JP6455256B2 - 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 layer having a first opening and a liquid inlet, and a second opening disposed at a position opposed to the first opening, the first layer being opposed to the first opening. A second layer having an intermediate layer disposed between the first layer and the second layer, and extending over a predetermined distance from an end of the liquid inlet, and the first opening and the second layer An intermediate layer forming a flow path space connecting the observation space between the second opening and the liquid inlet, and the second layer side of the first opening are closed from the inside of the opening, and at least the first The first thin film that is supported on the side surface of the opening and is transparent to the electron beam, and the first layer side of the second opening are closed from the inside of the opening, and are supported at least on the side of the second opening. And a second thin film that is transmissive to an electron beam. To provide Le. According to this, the convenience of the cell which accommodates a sample can be improved.

  The first layer and the second layer may be silicon, and the intermediate layer may be silicon oxide. According to this, a sample accommodation cell can be manufactured easily.

  The first layer and the second layer may be silicon having a (100) surface, and the first opening and the second opening may have a (111) surface inside the opening. According to this, a sample accommodation cell can be manufactured easily.

  According to one embodiment of the present invention, in the substrate having the first layer, the second layer, and the intermediate layer sandwiched between the first layer and the second layer, the region corresponding to the first opening and the liquid inlet The first layer is etched to expose the intermediate layer, the intermediate layer exposed in the region corresponding to the first opening is covered with a first thin film, and the second opening facing the first opening The intermediate layer is exposed by etching the second layer in a region corresponding to the portion, the intermediate layer exposed in the region corresponding to the second opening is covered with a second thin film, and the liquid injection port is interposed. The first etching solution is supplied to the intermediate layer to etch the intermediate layer, and the observation space between the first opening and the second opening, and the observation space and the liquid inlet are connected. Sample collection characterized by forming a channel space To provide a method of manufacturing a cell. According to this, the convenience of the cell which accommodates a sample can be improved.

  Etching the second layer to expose the intermediate layer is etching using a second etchant, and the first thin film is more resistant to the second etchant than the first etchant. You may provide a film | membrane and the film | membrane which is laminated | stacked on the said intermediate | middle layer side rather than the said film | membrane, and has tolerance to the said 1st etching liquid rather than the said 2nd etching liquid. According to this, the first thin film can be easily formed.

  Etching the first layer and the second layer to expose the intermediate layer forms a separation groove that does not reach the intermediate layer along the edge of the sample storage cell to be separated. May be included. According to this, it is possible to easily separate the sample storage cells without increasing the process load.

  Covering the intermediate layer exposed in the region corresponding to the first opening with the first thin film means that the intermediate layer exposed in the region corresponding to the first opening and the liquid inlet is the first thin film. The intermediate layer may be exposed by covering with a thin film and etching the first thin film in a region corresponding to the liquid inlet. According to this, a sample accommodation cell can be manufactured easily.

  Etching the first layer and the second layer to expose the intermediate layer is etching using a second etchant, and the first layer and the second layer are the second etchant. The intermediate layer may be more resistant to the second etching solution than the first etching solution. According to this, a sample accommodation cell can be manufactured easily.

  The first layer and the second layer may be silicon, and the intermediate layer may be silicon oxide. According to this, a sample accommodation cell can be manufactured easily.

  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 layer 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 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 sample storage cell in 1st Embodiment of this invention. It is a figure explaining the manufacturing process of the sample storage cell following FIG. It is a figure explaining the manufacturing process of the sample storage cell following FIG. It is a figure explaining the method of isolate | separating the some sample storage cell in 1st Embodiment of this invention. It is a figure explaining the method of sealing the sample storage cell in 2nd Embodiment of this invention. It is a figure explaining the shape of channel space FP of the sample storage cell in a 3rd embodiment of the present invention.

