JP2015031515A - Near-field optical observation apparatus, sample-containing environmental cell manufacturing method, scanning electron optical microscope, and method of using scanning electron optical microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a near-field optical observation apparatus capable of optically observing a non-dyed and non-fixed biological sample in liquid with high resolution, a sample-containing environmental cell manufacturing method, a scanning electron optical microscope, and a method of using the scanning electron optical microscope.SOLUTION: A near-field optical observation apparatus 11 comprises: an environmental cell 12 including a space part capable of hermetically holding a sample, an incidence window provided on one side of the space part, and an emission window provided on the other side of the space part; and a light emission measurement module 13. The incidence window includes a luminescent material that can emit light by electron beam excitation.

Description

  The present invention relates to a near-field optical observation apparatus, a sample-containing environment cell preparation method, a scanning electron optical microscope, and a method of using a scanning electron optical microscope.

Optical microscopes have been able to provide images containing extensive information on different objects in different environments and have played an important role in biology in particular.
Examples of the optical microscope include a phase contrast microscope and a fluorescence microscope. The phase-contrast microscope can observe living cells in the liquid without staining them. Fluorescence microscopy has made it possible to visualize only specific molecules and tissues, creating many biological discoveries. However, the image resolution of these optical microscopes is limited by the diffraction limit.
In the field of optical microscopes, the following new technologies are being actively developed.

<Trends in optical microscopes: (1) Near-field optical microscope>
In an optical microscope, without using a lens, the outer periphery of an optical fiber with a sharp tip is coated with a light-shielding coating with metal, and an optical fiber probe with a minute opening only on the tip is mechanically scanned to scan the object. Scanning Near-field Optical Microscopy (abbreviated as SNOM) breaks the diffraction limit wall and now observes the state of light localization in nanostructures Established as a general method.

  However, since it is difficult to control the distance between the probe and the object in the liquid, there are limited applications to cells in the liquid, and in most cases, the object is observed by fluorescent staining. The chip-enhanced Raman scattering method, which is a kind of near-field optical microscope technology, is expected as a unique technique that does not require staining, but its application to biological samples has just started and there are few cases.

<Trends in optical microscopes: (2) Far-field nano-resolution fluorescence optical microscope>
In the last decade or so, various far-field nano-resolution fluorescence optical microscopes utilizing the nonlinearity of fluorescence and single-molecule detection have been developed and traditional lenses are used. It became possible to observe living cells in the liquid three-dimensionally and super-resolution exceeding the diffraction limit with an optical system.

  However, these techniques in principle require fluorescent staining. How to observe unstained living cells by a method similar to these methods is considered as an important research topic in the future.

On the other hand, electron microscopes have provided resolutions that far exceed the resolution barrier of optical microscopes.
However, in an electron microscope, it is difficult to observe living cells in water because the sample must be placed in a vacuum environment. For example, in order to maintain the natural state of an object, a complicated sample preparation technique that is not necessary with an optical microscope is required. In addition, information such as fluorescence, light absorption, and refractive index, which have provided important information in the optical microscope, has been lost.
In the field of electron microscopes, the following new technologies are being actively developed.

<Trends of electron microscope: (1) Observation of optical information by electron beam>
In the electron microscope, a technique for observing optical characteristics with an electron beam has been developed. The cathodoluminescence method (CL, electron beam emission method) for detecting luminescence excited by an electron beam is a typical example, and is a stained or unstained biological sample (however, it is limited to a dry dead state placed under vacuum) ) For a long time.

Development of luminescent markers that emit light with an electron beam by using chemically bonded to specific molecules or tissues that want to observe minute molecules and particles is still in progress.
For non-biological samples that can be safely exposed to vacuum, a variety of other electron microscopy techniques are used to observe various optical properties and are becoming more common in the field of nanophotonics. Yes.

<Trends in electron microscopes: (2) Development of environmental cells>
Another notable advance in electron microscope technology is the development of environmental cells.
The observation of unstained, non-fixed biological samples in liquid has been an important issue since the early days of electron microscope history, but progress over the past decade has been remarkable. The non-fixed biological sample is a biological sample that has not been killed, that is, a living biological sample.
Using an environmental cell that shuts off the region surrounding the sample with a thin film from the vacuum environment, transmission electron microscope (TEM), scanning transmission electron microscope (STEM), scanning electron microscope (SEM), transmission electrons, scattered electrons, or CL Many cases of successful observation of objects in liquids have been reported.

