1502 and a film 3001 are formed so as to cover the recesses 1201Q, 1301Q, and 1501Q (FIG. 47B). A part of the film 3001 is etched to form the gap film 30Q. Further, a part of the film 1502 (portion where the openings 120 and 130 are disposed) is etched to expose the openings 1201Q and 1301Q, thereby forming a first thin film 150Q that covers the protrusion 1501RQ (FIG. 47C). ). The subsequent process proceeds in the same manner as in the first embodiment, and openings 110, 120, and 130 are formed in the first substrate 10Q (FIG. 47D). In the etching for forming the opening 110, the etching is stopped when the first thin film 150Q is exposed. In this way, the beam 150RQ having the first thin film 150Q disposed on the side surface is formed. The same applies to the second substrate 20Q.

<Nineteenth embodiment>
In the nineteenth embodiment, a sample storage cell 1S capable of increasing the thickness of a beam will be described. In the seventeenth embodiment, the thickening of the beam is realized by using a part of the substrate as the beam. However, in the sample storage cell 1S, other realization methods are illustrated.

  FIG. 48 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in a nineteenth embodiment of the present invention. In this example, the beam 150RS is disposed on the opening 110 side of the first thin film 150S. The beam 250RS is disposed on the opening 210 side of the second thin film 250S. Even in this configuration, as in the seventeenth embodiment, the observation space MS does not change even if the beam is thickened, which is effective in strengthening the beam. For example, the thickness of the beam can be about 200 nm.

  The manufacturing method for realizing the beams 150RS and 250RS of the sample storage cell 1S manufactures the substrate portion and the thin film portion separately. Hereinafter, the manufacturing method will be specifically described.

  FIG. 49 is a diagram illustrating a manufacturing process of the first substrate in the sample storage cell according to the nineteenth embodiment of the present invention. FIG. 50 is a diagram for explaining the manufacturing process of the first substrate following FIG. 49. First, the support substrate 90S is prepared. The support substrate 90S is a silicon substrate in this example. A film 1501S to be the first thin film 150S and a film 1502S to be the beam 150RS are formed on the support substrate 90S (FIG. 49A). In this example, the film 1501 is a silicon nitride film and has a thickness of 20 nm. The film 1502 is an amorphous silicon film and has a thickness of 200 nm.

  Subsequently, the beam 150RS is formed by etching the film 1502 by using a photolithography technique (FIG. 49B). Then, the film 1501 in the regions 125 and 135 corresponding to the positions of the openings 120 and 130 is etched to form the first thin film 150S (FIG. 49C).

  Subsequently, a first substrate 10S is prepared as a substrate portion (FIG. 50A). The first substrate 10S is a glass substrate in this example. Openings 110, 120, and 130 are formed in the first substrate 10S (FIG. 50B). When the openings 110, 120, and 130 are formed, for example, the etching may be performed after irradiating the opening with a laser to increase the etching rate for hydrofluoric acid.

  A structure including the first thin film 150S shown in FIG. 49C is bonded to the first substrate 10S in which the openings 110, 120, and 130 are neglected (FIG. 50C). In this example, gold (Au) may be formed on the bonding surface to be bonded, and Au—Au bonding may be realized. A support substrate 91S is bonded to the opposite side of the first substrate 10S from the support substrate 90S, and the support substrate 90S is etched (FIG. 50D). The support substrate 91S is, for example, a glass substrate, and the peelable adhesive layer described in the above-described embodiment is formed on the bonding surface.

  FIG. 51 is a diagram for explaining a manufacturing process for the second substrate in the sample storage cell according to the nineteenth embodiment of the present invention. First, the support substrate 95S is prepared. The support substrate 95S is a silicon substrate in this example. A film 3001S to be the gap film 30, a film to be the second thin film 250S, and a film 2502S to be the beam 250RS are formed on the support substrate 95S (FIG. 51A). In this example, the film 1501 is a silicon nitride film and has a thickness of 20 nm. The film 1502 is an amorphous silicon film and has a thickness of 200 nm. The film 3001S is gold (Au) and has a thickness of 200 nm.

  Subsequently, the beam 250RS is formed by etching the film 2502 using a photolithography technique (FIG. 51B). Subsequently, similarly to the first substrate 10S (FIGS. 50A to 50C), the second substrate 20S in which the opening 210 is formed includes the second thin film 250S shown in FIG. 51B. The body is bonded, the support substrate 96S is bonded, and the support substrate 95S is etched (FIG. 51C).

