JP2016213153A - Sample storage cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample storage cell that is enhanced in convenience.SOLUTION: A sample storage cell includes a first substrate having a first opening portion, a second substrate which is arranged to face the first substrate and has a second opening at a position facing the first opening, a first thin film which blocks the second substrate side of the first opening portion, a second thin film which blocks the first substrate side of the second opening portion, and a gap film which is disposed at least between the first opening portion and the second opening portion so as to be sandwiched between the first thin film and the second thin film. Between the first substrate and the second substrate are arranged an observation space surrounded by the first thin film, the second thin film, and the gap film, and a flow path space for connecting the observation space and the external space. The first thin film and the second thin film has transparency to electron beams.SELECTED DRAWING: Figure 3

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, and the gap control between the seat and the lid is difficult. 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, at least the first opening and the second opening A gap film disposed between the first thin film and the second thin film, and the first thin film and the second substrate between the first substrate and the second substrate. An observation space surrounded by two thin films and the gap film, and a flow path space for connecting the observation space and the external space, and the first thin film and the second thin film are And a sample storage cell characterized by having permeability. According to this, the convenience of the cell which accommodates a sample can be improved.

  At least one of the first substrate and the second substrate may be transparent to visible light. According to this, it is possible to easily confirm the position of the sample existing in the flow path space.

  At least one of the first substrate and the second substrate may be a silicon substrate. According to this, the manufacturing process of a sample storage cell can be made easy.

  The columnar gap film may be disposed in a region sandwiched between the first opening and the second opening. According to this, an observation space can be enlarged efficiently.

  The flow path space may be connected to the external space via at least two openings, and any one of the at least two openings may be disposed on the first substrate. According to this, the sample can be easily injected into the channel space.

  The first substrate may have at least two openings in addition to the first opening, and the two openings and the flow path space may be separated via the first thin film. . According to this, the manufacturing process of a sample storage cell can be made easy.

  The gap film may be made of a material different from that of the first thin film and the second thin film. According to this, the manufacturing process of a sample storage cell can be made easy.

  Gold may be formed between the gap film and at least one of the first thin film and the second thin film. According to this, the first thin film and the second thin film can be firmly bonded via the gap film. Moreover, even if a pressure difference arises between external space and observation space during a measurement, it can suppress that a 1st thin film and a 2nd thin film spread.

  The gap film may be formed with a layer containing at least gold. According to this, the first thin film and the second thin film can be firmly bonded via the gap film. Moreover, even if a pressure difference arises between external space and observation space during a measurement, it can suppress that a 1st thin film and a 2nd thin film spread.

  The gap film may have a recess formed on one side of the first thin film and the second thin film. According to this, the bonding strength between the first thin film and the second thin film can be improved via the gap film.

  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. It is a figure explaining the manufacturing process regarding joining of the 1st board | substrate and 2nd board | substrate in 1st Embodiment of this invention, and formation of opening. It is a figure explaining the manufacturing process regarding joining of the 1st board | substrate and 2nd board | substrate in 2nd Embodiment of this invention. It is a figure explaining the manufacturing process regarding joining of the 1st board | substrate and 2nd board | substrate in 3rd Embodiment of this invention. It is a figure explaining the manufacturing process regarding joining of the 1st board | substrate and 2nd board | substrate in 4th Embodiment of this invention. It is a figure explaining the transmittance | permeability characteristic of the 2nd thin film of the sample storage cell in 5th 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 6th 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 7th Embodiment of this invention. It is a figure explaining the surface treatment of a sample accommodation cell, sample injection processing, and cell sealing processing in an 8th embodiment of the present invention. It is a figure explaining the manufacturing process of the sample storage cell which has a gap film | membrane (especially pillar part) in 9th Embodiment of this invention. It is a figure explaining the manufacturing process of the sample storage cell in 10th Embodiment of this invention. It is a figure explaining the manufacturing process of the sample storage cell following FIG. It is a schematic diagram which shows the cross-sectional structure of the sample storage cell in 11th Embodiment of this invention. It is a schematic diagram which shows the example of various arrangement | positioning of observation space.