Non-Patent Document 3 has a configuration in which a SiN membrane, a spacer, and a SiN membrane are sandwiched between two Si substrates in this order, and cells are arranged together with Au particles in a space provided by the spacer. An environmental cell for STEM is described. It is not an optical microscope because it is configured to penetrate a high-density electron beam and measure scattered electrons. When cells are used as samples, they die by measurement. Further, since contrast is generally not obtained with unstained cells, the cells are labeled with Au particles, and only the Au particles are observed.
In Non-Patent Document 4, SiO ₂ (9 nm), spacer (Epoxy), SiO ₂ (9 nm) are sandwiched in this order between two Si substrates, and cells are placed in the space provided by the spacer. An environmental cell for a TEM having a configuration to be arranged is described. It is not an optical microscope because it is configured to penetrate a low-density electron beam and project a transmission electron image.

  In Patent Document 4 and Non-Patent Document 5, the electron beam side substrate and the optical fiber are arranged in this order, and the cells are arranged in a space provided between the electron beam side substrate and the optical fiber. An environmental cell for SEM having a configuration is described. The electron beam is reflected on the cell surface, and the reflected electrons are observed by a reflected electron detector, and CL is observed by a photodetector through an optical fiber.

  Regarding environmental cells, Protochips manufactures and ships STEM or TEM products (Non-Patent Document 6). In addition, Quantomix manufactures and ships products for SEM (Non-patent Document 7). However, there is no environmental cell for optical microscope applications.

  As another approach, in order to observe a sample in a liquid without using an environmental cell, a dedicated SEM made upside down is also developed.

  A serious problem in observing a biological sample in a liquid with an electron microscope is damage to an object due to an electron beam. As long as the principle of image acquisition is based on absorption, scattering, and CL of an electron beam by the object, irradiation of the object with an electron beam is inevitable in principle. In TEM, there are cases in which cells were alive after observation by minimizing the electron beam irradiation dose. However, in the method using a general STEM, the damage threshold value that has been conventionally known is exceeded. It has been reported that cells were not able to survive despite the suppression of electron beam dose. Even in the technique using SEM, there is no report that the biological sample in the liquid was alive after observation.

  Patent Document 1 describes an optical microscope having a configuration in which an “optical microscope section” and a “portion for performing optical excitation by an electron microscope imaging system” are connected. Non-Patent Document 1 is an implementation of the optical microscope according to Patent Document 1 using a special inverted SEM made upside down. However, a method for suppressing an electron beam irradiation to a sample and obtaining an image without damaging the sample with an electron beam is not shown. In addition, these documents do not describe a method for optically observing a biological sample using a general electron microscope that is not an inverted type. Patent Documents 2 and 3 disclose an X-ray microscope using an environmental cell. In this method, an X-ray transmission image of the sample is observed with characteristic X-rays generated from the observation sample support member. However, an optical microscopic image cannot be obtained.

International Publication No. 2009/148094 Japanese Patent No. 4565168 JP 2011-007766 A Special table 2008-510988 gazette

W. Inami, K .; Nakajima, A .; Miyakawa, and Y.M. Kawata, “Electron beam exclusion assisted optical microscope with ultra-high resolution”, Opt. Express 18, 12897 (2010) T. T. et al. Ogura, “Measurement of the Unstained Biological Sample by a Novel Scanning Electron Generation X-ray Microscope Based on SEM”, Biochem. Biophys. Res. Commun. 385, 624 (2009) D. B. Peckys, G.M. M.M. Veith, D.M. C. Joy, and N.J. de Jonge, “Nanoscale Imaging of Whole Cells Using a Liquor Enclosure and a Scanning Transmission Electron Microscope”, PLO SONE 9 (e8214). K. -L. Liu, C.I. -C. Wu, Y .; -J. Huang, H .; -L. Peng, W.M. -Y. Chang, P.A. Chang, L.M. Hsu, and T.M. -R. Yew, "Novel microchip for in situ TEM imaging of living organisms and bio-reactions in aquatic conditions", Lab Chip 8, 1915 (2008). S. Thiberge, A.M. Nechstan, D.C. Sprinzak, O .; Gileadi, V.M. Behar, O .; Zik, Y. et al. Cowers, S.M. Michaeli, J. et al. Schlessinger, and E.M. Moses, “Scanning electron microscopy of cells and tissues under fully hydrated conditions”, Proc. Natl. Acad. Sci. USA 101, 3346 (2004) Protochips product website http://www.protochips.com/ Quantomics product homepage http://www.wetsem.com/