<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 formed of a first layer 10, a second layer 20, and an intermediate layer 30 sandwiched between them. In this example, the first layer 10, the second layer 20, and the intermediate layer 30 constitute an SOI (Silicon on Insulator) substrate. The first layer 10 is silicon (SOI layer) and has a thickness of 1 μm or more and 100 μm or less (in this example, 5 μm). The second layer 20 is silicon (support layer, support substrate) and has a film thickness of several hundred μm (in this example, 300 μm). The intermediate layer 30 is silicon oxide (BOX layer), and has a film thickness of several hundred nm (200 nm in this example). By partially removing the intermediate layer 30, a space (flow path space FP and observation space MS) is formed between the first layer 10 and the second layer 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 or a rectangle having one side of about 0.5 mm to 3 mm, and in this example, one side is approximately 1.0 mm.

  Each of the first layer 10 and the second layer 20 has an opening. In this example, the openings 110, 120, and 130 are arranged in the first layer 10 that is thinner than the second layer 20. An opening 210 (see FIGS. 2 and 3) is arranged in the second layer 20. Next, a detailed structure including the internal structure of the sample storage cell 1 will be described with reference to FIGS.

  FIG. 2 is a plan view of the sample storage cell as viewed from the first layer side according to the first embodiment of the present invention. FIG. 3 is a schematic diagram illustrating a cross-sectional configuration (a cross-sectional structure taken along a cross-sectional line A-A ′ in FIGS. 1 and 2) of the sample storage cell according to the first embodiment of the present invention. FIG. 3 shows an end view corresponding to each cross-sectional line. The schematic views showing the following cross-sectional configurations are also shown as end views.

  The opening on the intermediate layer 30 (second layer 20) side of the opening 110 is closed by the first thin film 150 from the inside of the opening. That is, the first thin film 150 closes the opening while being in contact with at least a part of the side surface (inner surface) of the opening 110. In this example, the first thin film 150 is also formed on the mask layer 155 formed on the surface of the first layer 10 (surface on the external space 1000 side). The portion of the first thin film 150 formed on the mask layer 155 and the first thin film 150 blocking the opening 110 are separated in the example shown in FIG. It may be connected.

  The opening on the intermediate layer 30 (first layer 10) side of the opening 210 is closed by the second thin film 250 from the inside of the opening. In other words, the second thin film 250 closes the opening while being in contact with at least a part of the side surface (inner surface) of the opening 210. In this example, the first thin film 250 is also formed on the mask layer 255 formed on the surface of the second layer 20 (surface on the external space 1000 side). Although the portion of the second thin film 250 formed on the mask layer 255 and the second thin film 250 blocking the opening 210 are separated in the example shown in FIG. 3, they may be connected.

  The portion of the first thin film 150 that closes the opening 110 and the portion of the second thin film 250 that closes the opening 210 are opposed to each other. The first thin film 150 and the second thin film 250 are films that are permeable to electron beams. A region sandwiched between the portion of the first thin film 150 blocking the opening 110 and the portion of the second thin film 250 blocking the opening 210 is referred to as an observation space MS. In the observation space MS, a sample to be measured is arranged and an electron beam passes through.

  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, if it becomes thicker than 200 nm, the electron beam will not be transmitted. 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.

  The distance between the first thin film 150 and the second thin film 250 corresponds to the film thickness of the intermediate layer 30. Therefore, in this example, as described above, the thickness is 200 nm. This distance is desirably set depending on the size of the sample, that is, the observation object (for example, a cell), and needs to be 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 target, the observation target overlaps, making it difficult to obtain the observation results of the individual observation target or transmitting the electron beam. It becomes difficult to do. That is, the distance between the first thin film 150 and the second thin film 250 does not need to be greater than a distance that does not allow the electron beam to pass.

  The opening portion 110 and the opening portion 210 are disposed to face each other, and are designed to open with substantially the same size on the intermediate layer 30 side when viewed from the first layer 10 side. In this example, the shape of the opening 110 and the opening 210 (on the intermediate layer 30 side) is a square of 50 μm × 50 μm. The shape of the opening may be a rectangle such as 5 μm × 50 μm.

  The shape of the openings 120 and 130 is larger than that of the opening 110. In this example, the size of the opening on the intermediate layer 30 side is a square of 100 μm × 100 μm. The shape of the opening may also be a rectangle instead of a square. In each figure used for description, in order to express the structure in an easy-to-understand manner, the ratio between the components is adjusted and shown.