  FIG. 52 is a diagram illustrating a manufacturing process for bonding a first substrate and a second substrate in a nineteenth embodiment of the present invention. In FIG. 51C, the exposed film 3001S is etched to form a gap film 30S (FIG. 52A). Then, the peripheral thin film 151S around the first thin film 150S on the first substrate 10S formed in FIG. 50D and the gap film 30K formed in FIG. 51C are brought into contact with each other and bonded together (FIG. 52). (B)). Thereafter, the adhesive force of the adhesive layer between the support substrate 91S and the first substrate 10S and the adhesive layer between the support substrate 96S and the second substrate 20S is reduced by heating or the like. Then, the sample substrate cell 1S shown in FIG. 48 is manufactured by peeling the support substrate 91 from the first substrate 10SA and peeling the support substrate 96S from the second substrate 20S.

  The first substrate 10S may be a silicon substrate instead of a glass substrate. In this case, the support substrate 90S may be a glass substrate. The second substrate 20S may be a silicon substrate instead of a glass substrate. In this case, the support substrate 95S may be a glass substrate. One of the first substrate 10S and the second substrate 20S may be a glass substrate, and the other may be a silicon substrate.

<Beam arrangement example>
In the above embodiment, the beams 150R and 250R are arranged on the first thin film 150 and the second thin film 250 so as to have a lattice shape, but may be arranged so as to have other shapes. Examples of various shapes of the beam 150R will be described. The same applies to the beam 250R.

  FIG. 53 is a schematic diagram showing various arrangement examples of beams. The beams 150R may be arranged radially (FIG. 53 (a)). The beam 150R may not be arranged at the center portion of the first thin film 150 (the mesh pattern portion in the figure). In this case, for example, as shown in FIGS. 53 (b), (c), and (d), the beam 150R may be disposed.

  In the example shown in FIG. 53 (b), the beam 150R is arranged in a quadrilateral shape at the central portion, and is arranged so as to connect the quadrilateral to the four corners. In the example shown in FIG. 53 (c), the beam 150R is arranged in a quadrangular shape at the central portion, and is arranged so as to connect the quadrilateral to four sides (in this example, the center of the side). In the example shown in FIG. 53 (d), the beam 150R is arranged in a circular shape in the central portion, and is arranged so as to connect the circular shape to the four corners.

<Example of beam shape>
In the above embodiment, the beams 150R and 250R are arranged so that the first thin film 150 and the second thin film 250 are exposed in a square shape, but may be arranged so as to have other shapes. The example of the shape which can take various of the shape which the 1st thin film 150 exposes is demonstrated. The same applies to the beam 250R.

  FIG. 54 is a schematic diagram showing examples of various shapes of beams. In the example shown in FIG. 54A, the beam 150R is arranged so that the first thin film 150 (the mesh pattern portion in the figure) is exposed in a circle (exposed region 150HA1). The exposed portion is not limited to a quadrangle and may be a circle. Further, the thin film 150 may be exposed in various shapes such as a polygon and a figure surrounded by a curve.

  Further, the thin film 150 may be exposed in a plurality of types of shapes. In other words, a plurality of exposed areas are formed by the beam, and at least one exposed area is different from the other exposed areas. For example, in the example shown in FIG. 54B, the beam 150R is arranged so that the first thin film 150 is exposed in various shapes. In particular, as shown in FIG. 54 (b), when the shape of an arrow (exposed area 150HA2), isosceles triangle (exposed area 150HA3) or the like that can indicate directionality is observed with an electron microscope. Easy to locate. This shows the same effect as the marker (particularly the marker M5) as shown in the seventh embodiment. In other words, in order to specify the observation position, it is only necessary that either the shape of the beam or the shape of the exposed region of the thin film has directivity.

<Other embodiments>
The first thin film 150 and the second thin film 250 are not limited to a silicon nitride film, and may be a silicon nitride oxide film, a silicon oxide film, or an amorphous silicon film, and further, a metal film, a metal nitride film, a metal oxide film, and a metal A nitrided oxide film may be used. As described above, these thin films need to be permeable to electron beams. And since it exposes in a vacuum at the time of observation with an electron microscope, it is desirable for these thin films to have barrier property with respect to air | atmosphere or a water | moisture content.