  The present invention relates to a near-field optical observation apparatus capable of optically observing an unstained, non-fixed biological sample in a liquid with high resolution, a sample-containing environment cell preparation method, a scanning electron optical microscope, and a method of using the scanning electron optical microscope It is an issue to provide.

  In view of the above circumstances, the present inventor fused the near-field optical observation apparatus equipped with the environmental cell and the electron microscopic method by trial and error, so that there is no staining in the liquid in the environmental cell. Instead of a sharp optical fiber probe that mechanically scans while holding a non-fixed biological sample and placed inside a vacuum chamber of an electron microscope, it illuminates with a fluorescent dye that emits light when irradiated with an electron beam. A scanning electron optical microscope (abbreviated as SEOM) for use as a probe (illumination light source) was developed. This is also a near-field optical microscope (SNOM).

  Specifically, a window that emits light with an electron beam having a thickness of 50 nm in an environmental cell is scanned with a low-energy electron beam, and the generated photons are counted with a photomultiplier tube (PMT) disposed below. The near-field optical image of the sample attached to the opposite surface of the material is mechanically scanned by a photomultiplier tube with an optical image having a high resolution exceeding 100 nm disposed below, without being affected by the diffraction limit of the sample. I was able to get it without relying on. According to the conventional theory, it is considered that the light of 240 nm or less cannot be identified by the optical microscope due to the diffraction limit. However, an image having a small structure of 62 nm can be observed due to the formation of dipole radiation enhancement or energy transfer phenomenon.

  This near-field optical observation apparatus can be easily attached to the sample chamber of an existing scanning electron microscope, and a scanning electron optical microscope (SEOM) can be realized only by combining the near-field optical observation apparatus and the electron microscope.

In the environment cell of the near-field optical observation apparatus, the sample could be held in any environment such as air, water, physiological saline, or culture solution.
Cells and nanoparticles could be used as the sample, and the surface structure of human lung epidermis cells that remained alive without staining in the liquid and cytotoxic nanoparticles incorporated into the cells could be observed.

  As described above, the image is formed by dipole radiation enhancement according to the refractive index of the object, and when the object is an absorptive substance, the quenching phenomenon of CL due to energy transfer also contributed to the contrast formation. .

  General electron beam energy is 5 to 30 kV in SEM and 100 to 200 kV in TEM and STEM, but it uses a low energy electron beam of 0.8 to 1.2 kV. And the sample adhered to the opposite side of the window material was not damaged.

As explained above, the scanning electron optical microscope (SEOM) enables unstained and resolved optical observation of living cells in the liquid, specifically, the adhesion interface of living human lung epidermal cells. The present invention has been completed by finding that it can be used for observation of the microstructure and the intracellular uptake process of toxic nanoparticles.
The present invention has the following configuration.

(1) An environmental cell including a space part capable of sealingly holding a sample, an incident window provided in one of the space parts, an emission window provided in the other of the space parts, and a luminescence measurement module. A near-field optical observation apparatus comprising: a light emitting material capable of emitting light by electron beam excitation.

(2) The near-field optical observation apparatus according to (1), wherein a frame portion is provided in contact with the incident window.
(3) The near-field optical observation apparatus according to (2), wherein a hole is formed in the frame portion so as to communicate one surface side and the other surface side.
(4) The near-field optical observation apparatus according to (3), wherein the entrance window is provided on one surface side of the frame portion.

(5) The near-field optical observation apparatus according to (3) or (4), wherein the hole is a part of the space.
(6) The near-field optical observation apparatus according to (3), wherein the entrance window is provided on the other surface side of the frame portion.
(7) The near-field optical observation apparatus according to any one of (1) to (6), wherein an exit window is provided on the other surface side of the frame portion or spaced apart from the other surface side. .