<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 a first substrate 10 and a second substrate 20 via a gap film 50. The first substrate 10 is, for example, a silicon substrate. The second substrate 20 is a substrate that is transparent to visible light, and is, for example, a glass substrate. The first substrate 10 may also be a substrate (for example, a glass substrate) that is transparent to visible light, like the second substrate 20. Further, both the first substrate 10 and the second substrate 20 may be silicon substrates.

  The gap film 50 is a film that joins the first substrate 10 and the second substrate 20 to form a space (flow path space FP and observation space MS) between the first substrate 10 and the second substrate 20. The gap film 50 includes a peripheral part 510, a flow path forming part 530, and a pillar part 550, which will be described later. These may be collectively expressed as a gap film 50.

  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.

  As shown below, a gap film 50 connected to both of the first thin film 150 and the second thin film 250 partially exists. The gap film 50 extends from an outer portion of the opening 110 and the opening 210 (a portion where there is no opening between the first substrate 10 and the second substrate 20). Therefore, the gap film 50 (the flow path forming portion 530 and the column portion 550) has a beam-like function, and can support the first thin film 150 and the second thin film 250.

  When the first thin film 150 and the second thin film 250 are not supported by the gap film 50, these thin films need to be supported outside the openings 110 and 210. That is, since the region (for example, near the center of the opening) away from the portion supporting the thin film in the region through which the electron beam passes increases, it is necessary to increase the strength of the thin film. On the other hand, by supporting the first thin film 150 and the second thin film 250 with the gap film 50 as in this example, it is possible to secure a region through which the electron beam passes and reduce a region away from the supporting portion. it can. Therefore, when the thin film is supported by the gap film, it is possible to keep the thin film from being damaged even if the strength of the thin film is lowered as compared with the case where the thin film is not supported by the gap film. Conversely, it can be interpreted that the strength of the thin film is increased even at the same film thickness. As a result, the thin film can be further thinned.

  A gap film 50 is disposed between the first thin film 150 and the second thin film 250. As shown in FIGS. 2, 3, and 4, the gap film 50 includes a peripheral portion 510, a flow path forming portion 530, and a pillar portion 550. The peripheral portion 510 is a portion that faces the end portion of the sample storage cell 1. The flow path forming part 530 is a part arranged so as to determine the position of the observation space MS between the opening part 110 and the opening part 210. The column part 550 is a part surrounded by the observation space MS. The column part 550 may not exist. The peripheral portion 510 and the flow path forming portion 530 are not clearly separated from each other, but are defined separately for convenience. On the other hand, the column part 550 is surrounded by the observation space MS and exists independently of the surroundings.

  In this example, the gap film 50 is made of gold (Au). The gap film 50 is bonded by Au—Au bonding near the center of the film. That is, before this bonding, the gap film 50 is divided into a layer on the first thin film 150 side and a layer on the second thin film 250 side. Then, the gap film 50 is formed by bonding. In the following description of the manufacturing method, details of the formation process of the gap film 50 will be shown. Note that a laminated film in which gold is formed on the bonding surface and the other part is formed of a silicon oxide film, a silicon nitride film, or the like may be used. In this example, gold functions as an adhesive layer during bonding. On the other hand, gold may not be formed. The thickness of the gap film 50 is not less than 10 nm and not more than 400 nm, desirably, not less than 50 nm and not more than 300 nm, and in this example, is 200 nm. This thickness corresponds to the distance between the first thin film 150 and the second thin film 250.

  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 (the film thickness of the gap film 50) is set depending on the size of the sample, that is, the observation object (for example, a cell), it is at least larger than the observation object. Need to be. 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.

  The gap film 50 may or may not transmit an electron beam. The electron beam only needs to pass through the first thin film 150 and the second thin film 250 in the region where the gap film 50 is not disposed. 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, it is not necessary to set the distance between the first thin film 150 and the second thin film 250 (the film thickness of the gap film 50) to be equal to or longer than the distance through which the electron beam cannot pass.