  It is also desirable to prevent the membrane from sagging. Furthermore, it is desirable that the material is not easily corroded by an acid typified by hydrofluoric acid during the production process (even if etched, the surface is hardly roughened and is not easily discolored). In view of these factors, it is desirable in manufacturing to select materials for the first thin film, the second thin film, and the beam. In addition, since it is not necessary to pass an electron beam in a beam as mentioned above, the range of material selection is wider than a 1st thin film and a 2nd thin film.

  Further, the first thin film (or the beam) and the second thin film (or the beam) are used as a contact surface (for example, the area CA shown in the above-described embodiment) when the first substrate and the second substrate are joined. By forming these beams and using these materials as predetermined materials, the bonding strength can be improved and the bonding process can be facilitated. For example, if gold (Au) is used for the beam of at least one substrate and this gold is also formed on the contact surface, it can be firmly bonded even at a low temperature of about 200 ° C. and a low pressure of about 100 MPa. The load on the first thin film and the second thin film can be reduced. A gold paste or the like may be used for the contact surface, and gold may be used for the gap film.

  The first thin film and the second thin film are not limited to a single layer, and may be a film in which a plurality of types of films are stacked. When the first thin film 150 is a silicon oxide film, the thermal oxide film of the first substrate 10 may be used. When the first thin film 150 and the second thin film 250 are formed of a silicon nitride film, the first thin film 150 and the second thin film 250 are easily processed thinly, and the strength of the film is easily secured. Furthermore, the stress can be easily adjusted by controlling the film quality, and the film can be formed so as not to loosen. Further, by arranging the beam, the strength and stress adjustment of the entire film can be further facilitated.

  In order to prevent charge-up of the sample storage cell during observation with an electron microscope, a conductive film such as carbon of several nm is applied to the outer surface of the cell (at least the surface of the first substrate, the second substrate, the first thin film, and the second thin film). It may be arranged in a part). Further, the beams 150R and 250R may be formed of a conductive material.

  In each of the above embodiments, the second substrate has been described as a substrate that is transparent to visible light. However, like the first substrate, the second substrate is a substrate that is not transparent to visible light, such as a silicon substrate. Also good. In this case, both the first substrate and the second substrate are substrates that do not transmit visible light, but the connection between the first substrate and the second substrate, the ease of manufacturing, etc. Considering this, a highly convenient cell can be realized.

  Each of the above embodiments is not a configuration that is applied independently, and may be applied redundantly. For example, a sample storage cell in which the opening 135 in the third embodiment is formed instead of the opening 130 in the first embodiment may be realized. Further, in one sample storage cell, the beam 150R and the beam 250R may be beams described in different embodiments.

DESCRIPTION OF SYMBOLS 1 ... Sample accommodation cell, 10 ... 1st board | substrate, 12 ... Thermal oxide film, 14 ... Stopper film | membrane, 16, 26 ... Support substrate, 20 ... 2nd board | substrate, 30 ... Gap film | membrane, 40 ... Adhesive agent, 41 ... Glass filler 101 ... cavity, 110, 120, 125, 130, 135, 210, 220, 230 ... opening, 150 ... first thin film, 150R, 250R ... beam, 151, 251 ... peripheral thin film, 155, 255 ... projection, 250 ... second thin film, 300 ... resin before curing, 320,330 ... sealing material, 700 ... sample-containing liquid, 800 ... observation cell preparation device, 810 ... sample injector, 811 ... chip mounting portion, 813 ... support portion, 815 ... Control unit, 817 ... Chip removal unit, 820 ... Chip, 825 ... Chip table, 830,840 ... Sample cup, 835,845: Cup table, 850 ... Sample table, 860 ... V illuminator, 870 ... disposal outlet, 888 ... stage, 1000 ... outer space, 1501,1501R, 1502,1503,2501,2501R, 2502,3001 ... film

Claims (17)