(8) The near-field optical observation according to (3), wherein the hole portion that communicates the one surface side and the other surface side of the frame portion is formed so as to expand from the incident window side toward the emission window side. apparatus.
(9) The near-field optical observation apparatus according to any one of (8), wherein a wall surface of the frame portion facing the hole is covered with a highly light-reflective material.
(10) The incident window is formed of two or more layers, a structural layer made of a high-strength material, and a light-emitting layer that is laminated on the structural layer and has a light-emitting material that can emit light by electron beam excitation; The near-field optical observation apparatus according to any one of (1) to (9), wherein

(11) The near-field optical observation apparatus according to (10), wherein the light emitting layer is made of a light emitting material capable of emitting light by electron beam excitation.
(12) The near-field optical observation apparatus according to (11), wherein the light-emitting layer is formed by adding another light-emitting material capable of emitting light by electron beam excitation to the light-emitting material.
(13) The structure layer is a membrane made of a high-strength material of any one of SiN, SiO ₂ , SiC, Si, polyimide, parylene, carbon, collodion, formval, and triacetylcellulose (10 ) To (12) The near-field optical observation device according to any one of the above.

(14) The near-field optical observation apparatus according to any one of (1) to (13), wherein the incident window has a thickness of 5 nm to 500 nm.
(15) The exit window is made of any one of glass, polyester, polyimide, SiN, SiO ₂ , SiC, Si, parylene, carbon, collodion, formval, and triacetylcellulose (1) The near-field optical observation apparatus according to any one of to (14).
(16) The near-field optical observation apparatus according to any one of (1) to (15), wherein a plurality of the incident windows are provided.

(17) The near-field optical observation apparatus according to any one of (1) to (16), wherein the light emission measurement module is disposed on an exit window side of the environmental cell.
(18) The light emission measurement module is disposed apart from the environmental cell, and an optical path system that connects an emission window side of the environmental cell and a photoelectric surface of the light emission measurement module is provided. The near-field optical observation apparatus according to any one of (1) to (16).
(19) The near-field optical observation apparatus according to (18), wherein the optical path system is formed by combining two or more optical components selected from a group of a lens, a mirror, and an optical fiber. .
(20) The near-field optics according to any one of (1) to (19), wherein the light emission measurement module is any one of a module having a photomultiplier tube, a spectroscopic measurement system, and a multiwavelength light emission measurement module. Observation device.

(21) Prepare a first substrate having an entrance window on one side or the other side of the frame part having a hole communicating between the one side and the other side, and attach a sample to the other side of the entrance window And then dropping the filling medium so as to cover the sample, preparing a second substrate having an exit window, dropping the filling medium so as to cover the exit window, and the first substrate And a step of superposing the second substrate so that the filling medium is mixed therewith.

(22) The sample containing according to (21), wherein the light emitting layer is dropped and coated on a structural layer made of a high-strength material to form an incident window as a pre-step of the step of dropping the filling medium. Environmental cell fabrication method.
(23) In the superimposing step, excess filling medium is discharged and removed from the filling medium discharging hole provided in the first substrate or the second substrate, and then the filling medium discharging hole is filled. The method for preparing a sample-containing environmental cell according to (21) or (22), wherein another sealing seal is overlapped and adhered to the sealing seal provided on the medium discharge side.
(24) The sample-containing environmental cell according to any one of (21) to (23), wherein the first substrate and the second substrate are screwed and fixed to the third substrate after the overlapping step. Method.

(25) A scanning electron optical microscope comprising the near-field optical observation apparatus according to any one of (1) to (20) and an electron microscope.
(26) The scanning electron optical microscope according to (25), wherein the near-field optical observation device is arranged in a vacuum chamber of an electron microscope.
(27) The environmental cell of the near-field optical observation device is held by an environmental cell holding unit, and the light emission measurement module of the near-field optical observation device is held by the holding unit, with respect to the holding unit The scanning electron optical microscope according to (25) or (26), further comprising an alignment movement mechanism for the environmental cell holding unit.
(28) According to (27), the environmental cell holding unit can be detached from the holding unit and moved to another vacuum chamber connected to a vacuum chamber of an electron microscope by the alignment moving mechanism. The scanning electron optical microscope as described.