  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, by adjusting the arrangement pattern of the gap film 50 (the flow path forming unit 530), that is, the arrangement pattern of the flow path space FP, the opening 110 and the opening 210 are formed without improving the strength of the thin film. The shape of the opening can be set larger.

  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 bonded via the first thin film 150 and the second thin film 250 with the gap film 50 (peripheral portion 510, flow path forming portion 530, and column portion 550) interposed therebetween. A space surrounded by the first thin film 150, the second thin film 250, and the flow path forming part 530 is formed between the first substrate 10 and the second substrate. 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 surrounded by the first thin film 150, the second thin film 250, and the flow path forming unit 530, and is used for an electron microscope. The space through which the electron beam passes.

  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. The channel width of the channel space FP is 0.2 μm or more and 500 μm or less, and preferably 100 μm or more and 400 μm or less. On the other hand, the flow path width of the observation space MS (the region where the first substrate 10 and the second substrate 20 are open) is 0.2 μm or more and 50 μm or less, and desirably 2 μm or more and 40 μm or less. It is necessary to increase the strength of the thin film as the flow path width becomes wider in the observation space MS. On the other hand, if the flow path space FP and the observation space MS are both narrowed, for example, narrower than the distance (the thickness of the gap film 50) between the first thin film 150 and the second thin film 250, the sample is removed. It may become impossible to pass. For example, when the gap film 50 is formed as thin as possible according to the size of the sample, the sample cannot pass if the channel width is narrower than this. Therefore, the first thin film 150 and the second thin film 250 can be made thinner by narrowing the channel width so that the sample can pass through. In this example, by forming the column portion 550, the observation space MS can be efficiently increased while increasing the portion supporting the thin film.

  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. One opening (for example, the opening 130) may be formed in another part of the first substrate 10, or may be formed in the second substrate 20. In addition, an opening may be formed between the first substrate 10 and the second substrate 20 so that the channel space FP reaches the side surface of the sample storage cell.

  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 curable type or a one-component low-temperature curable 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 disposed 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.

  In addition, since the gap film 50 is disposed between the first thin film 150 and the second thin film 250, the strength of the thin film itself can be reduced. As a result, the area of the observation space MS can be increased or the thin film can be made thinner than when the gap film 50 is not disposed. Furthermore, since the external space 1000 is in a decompressed state with respect to the observation space MS during observation with an electron microscope, the observation space MS can be expanded by applying force so that the first thin film 150 and the second thin film 250 expand toward the external space 1000 side. However, if the first thin film 150 and the second thin film 250 are connected by the gap film 50, the distance between the first thin film 150 and the second thin film 250 is at least in a portion in contact with the gap film 50. Is fixed, it is possible to suppress the observation space MS from expanding.

  Therefore, 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 can be kept large enough to be transmissive to the electron beam. it can. 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). Subsequently, it is dropped into the opening 130 (FIG. 8E). 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 via a gap film 50. Each of the first substrate 10 and the second substrate 20 is bonded after undergoing a predetermined manufacturing process described below.

  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. Note that the use of a silicon substrate having a thickness of about 300 μm eliminates the need for a thinning process in FIG. 19D described later, and thus a support substrate for the thinning process may not be used.

  A first thin film 150 and a film 5001 to be a part of the gap film 50 are formed on the first substrate 10 (FIG. 9B). The first film 150 is a silicon nitride film and has a thickness of 20 nm. The film 5001 is gold (Au) and has a thickness of 100 nm. These films may be formed by vapor deposition such as CVD (Chemical Vapor Deposition), PVD (Physical Vapor Deposition), or plating. When the first film 150 is not a silicon nitride film but a silicon oxide film, it may be a thermal oxide film formed by thermally oxidizing the first substrate 10 of the silicon substrate. The same applies to various films formed below.