  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. However, when the etching of the first layer 10 and the second layer 20 is performed by crystal anisotropic etching, it is desirable that the shape of the opening is a quadrangle. Further, the shape of the opening 120 and the shape of the opening 130 may be different. For example, the opening on the side where the sample-containing liquid is injected may be large.

  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. As will be described later, crystal anisotropic etching is performed using an etching solution such as a TMAH (Tetramethylammonium hydroxide) aqueous solution using the first layer 10 and the second layer 20 which are single crystal silicon having a (100) plane as a surface. By doing so, since the (111) plane appears on the side surface of the opening, it is easy to control the taper shape.

  Note that 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 intermediate layer 30 (second layer 20) side smaller than an opening area on the external space 1000 side. Further, the opening 210 has an opening area on the intermediate layer 30 (first layer 10) side 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.

  A space surrounded by the intermediate layer 30 is formed between the first layer 10 and the second layer 20. This space includes the observation space MS and the flow path space FP, and includes a first range extending from the opening end of the opening 120 and a second range expanding from the opening end of the opening 130. From each opening end portion to the space end portion, the opening extends almost equidistantly (in this example, about 150 μm). The first range and the second range partially overlap. In this example, the observation space MS is arranged in the overlapping portion.

  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.

  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. 4 is a diagram illustrating a method for 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 flow path space FP from the opening 120, the sample-containing liquid 700 moves through the flow path space FP to the observation space MS, and further reaches the opening 130. Note that the sample-containing liquid 700 may be injected into the opening 130 instead of the opening 120, but in the following description, the description will be made assuming that the sample-containing liquid 700 is injected into the opening 120. In this case, the opening 130 functions as a discharge port for discharging the gas in the flow path space FP when the sample-containing liquid 700 is injected.

  FIG. 5 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. 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. 5) 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. can do. The direction of the electron beam EB may be opposite to the direction shown in FIG.

  The sample injection process in FIG. 4 and the cell sealing process in FIG. 5 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. 6 is a diagram 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. 6, hereinafter referred to as the X direction). Further, the sample injector 810 has a horizontal direction perpendicular to the moving direction of the stage 888 (depth direction in FIG. 6, hereinafter referred to as Y direction) and vertical direction (vertical direction in FIG. 6, hereinafter referred to as Z direction). 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. 6A 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. 6B). 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. 6C). 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. 6 (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. Subsequently, a sealing process of the observation cell manufacturing apparatus 800 will be described.

  FIG. 7 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. 7A). The chip 820 holding the pre-curing resin 300 is moved onto the opening 120 of the sample storage cell 1, and the pre-curing resin 300 in the chip 820 is discharged and dropped into the opening 120 (FIG. 7D). Then, it is dripped at the opening part 130 (FIG.7 (e)). Note that the resin 300 before curing may be sucked into the chip 820 again before dropping into the opening 130. This is the same 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, and 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. 6A). 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.

[Method for manufacturing sample storage cell]
As described above, the sample storage cell 1 is manufactured using an SOI substrate. Then, by etching a part of the intermediate layer 30 made of silicon oxide, a flow path space FP and an observation space MS are formed between the first layer 10 and the second layer 20 made of silicon. Hereinafter, the manufacturing method of the sample storage cell 1 will be specifically described.

  FIG. 8 is a diagram for explaining a manufacturing process of the sample storage cell in the first embodiment of the present invention. FIG. 9 is a diagram for explaining the manufacturing process of the sample storage cell subsequent to FIG. FIG. 10 is a diagram for explaining the manufacturing process of the sample storage cell subsequent to 9. Each figure shows a cross-sectional structure corresponding to FIG. First, as shown in FIG. 8A, mask layers 155 and 255 are formed on both surfaces of the SOI substrate (the first layer 10, the second layer 20, and the intermediate layer 30). The mask layers 155 and 255 are desirably films having resistance (low etching rate) to an etching solution (in this example, a TMAH aqueous solution) for silicon, which will be described later, and is, for example, a silicon nitride film. The film thickness of the mask layers 155 and 255 is, for example, not less than 50 nm and not more than 2 μm.