A first substrate having a first opening;
A second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening;
A first thin film that closes the second substrate side of the first opening;
A second thin film that closes the first substrate side of the second opening;
A beam disposed on the first thin film;
With
Between the first substrate and the second substrate, there is a flow for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and the external space. Road space is arranged,
At least one of the first substrate and the second substrate has transparency to visible light,
The first thin film and the second thin film are transmissive to an electron beam,
The beam is disposed on the first opening side of the first thin film,
A protrusion is arranged on the surface of the first thin film on the observation space side,
The sample storage cell, wherein a region of the first thin film where the protruding portion is not disposed and a region of the first thin film where the beam is not disposed are opposed at least in part. . A first substrate having a first opening;
A second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening;
A first thin film that closes the second substrate side of the first opening;
A second thin film that closes the first substrate side of the second opening;
A beam disposed on the first thin film;
With
Between the first substrate and the second substrate, there is a flow for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and the external space. Road space is arranged,
At least one of the first substrate and the second substrate is a silicon substrate;
The first thin film and the second thin film are transmissive to an electron beam,
The beam is disposed on the first opening side of the first thin film,
A protrusion is arranged on the surface of the first thin film on the observation space side,
The sample storage cell, wherein a region of the first thin film where the protruding portion is not disposed and a region of the first thin film where the beam is not disposed are opposed at least in part. . The protrusion is disposed on the observation space side of the first thin film and the second thin film,
3. The sample storage cell according to claim 1, wherein the protrusion disposed on the first thin film and the protrusion disposed on the second thin film face each other. A first substrate having a first opening;
A second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening;
A first thin film that closes the second substrate side of the first opening;
A second thin film that closes the first substrate side of the second opening;
A beam disposed on the first thin film;
With
Between the first substrate and the second substrate, there is a flow for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and the external space. Road space is arranged,
At least one of the first substrate and the second substrate has transparency to visible light,
The first thin film and the second thin film are transmissive to an electron beam,
The sample storage cell, wherein the first thin film has a portion in contact with a side surface of the beam. A first substrate having a first opening;
A second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening;
A first thin film that closes the second substrate side of the first opening;
A second thin film that closes the first substrate side of the second opening;
A beam disposed on the first thin film;
With
Between the first substrate and the second substrate, there is a flow for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and the external space. Road space is arranged,
At least one of the first substrate and the second substrate is a silicon substrate;
The first thin film and the second thin film are transmissive to an electron beam,
The sample storage cell, wherein the first thin film has a portion in contact with a side surface of the beam. Furthermore, a beam is also arranged on the second thin film,
The sample storage cell according to claim 4 or 5, wherein the second thin film has a portion in contact with a side surface of the beam. A first substrate having a first opening;
A second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening;
A first thin film that closes the second substrate side of the first opening;
A second thin film that closes the first substrate side of the second opening;
A beam disposed on the first thin film;
With
Between the first substrate and the second substrate, there is a flow for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and the external space. Road space is arranged,
At least one of the first substrate and the second substrate has transparency to visible light,
The first thin film and the second thin film are transmissive to an electron beam,
The sample receiving cell, wherein the beam is arranged as a part of the first substrate. A first substrate having a first opening;
A second substrate disposed opposite to the first substrate and having a second opening at a position facing the first opening;
A first thin film that closes the second substrate side of the first opening;
A second thin film that closes the first substrate side of the second opening;
A beam disposed on the first thin film;
With
Between the first substrate and the second substrate, there is a flow for connecting an observation space between the first thin film and the second thin film on which a sample is arranged, and the observation space and the external space. Road space is arranged,
At least one of the first substrate and the second substrate is a silicon substrate;
The first thin film and the second thin film are transmissive to an electron beam,
The sample receiving cell, wherein the beam is arranged as a part of the first substrate. Furthermore, a beam is also arranged on the second thin film,
2. The region of the first thin film where the beam is not disposed and the region of the second thin film where the beam is not disposed are opposed at least in part. The sample storage cell according to 2, 4, 5, 7 or 8.   The sample storage cell according to claim 1, wherein the beam is made of a material different from that of the first thin film. The beam is an amorphous silicon film,
The sample storage cell according to claim 10, wherein the first thin film is a silicon nitride film. The beam is a silicon nitride film,
The sample storage cell according to claim 10, wherein the first thin film is a silicon nitride film having a nitrogen content higher than that of the beam.   6. The sample storage cell according to claim 1, 2, 4, or 5, wherein the beam is made of the same material as the first thin film. The region of the first thin film where the beam is not arranged is divided into a plurality of divided regions,
The sample storage cell according to claim 1, 2, 4, 5, 7, or 8, wherein at least one of the divided regions has a shape different from that of the other divided regions.   The sample storage cell according to claim 1, 2, 4, 5, 7, or 8, wherein the beam is thicker than the first thin film. The first substrate is a silicon substrate;
The sample storage cell according to claim 1, 4 or 7, wherein the second substrate is a glass substrate. The sample storage cell according to claim 1, 2, 4, 5, 7, or 8, wherein the first substrate is transmissive to visible light.

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