  A part of the film 5001 is removed by using a photolithography technique (FIG. 9C). The area to be removed is an area corresponding to the flow path space FP and the observation space MS. As a result, the peripheral part 511, the flow path forming part 531 and the column part 551 are formed. 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. Alternatively, the peripheral portion 511, the flow path forming portion 531 and the column portion 551 may be formed by screen printing using a paste-like material in which an organic solvent is mixed with gold particles having a particle size of about several tens of nm.

  Here, a part of the first film 150 may be removed. In the case of removal, the removed region is a region corresponding to the position where the openings 120 and 130 are formed. In this example, this step does not exist. The case of removing a part of the first film 150 will be described in another embodiment.

  Thereafter, a support substrate including an adhesive layer is attached to the side of the first substrate 10 where the first thin film 150 is formed. The pressure-sensitive adhesive layer of the support substrate 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.

  The first substrate 10 supported by the support substrate is thinned (FIG. 9D). 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, 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. As described above, the first substrate 10 on which the gap film and the first thin film 150 are arranged is manufactured. In this example, the second substrate 20 is formed in the same manner. In this example, the 2nd board | substrate 20 is a board | substrate which has the transmittance | permeability with respect to visible light, as mentioned above, Comprising: In this example, it is a glass substrate, and has a thickness of 700 micrometers. Note that by using a glass substrate of about 300 μm, it is not necessary to perform the thinning process.

  FIG. 10 is a diagram for explaining a manufacturing process related to the bonding of the first substrate and the second substrate and the formation of the opening in the first embodiment of the present invention. As described above, the first substrate 10 and the second substrate 20 on which a thin film or the like is formed are arranged such that the first thin film 150 and the second thin film 250 face each other (FIG. 10A), and the first thin film 150 is formed. The gap film (peripheral part 511, flow path forming part 531 and column part 551) arranged on the upper side and the gap film (peripheral part 511, flow path forming part 531 and column part 551) arranged on the second thin film 250 Are brought into contact with each other and joined (FIG. 10B). Each surface is gold (Au), and Au-Au bonding is realized. That is, gold functions as an adhesive layer. Any material that functions as an adhesive layer is not limited to gold.

  By this bonding, a gap film 50 (peripheral portion 510, flow path forming portion 530 and column portion 550) is formed between the first substrate 10 and the second substrate 20, and the first thin film 150 is interposed via the gap film 50. And the second thin film 250 are firmly bonded, and a space including the flow path space FP and the observation space MS is formed between them. In addition, according to the Au—Au bonding, a strong bonding can be performed even at a low temperature of about 200 ° C. and a low pressure of about 100 MPa, so that the load on the first thin film 150 and the second thin film 250 can be reduced.

  Subsequently, using the photolithography technique, openings 110, 120, and 130 are formed in the first substrate 10, and openings 210 are formed in the second substrate 20 (FIG. 10C). In this etching step, D-RIE (Deep Reactive Ion Etching) may be used, or wet etching may be 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. Similarly, the inner wall of the opening 210 may be processed so as to 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.

  In this way, the sample storage cell 1 is manufactured. Although the sample storage cell 1 has been described as one cell in each drawing, a plurality of sample storage cells 1 are simultaneously formed on one substrate in an actual manufacturing process. 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.

  In this state, in the sample storage cell 1, not only the first thin film 150 blocks the opening 110 but also the openings 120 and 130. This is because the film corresponding to the openings 120 and 130 is not removed before the first substrate 10 and the second substrate 20 are bonded together via the gap film 50. In the sample storage cell 1 configured as described above, the channel space FP and the external space 1000 are separated by the first thin film 150. Therefore, the sample-containing liquid cannot be injected into the observation space MS as it is.