  These films may be formed by vapor deposition such as CVD (Chemical Vapor Deposition) and PVD (Physical Vapor Deposition). Further, if the mask layers 155 and 255 are not a silicon nitride film but a silicon oxide film, a thermal oxide film formed on the surface by thermally oxidizing the first layer 10 and the second layer 20 may be used. The same applies to various films formed below.

  A part of the mask layer 155 is etched using a photolithography technique (FIG. 8B). The region to be etched is a region corresponding to the openings 110, 120, and 130. For etching the mask layer 155, RIE (Reactive Ion Etching) is used. At this time, the first layer 10 may also be partially etched (overetched). For the etching of the film, both dry etching and wet etching can be applied, and the same applies to the following description unless otherwise specified.

  Next, using the mask layer 155 as an etching mask, the first layer 10 is etched to expose the intermediate layer 30 (FIG. 8C). This etching is crystal anisotropic etching, and a TMAH aqueous solution is used as an etchant. In this example, the surface of the first layer 10 (silicon) is the (100) plane, and the side surfaces of the openings 110, 120, and 130 are formed along the (111) plane with a low etching rate. The intermediate layer 30 serves as an etching stopper.

  Next, the first thin film 150 is formed so as to cover the openings 110, 120, and 130 (FIG. 9A). The first thin film 150 is silicon nitride and has a thickness of 20 nm. At this time, the first thin film 150 is preferably formed so as to have a tensile stress. The first thin film 150 preferably has resistance to a TMAH aqueous solution and BHF (buffered hydrofluoric acid: a mixed aqueous solution of hydrofluoric acid and ammonium fluoride). However, since it is difficult to have complete resistance, the film thickness may be increased in advance in consideration of the film thickness that decreases in etching using a TMAH aqueous solution or BHF described later.

  In the first thin film 150, the portion formed in the openings 110, 120, and 130 and the portion formed on the mask layer 155 are separated from each other because the mask layer 155 is formed at the end of the opening. This is because it protrudes like a spear. This wrinkle is often about 5% of the etching amount in the depth direction of the first layer 10. Therefore, if the wrinkles are small, depending on the manufacturing conditions, the portion formed in the openings 110, 120, and 130 may be connected to the portion formed on the mask layer 155.

  Next, a part of the first thin film 150 is etched using a photolithography technique (FIG. 9B). The first thin film 150 is etched so that at least a portion formed in the opening 110 is left and at least a part of the intermediate layer 30 is exposed from the openings 120 and 130. In the example shown in FIG. 9B, the mask layer 155 in the portion where the first thin film 150 is etched and the portion where it is laminated are also etched. Thereby, the wrinkles formed at the ends of the openings 120 and 130 may be etched. By this step, the formation of the pattern on the first layer 10 side is completed. In the step of forming (depositing) the first thin film 150 (FIG. 9A), the first thin film 150 may not be formed in the openings 120 and 130 using a mask or the like. That is, the first thin film 150 may be formed so as to cover the exposed intermediate layer 30 inside the opening 110.

  Next, a part of the mask layer 255 is etched using a photolithography technique (FIG. 9C). The region to be etched is a region corresponding to the opening 210. Then, the second layer 20 is etched using the mask layer 255 as an etching mask (FIG. 10A). This etching is a crystal anisotropic etching similar to the etching of the first layer 10, and a TMAH aqueous solution is used as an etching solution. In this example, the surface of the second layer 20 (silicon) is the (100) plane, and the side surface of the opening 210 is formed along the (111) plane with a low etching rate. The intermediate layer 30 serves as an etching stopper. The second layer 20 is thicker than the first layer 10 by etching. Therefore, the region where the mask layer 255 is etched in FIG. 9C is larger than the region where the mask layer 155 is etched corresponding to the position of the opening 110 in FIG.

  Next, the second thin film 250 is formed so as to cover the opening 210 (FIG. 10B). The second thin film 250 is silicon nitride and has a thickness of 20 nm. At this time, the second thin film 250 is preferably formed so as to have a tensile stress. The second thin film 250 is preferably resistant to BHF. However, since it is difficult to have complete resistance, the film thickness may be increased in advance in consideration of a film thickness that decreases in etching using BHF, which will be described later.