(29) A method for using the scanning electron optical microscope according to any one of (24) to (28), wherein an electron beam having an energy at which an electron beam transmittance of an incident window of the environmental cell is 1% or less is applied to the environmental cell. A method of using a scanning electron optical microscope characterized by irradiating an incident window of a scanning electron microscope.
(30) The method of using the scanning electron optical microscope according to (29), wherein an electron beam having an energy of 0.8 kV or more and 1.2 kV or less is irradiated to an incident window of an environmental cell.

  The near-field optical observation apparatus according to the present invention includes an environment cell including a space part capable of sealingly holding a sample, an incident window provided in one of the space parts, and an exit window provided in the other of the space parts. And a light emission measuring module, and the incident window has a light emitting material capable of emitting light by electron beam excitation. Non-fixed biological sample can be stably held as a measurement target, and the fluorescent material in the window of the environmental cell emits light by electron beam excitation. The light source can be used as a near-field light source, so that optical observation can be performed with high resolution exceeding the diffraction limit.

  In the sample-containing environment cell manufacturing method of the present invention, a first substrate having an entrance window provided on one side of a frame part having a hole communicating with one side and the other side is prepared, and the other side of the entrance window is provided. A step of dropping a filling medium so as to cover the sample, a step of preparing a second substrate having an exit window, and dropping a filling medium so as to cover the exit window; Since the first substrate and the second substrate are superposed so that the filling medium is mixed, the sample is attached to the incident window, the inside of the environmental cell is filled with the filling medium, and the inside is closed. As a system, an environmental cell containing an unstained, non-fixed biological sample in a liquid can be easily prepared.

  Since the scanning electron optical microscope of the present invention has the above-mentioned near-field optical observation apparatus and the electron microscope, an environmental cell containing an unstained, non-fixed biological sample in the liquid is used as an electron microscope. The inside of the vacuum chamber is arranged, and an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  The method of using the scanning electron optical microscope of the present invention is the above-described method of using the scanning electron optical microscope, wherein an electron beam having an energy at which the electron beam transmittance of the incident window of the environmental cell is 1% or less is applied to the environmental cell. The environment cell containing the unstained, non-fixed biological sample in the liquid is placed in the vacuum chamber of the electron microscope, and the non-stained, non-fixed organism in the liquid. Optical observation can be performed with high resolution without damaging or destroying the sample.

1A is a schematic diagram illustrating an example of a scanning electron optical microscope according to a first embodiment of the present invention, and FIG. It is a schematic diagram which shows an example of the near-field optical observation apparatus which is embodiment of this invention. It is a schematic diagram which shows an example of the state which filled the sample in the environmental cell of the near-field optical observation apparatus which is embodiment of this invention. It is explanatory drawing of an example of the principle of the near-field optical observation apparatus which is embodiment of this invention. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a nanoparticle.

It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a living cell. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a living cell. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a living cell. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a living cell. It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a living cell.

It is process drawing which shows an example of the sample containing environmental cell preparation method which is embodiment of this invention, Comprising: It is a case where a sample is a living cell. It is a schematic diagram which shows an example of the scanning electron optical microscope which is the 2nd Embodiment of this invention. It is a schematic diagram which shows an example of a multiwavelength light emission measurement module. It is a schematic diagram which shows an example of the environmental cell with which the scanning electron optical microscope which is the 3rd Embodiment of this invention is equipped. It is a schematic diagram which shows an example of the environmental cell with which the scanning electron optical microscope which is 1st, 4-8 embodiment of this invention is equipped. It is a schematic diagram which shows an example of the combination of the structure layer and frame part of an environmental cell with which the scanning electron optical microscope which is 1st, 9th, 10th embodiment of this invention is equipped. It is a graph which shows a light reflection effect evaluation result. It is a simulation result of the electron beam diffusion region in the incident window. It is the external appearance photograph (a) of drop coating, and the enlarged photograph (b) of the pipette tip part used for drop coating. It is the operation photograph (a) of drop coating, and the smoothness evaluation photograph (b) of a drop coating surface. It is a graph which shows the evaluation result of the relationship between electron beam energy and the transmittance | permeability of an electron beam. A photograph (a) of the produced first to third substrates and a cell culture silicone ring, a combination photograph (b) of the first to third substrates, and an explanatory photograph (c) of the first to third substrates. is there. It is the photograph (a) of the electric / optical feedthrough terminal of an electron microscope, and the photograph (b) inside a vacuum chamber. It is the photograph (a) of an environmental cell holding | maintenance part, and its enlarged photograph (b). It is the photograph (a) and the enlarged photograph (b) of the environmental cell holding | maintenance part pulled out.