  Therefore, in this example, the first thin film 150 that closes the openings 120 and 130 is broken by physical force. The flow path space FP is connected to the external space 1000 through the hole generated thereby. An example of the physical force is a method of forming a hole by pressing a needle-like object against the first film 150 positioned at the bottom BA of the openings 120 and 130. Note that the physical force is not limited to a method of transmitting force by contacting the first thin film 150, but a method of indirectly transmitting force to the first thin film 150 by strongly blowing air or the like. There may be. For example, in the observation cell manufacturing apparatus 800, by forming a hole in the first thin film 150 at the bottom BA, the observation space MS is not exposed to the external space 1000 until the sample-containing liquid is injected, and the inside is cleaned. Can also be kept. 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 via the gap film 50, the sample-containing liquid 700 injected into the sample containing cell 1 There is almost no leak. 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. You can also. In addition, since the gap film 50 (in this example, the flow path forming portion 530 and the column portion 550) connected between the first thin film 150 and the second thin film 250 is disposed, the strength of the thin film itself is reduced. You can also As a result, the area of the observation space MS can be made larger or the thin film can be made thinner than when the flow path forming part 530 and the column part 550 are not present.

  Further, during observation with an electron microscope, the external space 1000 is in a decompressed state relative to the observation space MS, so that the observation space MS can be expanded by applying force so that the first thin film 150 and the second thin film 250 expand toward the external space 1000. There is sex. As in this example, if the first thin film 150 and the second thin film 250 are connected by the gap film 50, the first thin film 150 and the second thin film 250 are at least in contact with the gap film 50. Since the distance is fixed, the observation space MS can be prevented from expanding. For the same reason, a phenomenon (sticking phenomenon) in which the first thin film 150 and the second thin film 250 approach and adhere to each other can also be suppressed. Thus, according to the sample storage cell 1, convenience can be improved.

<Second Embodiment to Fourth Embodiment>
Subsequently, various examples of the state of the first substrate and the second substrate when the first substrate 10 and the second substrate 20 are bonded will be described. Of various examples, three examples (FIGS. 11 to 13) will be described as the second to fourth embodiments.

  FIG. 11 is a diagram for explaining a manufacturing process related to bonding of the first substrate and the second substrate in the second embodiment of the present invention. In the sample storage cell 1A of this example, the gap film 50 (the peripheral portion 510, the flow path forming portion 530, and the column portion 550) is formed on the first substrate 10 side, but not on the second substrate 20 side. That is, the second thin film 250 is formed on the surface on the second substrate 20 side. Therefore, the gap film 50 and the second thin film 250 become a bonding surface.

  As in the first embodiment, in order to realize Au—Au bonding, the bonding surface side of the gap film 50 needs to be formed of gold, and the bonding surface side of the second thin film 250 needs to be formed of gold. On the other hand, bonding may be performed by a method other than Au-Au bonding. In this case, the bonding force between the second thin film 250 and the gap film 50 (particularly in the observation space MS) may be weak or may not be bonded. In this case, in the measurement environment, that is, when the external space 1000 is a vacuum, the second thin film 250 and the gap film 50 are separated from each other in the observation space MS, and the first thin film 150 and the second thin film 250 are located on the external space 1000 side. It will spread. On the other hand, even if the second thin film 250 and the gap film 50 are not joined in the observation space MS, the gap film 50 has an effect of supporting the second thin film 250 in the manufacturing process, so that it is not wasted.

  FIG. 12 is a diagram for explaining a manufacturing process related to the joining of the first substrate and the second substrate in the third embodiment of the present invention. In the sample storage cell 1B of this example, compared with the sample storage cell 1A in the second embodiment, the first substrate 10 and the second substrate 20 have the openings 110, 120, 130, and 210 formed in advance. Be joined.

  FIG. 13 is a diagram illustrating a manufacturing process related to the bonding of the first substrate and the second substrate in the fourth embodiment of the present invention. In the sample storage cell 1C of this example, the first thin film 150 at the bottom of the openings 120 and 130 is removed in advance as compared with the sample storage cell 1B in the third embodiment. The step of removing a part of the first thin film 150 may be performed after the step described in FIG. If it does in this way, the opening parts 120 and 130 will be connected with the flow-path space FP at the time of 1 C of sample accommodation cells being formed.