  In the second thin film 250, the part formed inside the opening 210 and the part formed on the mask layer 255 are separated because the mask layer 255 is like a ridge at the end of the opening. This is because it protrudes. By this step, the formation of the pattern on the second layer 20 side is completed. Note that the ridge portion may be etched using a photolithography technique.

  Next, the intermediate layer 30 is etched by supplying an etching solution to the intermediate layer 30 through the openings 120 and 130 (FIG. 10C). In this example, the etching solution is BHF. Since this etching is isotropic etching, the intermediate layer 30 is etched so as to spread from the opening ends of the openings 120 and 130 with the passage of the etching time. On the other hand, the first layer 10 and the second layer 20 are hardly etched because they are resistant to BHF. Therefore, a space is formed between the first layer 10 and the second layer 20.

  Then, after the space extending from both of the openings 120 and 130 is connected and the region (observation space MS) where the first thin film 150 and the second thin film 250 face each other is formed, the etching is finished. As a result, a flow path space FP and an observation space MS are formed between the first layer 10 and the second layer 20. When the intermediate layer 30 is etched, bubbles may be generated in the etching solution. In such a case, the etching process may be performed while performing the deaeration process.

  Although the sample storage cell 1 described above 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, the process for separating each sample accommodation cell 1 into pieces is performed. This process may be cut by dicing or the like, but in this example, individualization is realized by another method.

  FIG. 11 is a diagram illustrating a method for separating a plurality of sample storage cells in the first embodiment of the present invention. FIG. 11A is a plan view of a part of the substrate before the sample storage cell 1 is separated from the first layer 10 side. FIG. 11B is a schematic diagram illustrating a cross-sectional configuration of the dividing groove portion (a cross-sectional structure taken along a cross-sectional line B-B ′ in FIG. 11). In addition, in FIG. 11, although the example which has arrange | positioned the sample accommodation cell 1 by 3x3 is shown, this extracts and illustrates a part of the whole board | substrate.

  A dividing groove SL1 is formed between the sample storage cells 1. In this example, the dividing groove SL1 is provided on the first layer 10 side. The dividing groove SL2 is also provided on the second layer 20 side at a position facing the dividing groove SL1. None of the dividing grooves SL1 and SL2 reaches the intermediate layer 30.

  This dividing groove SL1 is formed together when the openings 110, 120, and 130 shown in FIG. 8C are formed. This etching is crystal anisotropic etching, and the mask layer 155 is formed in a narrow slit shape, so that the dividing groove SL1 can be prevented from reaching the intermediate layer 30. For example, when the depth of the dividing groove SL1 is set to about 3 μm, the slit of the mask layer 155 may be set to a width of about 4 μm.

  Similarly, the dividing groove SL2 is formed together when the opening 210 shown in FIG. 10A is formed. This etching is a crystal anisotropic etching, and the mask layer 255 is formed in a slit shape having a narrow width, so that the dividing groove SL2 can be prevented from reaching the intermediate layer 30. For example, when the depth of the dividing groove SL2 is about 200 μm, the slit of the mask layer 255 may be set to a width of about 300 μm.

  Since the dividing grooves SL1 and SL2 do not reach the intermediate layer 30, it is possible to prevent the intermediate layer 30 from being etched from the dividing grooves SL1 and SL2 when the intermediate layer 30 shown in FIG. 10C is etched. . And it can isolate | separate from the adjacent sample accommodation cell 1 easily in parting groove | channel SL1, SL2 by giving a light impact to the sample accommodation cell 1. FIG.

  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.

  Since the sample storage cell 1 according to the above-described embodiment is formed using an SOI substrate, the first layer 10 and the second layer 20 are bonded together by the intermediate layer 30 in advance. Therefore, it is not necessary to join the two substrates, and the sample-containing liquid 700 injected into the space formed inside hardly leaks. In addition, since the distance between the first layer 10 and the second layer 20 is determined by the thickness of the intermediate layer 30, accurate gap control is possible. Moreover, according to the sample storage cell 1, the convenience can be improved.