<Fifth 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 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. 14 is a diagram for explaining the transmittance characteristics of the second thin film of the sample storage cell according to the fifth embodiment of the present invention. In FIG. 14, 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.

  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 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.

<Sixth and Seventh Embodiments>
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.

  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.

  In the sixth and seventh embodiments, sample storage cells 1D and 1E 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 1D and 1E 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. 15 is a diagram for explaining the shape of the reserve space provided in the flow path space of the sample storage cell in the sixth 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.

  When there are bubbles remaining in the flow path space FP, the sample storage cell 1B 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. 16 is a diagram for explaining the shape of the reserve space provided in the flow path space of the sample storage cell in the seventh 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 10C 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.

  When there are bubbles remaining in the flow path space FP, the sample storage cell 1E 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 1E so that the preliminary 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.

<Eighth 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, in the case of hydrophilic treatment, as long as the silicon substrate surface is exposed, it may be covered with an oxide film, may be formed by plasma treatment with fluorine and oxygen, or in an oxygen-containing atmosphere. You may form by the heat processing of. 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 part where the silicon substrate is exposed on the surface has been described as an example, when there is a part where the glass substrate is exposed on the surface, the hydrophilic treatment is performed on the surface of the substrate such as a hydroxyl group, a carboxyl group, or an amino group. The treatment to form a group, and the lipophilic (hydrophobic) treatment may be a treatment to form a hydrocarbon group such as a methyl group, a vinyl group, or an alkyl group on the substrate surface.

  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. 17 is a diagram for explaining the surface treatment, sample injection treatment, and cell sealing treatment of the sample storage cell in the eighth embodiment of the present invention. In this example, the sample storage cell 1F 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 1F 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.17 (c), (d), the resin 300 before hardening dripped from the chip | tip 820 will become difficult 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. 17A and 17B, 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. 17A and 17B, 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 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. As described in the first embodiment, when the first thin film 150 that closes the openings 120 and 130 is present, the first thin film 150 can be broken by this pressure injection.

<Ninth Embodiment>
In the ninth embodiment, an example will be described in which the gap film is formed on the first thin film 150 and the recess is formed on the second thin film 250 side. The gap film and the second thin film 250 are joined to each other by the effect of the concave portion like a sucker. Hereinafter, a sample storage cell 1G having a gap film (a column part 550 as an example) in which a recess is formed will be described.

  FIG. 18 is a diagram for explaining a manufacturing process of a sample storage cell having a gap film (particularly, a column) in the ninth embodiment of the present invention. First, after the step of FIG. 9C or FIG. 9D, a substrate surface fluoride treatment is performed to form a fluoride film 575 (FIG. 18A). As a result, the column portion 550 (gap film) is covered with the fluoride film 575. Thereafter, anisotropic dry etching is performed to remove the fluoride film 575 other than the side surface of the column portion 550. As a result, the side wall 570 which is a part of the fluoride film 575 remains on the side surface, and the upper surface of the column portion 550 is exposed (FIG. 18B).

  Subsequently, soft wet etching is performed on the column portion 550 using an etching solution. As a result, the exposed upper surface of the column portion 550 is slightly etched (FIG. 18C). The etching amount may be, for example, 1 nm to 30 nm, preferably 5 nm to 20 nm. Further, the side wall 570 is hardly etched by this etching solution. Therefore, as shown in FIG. 18C, the concave portion QA surrounded by the side wall 570 with the top surface of the column portion 550 as the bottom portion is formed. Then, as shown in FIG. 12 or FIG. 13, when the first substrate 10 and the second substrate 20 are joined, the second thin film 250 enters along the recess QA, and the second thin film 250 and the column portion 550 Are joined.

<Tenth Embodiment>
In the tenth embodiment, a sample storage cell 1H in which the flow path space FP and the observation space MS are formed after the first substrate 10 and the second substrate 20 are joined will be described.