Second Embodiment
In the first embodiment, the openings 120 and 130 are closed by the sealing materials 320 and 330 in order to separate the flow path space FP and the observation space MS from the external space 1000. In the second embodiment, an example in which this is covered with a cover plate will be described.

  FIG. 12 is a diagram illustrating a method for sealing a sample storage cell in the second embodiment of the present invention. In this example, the openings 120 and 130 are closed by the lid plate 370. The lid plate 370 is, for example, glass. An adhesive 360 may be formed between the lid plate 370 and the first layer 10 as shown in FIG. The lid plate 370 may have a size that closes the openings 120 and 130 (for example, a square that is about 200 μm larger than the openings), but has a larger shape in consideration of the ease of the sealing process. It may be a shape other than a square.

<Third Embodiment>
In the third embodiment, a case where there are three or more openings that connect the external space 1000 and the flow path space FP will be described.

  FIG. 13 is a diagram illustrating the shape of the flow path space FP of the sample storage cell in the third embodiment of the present invention. In this example, in addition to the openings 120 and 130 described in the first embodiment, an opening 121 having the same shape as these is formed. Thus, the number of openings that connect the external space 1000 and the flow path space FP is not limited to two, and may be larger. In this way, for example, the opening 121 and the opening 110 can be separated, and the sample-containing liquid 700 can be easily injected. However, in this case, it is necessary to seal an opening other than the opening 110 before observation with an electron microscope.

  In this case, the openings 120 and 130 may be smaller than the opening 121. That is, the opening for injecting the sample-containing liquid 700 may be enlarged, and the opening for introducing the etching solution when forming the flow path space FP may be reduced.

<Other embodiments>
[surface treatment]
The outer surface of the sample storage cell 1 (particularly, the outer surface of the first layer 10) may be subjected to a lipophilic (hydrophobic) treatment. In this way, if the sample-containing liquid 700 is a water-based one, 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. If the sample-containing liquid 700 is alcoholic (oil-based), the outer surface of the sample storage cell 1 may be subjected to a reverse process, that is, a hydrophilic process.

  For example, as long as it is a hydrophilic treatment, it may be a treatment covered 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.

[Types of first thin film and second thin film]
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. And it is desirable to have resistance to BHF so that it is not etched together when the intermediate layer 30 is etched. Moreover, the 1st thin film 150 and the 2nd thin film 250 are not restricted to a single layer, The film | membrane which laminated | stacked multiple types of film | membrane may be sufficient.

  Further, in the manufacturing process, a plurality of types of films may be stacked, and finally a single layer may be formed. For example, the first thin film 150 is a silicon nitride film (TMAH resistant film) that is more resistant to a TMAH aqueous solution than BHF, and a silicon nitride that is disposed closer to the intermediate layer 30 than the film and is more resistant to BHF than a TMAH aqueous solution. A film laminated with a film (BHF resistant film) may be used. The TMAH resistant film has a relatively lower silicon content than the BHF resistant film. For example, when expressed as SixNy, the TMAH resistant film has x = 3 and y = 4, and the BHF resistant film has x = 5 and y = 4. The TMAH resistant film may be a silicon oxide film. The BHF resistant film may be an amorphous silicon film, a polycrystalline silicon film, or a single crystal silicon film.

  In this way, in the step shown in FIG. 10A, the first thin film 150 is hardly etched due to the presence of the TMAH resistant film. In the process shown in FIG. 10C, the TMAH resistant film is etched while the first thin film 150 remains due to the presence of the BHF resistant film. In this case, since the first thin film is formed in advance as a thick film and can be finally thinned to a desired thickness, a stable film can be formed. In addition, the stress balance can be easily adjusted.

[Charge-up prevention]
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 (first layer 10, second layer 20, first thin film 150, and second thin film 250). It may be arranged on at least a part of the surface of the substrate.