  FIG. 19 is a diagram for explaining a manufacturing process of the sample storage cell according to the tenth embodiment of the present invention. FIG. 20 is a diagram for explaining the manufacturing process of the sample storage cell subsequent to FIG. First, a glass substrate 5010 is prepared (FIG. 19A). The glass substrate 5010 is irradiated with a laser to increase the etching rate of the irradiated area LA (FIG. 19B). The region LA irradiated with the laser is a region that finally becomes the channel space FP and the observation space MS.

  Subsequently, the second thin film 250 is formed on one surface side of the glass substrate 5010, and the second substrate 20 is further bonded (FIG. 19C). This bonding is realized by anodic bonding or using an adhesive layer. Then, a thinning process is performed on the glass substrate 5010 to form a glass layer 5020 having a thickness of about 200 nm (FIG. 19D). The thinning process is performed by CMP or the like. Then, the 1st thin film 150 is formed on the glass layer 5020, and also the 1st board | substrate 10 is joined (FIG.19 (e)). This bonding is also realized by anodic bonding or using an adhesive layer as described above.

  Subsequently, the openings 110, 120, and 130 are formed in the first substrate 10 and the opening 250 is formed in the second substrate 20 (FIG. 20A). Then, the first thin film 150 present at the bottom BA of the openings 120 and 130 is removed. At this time, if an adhesive layer is provided between the first thin film and the first substrate, the adhesive layer is also removed (FIG. 20B). As a result, the glass layer 5020 is exposed at the bottom BA of the openings 120 and 130. The exposed glass layer 5020 is a region irradiated with a laser.

  Subsequently, the glass layer 5020 is etched by introducing an etchant through the openings 120 and 130. The glass layer 5020 has a high etching rate in the region irradiated with the laser. Accordingly, the etching of the glass layer 5020 in the region irradiated with the laser proceeds (FIG. 20C). The etching solution continues to be introduced into the inside through the openings 120 and 130, and the region other than the region irradiated with the laser remains. As a result, a gap film (peripheral portion 510, flow path forming portion 530, and column portion 550) is formed between the first substrate 10 and the second substrate 20 (FIG. 20D). In this way, the sample storage cell 1H is manufactured.

<Eleventh embodiment>
In the eleventh embodiment, in order to improve the bonding strength between the first substrate and the second substrate, a paste-like material in which an organic solvent is mixed with gold particles having a particle size of about a submicron (hereinafter simply referred to as an adhesive). ) Will be described with reference to FIG. In addition, since the particle diameter of the gold particles is about submicron, the first thin film 150 and the second thin film 250 cannot pass the electron beam simply by being sandwiched between the first substrate 10 and the second substrate 20. It may spread to a certain distance. Therefore, the sample storage cell 1J has a structure in which the first thin film 150 and the second thin film 250 can maintain a distance equivalent to that of the above embodiment even when such an adhesive is used.

  FIG. 21 is a schematic diagram showing a cross-sectional configuration of a sample storage cell in an eleventh embodiment of the present invention. As shown in FIG. 21A, the first substrate 10J includes a recess 14 that forms a space into which the adhesive 410 enters. The concave portion 14 is formed along the end portion of the first substrate 10J so as to correspond to the region where the peripheral portion 510 was present in the above embodiment. The depth of the recessed part 14 should just be a grade which the particle size of the gold particle in the adhesive agent 410 is embedded at least. Therefore, for example, the recess 14 may be formed with a depth of several μm.

  The adhesive 410 is formed by screen printing or the like so as to be embedded in the recess 14. Then, as shown in FIG. 21B, the first substrate 10 </ b> J and the second substrate 20 are bonded via an adhesive 410. Since most of the adhesive 410 is embedded in the recess 14, the gap between the first substrate 10 </ b> J and the second substrate 20 is determined by the height of the flow path forming unit 530. According to this bonding, as described above, strong bonding can be performed even at a low temperature of about 200 ° C. and a low pressure of about 100 MPa.

<Example of arrangement of observation space MS>
In the above embodiment, the observation space MS is formed as two flow paths parallel to the opening 130 from the opening 120 by the gap film 50. Here, an example of the observation space MS having another shape will be described.