[Manufacture of sample storage cells using materials other than SOI substrates]
In the embodiment described above, the sample storage cell 1 is manufactured using the SOI substrate formed by the first layer 10, the second layer 20, and the intermediate layer 30, but may be other than the SOI substrate. That is, it is only necessary that the first layer 10 and the second layer 20 have resistance to an etching solution for etching the intermediate layer 30. On the other hand, it is desirable that the intermediate layer 30 has resistance to an etching solution used for etching when the openings are formed in the first layer 10 and the second layer 20. As long as the substrate is realized by a combination of the materials of the first layer 10, the second layer 20, and the intermediate layer 30, the SOI substrate may not be used.

DESCRIPTION OF SYMBOLS 1 ... Sample accommodation cell, 10 ... 1st layer, 20 ... 2nd layer, 30 ... Intermediate | middle layer, 110, 120, 130, 210 ... Opening part, 150 ... 1st thin film, 155, 255 ... Mask layer, 250 ... 1st 2 thin film, 300 ... resin before curing, 320, 330 ... sealing material, 360 ... adhesive, 370 ... lid plate, 700 ... sample-containing liquid, 800 ... observation cell preparation device, 810 ... sample injector, 811 ... chip 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 irradiation 870 ... Disposal port, 888 ... Stage, 1000 ... External space

Claims (9)

A first layer having a first opening and a liquid inlet;
A second layer disposed opposite the first layer and having a second opening at a position facing the first opening;
An intermediate layer disposed between the first layer and the second layer, extending over a predetermined distance from an end of the liquid inlet, and the first opening and the second opening An intermediate layer that forms a flow path space connecting the observation space between the liquid injection port,
A first thin film that closes the second layer side of the first opening from the inside of the opening, is supported on at least a side surface of the first opening, and is transmissive to an electron beam;
A second thin film that closes the first layer side of the second opening from the inside of the opening, is supported on at least a side surface of the second opening, and is transmissive to an electron beam;
A sample storage cell comprising: The first layer and the second layer are silicon;
The sample storage cell according to claim 1, wherein the intermediate layer is silicon oxide. The first layer and the second layer are silicon having a (100) surface,
3. The sample storage cell according to claim 2, wherein the first opening and the second opening have a (111) plane inside the opening. In the substrate having the first layer, the second layer, and the intermediate layer sandwiched between the first layer and the second layer, the first layer in the region corresponding to the first opening and the liquid inlet is etched. Exposing the intermediate layer;
Covering the intermediate layer exposed in the region corresponding to the first opening with a first thin film;
Etching the second layer in a region corresponding to the second opening facing the first opening to expose the intermediate layer;
Covering the intermediate layer exposed in the region corresponding to the second opening with a second thin film;
A first etching solution is supplied to the intermediate layer through the liquid inlet to etch the intermediate layer, and an observation space between the first opening and the second opening, and the observation space and the A method for manufacturing a sample storage cell, comprising: forming a flow path space connecting the liquid injection port. Etching the second layer to expose the intermediate layer is etching using a second etchant,
The first thin film is formed by laminating a film that is more resistant to the second etching solution than the first etching solution and the intermediate layer side of the film, and the first etching solution is more than the first etching solution. The method for producing a sample storage cell according to claim 4, further comprising a film resistant to liquid.   Etching the first layer and the second layer to expose the intermediate layer forms a separation groove that does not reach the intermediate layer along the edge of the sample storage cell to be separated. The method for manufacturing a sample storage cell according to claim 4, comprising: Covering the intermediate layer exposed in the region corresponding to the first opening with a first thin film,
Covering the intermediate layer exposed in the region corresponding to the first opening and the liquid inlet with the first thin film;
5. The method for manufacturing a sample storage cell according to claim 4, further comprising exposing the intermediate layer by etching the first thin film in a region corresponding to the liquid inlet. Etching the first layer and the second layer to expose the intermediate layer is etching using a second etchant;
The first layer and the second layer are more resistant to the first etchant than the second etchant,
5. The method for manufacturing a sample storage cell according to claim 4, wherein the intermediate layer is more resistant to the second etching solution than the first etching solution. The first layer and the second layer are silicon;
9. The method for manufacturing a sample storage cell according to claim 8, wherein the intermediate layer is silicon oxide.

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