  FIG. 22 is a schematic diagram illustrating various arrangement examples of the observation space. FIG. 22A is an arrangement example of the observation space MS in which two parallel flow paths are connected as in the above embodiment. FIG. 22B is an arrangement example of the observation space MS in which a curve is included in the shape of the flow channel in the example shown in FIG. If the channel shape does not have an angle (corner) of 90 degrees or less, the sample-containing liquid can be more easily injected, and the generation of bubbles can be suppressed. In the example shown in FIG. 22B, such a corner is formed into an arc.

  FIG. 22C shows an arrangement example of the observation space MS in which a plurality of flow paths are joined and then separated into a plurality of flow paths again. FIG. 22D is an arrangement example of the observation space MS in which a plurality of flow paths are formed in parallel, and portions where the flow paths are expanded are alternately formed. Here, four arrangement examples have been described, but various other arrangement examples are possible. Moreover, in any arrangement example, the flow path has a portion that has expanded, but the width of the flow path may be constant in the observation space MS. Further, in any arrangement example, the column portion 550 is arranged at the portion where the flow path is expanded, but the column portion 550 is not necessarily arranged.

<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 consideration of these factors, it is desirable in manufacturing to select materials for the first thin film and the second thin 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 disposing the gap film 30 (the flow path forming portion 330 and the column portion 350), the strength of the entire film, stress adjustment, and the like are 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).

DESCRIPTION OF SYMBOLS 1 ... Sample accommodation cell, 10 ... 1st board | substrate, 12 ... Thermal oxide film, 14 ... Recessed part, 20 ... 2nd board | substrate, 50 ... Gap film | membrane, 110, 120, 130, 210 ... Opening part, 150 ... 1st thin film, 250 ... second thin film, 300 ... resin before curing, 320, 330 ... sealing material, 410 ... adhesive, 510, 511 ... peripheral part, 530, 531 ... flow path forming part, 550, 551 ... pillar part, 570 ... Side wall, 575 ... Fluoride film, 700 ... Sample-containing liquid, 800 ... Observation cell preparation device, 810 ... Sample injector, 811 ... Tip attachment part, 813 ... Support part, 815 ... Control part, 817 ... Chip removal part, 820 ... chip, 825 ... chip base, 830, 840 ... sample cup, 835, 845 ... cup base, 850 ... sample base, 860 ... UV irradiator, 870 ... waste port, 888 ... stage, 1000 ... external space, 1 01,5001 ... film, 5010 ... glass substrate, 5020 ... glass layer

Claims (10)

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 gap film disposed between the first thin film and the second thin film at least between the first opening and the second opening;
With
Between the first substrate and the second substrate, there is a flow for connecting the observation space surrounded by the first thin film, the second thin film, and the gap film, and the observation space and the external space. Road space is arranged,
The sample storage cell, wherein the first thin film and the second thin film are transparent to an electron beam.   2. The sample storage cell according to claim 1, wherein at least one of the first substrate and the second substrate is transmissive to visible light.   The sample storage cell according to claim 1, wherein at least one of the first substrate and the second substrate is a silicon substrate.   2. The sample storage cell according to claim 1, wherein the columnar gap film is disposed in a region sandwiched between the first opening and the second opening. The flow path space is connected to the external space through at least two openings,
2. The sample storage cell according to claim 1, wherein any one of the at least two openings is disposed on the first substrate. In the first substrate, at least two openings are arranged in addition to the first opening,
2. The sample storage cell according to claim 1, wherein the two openings and the flow path space are isolated via the first thin film.   The sample storage cell according to claim 1, wherein the gap film is made of a material different from that of the first thin film and the second thin film.   2. The sample storage cell according to claim 1, wherein gold is formed between the gap film and at least one of the first thin film and the second thin film.   2. The sample storage cell according to claim 1, wherein the gap film is formed with a layer containing at least gold.   2. The sample storage cell according to claim 1, wherein the gap film has a recess formed on one side of the first thin film and the second thin film.

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