It is a photograph of a holding part and an environmental cell holding part. It is the photograph which attached the silicone ring for cell cultures to the 1st board | substrate, and arrange | positioned in the petri dish. It is the schematic image (b) which extracted the main structure from the microscope image (a) which shows the observation result of the non-stained non-fixed cell in a liquid, and a filtering image. It is the graph (c) of the half value width obtained from the microscope image (a), (b) and photograph which show a high-resolution evaluation result. It is a graph which shows an optical spectrum evaluation result. It is a graph which shows the optical spectrum evaluation result of the sample which consists of inorganic fluorescent substance and organic fluorescent substance. It is a microscope image which shows the observation result of a gold nanoparticle.

(First embodiment of the present invention)
Hereinafter, a near-field optical observation apparatus, a sample-containing environment cell preparation method, a scanning electron optical microscope, and a method of using the scanning electron optical microscope, which are embodiments of the present invention, will be described with reference to the accompanying drawings.

<Scanning electron optical microscope>
First, the scanning electron optical microscope which is the 1st Embodiment of this invention is demonstrated.
FIG. 1 is a schematic diagram (a) illustrating an example of a scanning electron optical microscope according to the first embodiment of the present invention, and a block diagram (b) of the entire system.
As shown in FIG. 1A, a scanning electron optical microscope 101 according to a first embodiment of the present invention is roughly configured to include a near-field optical observation device 11 and an electron microscope.
Specifically, a substantially cylindrical vacuum chamber 103 capable of reducing the pressure inside, an electron source (electron gun) 102 disposed on the tip side inside the vacuum chamber 103, and an electron beam 91 emitted from the electron source 102 It has a scanning coil 104 that controls the traveling direction, and a near-field optical observation device 11 that is disposed on the proximal end side inside the vacuum chamber 103.
Another vacuum chamber 105 is connected to the vacuum chamber 103. The vacuum chamber 103 and the vacuum chamber 105 can be freely opened and closed.
A shutter 108 is disposed between the environmental cell 12 and the electron gun 102. The shutter 108 is a device that electromagnetically or mechanically opens and closes the electron beam 91 so that the electron beam 91 is not irradiated onto the environmental cell 12 as necessary. A beam blanker that generates an electric field between the electrodes and blocks the electron beam. Is common. In the present apparatus, a mechanical shutter is used instead of the beam blanker.
As the electron beam 91, a low energy electron beam of 0.8 to 1.2 kV is used. Thereby, the damage given to a sample can be reduced.

The near-field optical observation device 11 includes an environmental cell 12 and a light emission measurement module 13.
The environmental cell 12 is held in the environmental cell holding unit 110, and the luminescence measurement module 13 is held in the luminescence measurement module holding unit 109. An alignment moving mechanism (not shown) for the environmental cell holding unit 110 is provided with respect to the holding unit 109.
The environmental cell holding unit 110 is detached from the holding unit 109 and can be moved to another vacuum chamber 105 connected to the vacuum chamber 103 of the electron microscope by the alignment moving mechanism (not shown). Thereby, the sample can be exchanged without breaking the vacuum in the vacuum chamber 103.

  As shown in FIG. 1B, the scanning electron optical microscope system according to the embodiment of the present invention includes an electron gun control circuit 121, a scanning signal control circuit 122, an electron beam irradiation control circuit 123, and a photodetector. A power supply circuit 124, an optical signal shaping circuit 125, a secondary electron signal shaping circuit 126, a secondary electron detector 127 connected to the secondary electron signal shaping circuit 126, and a control PC 128 connected to these circuits , And a monitor 129 connected to the control PC 128.

  Here, the electron gun control circuit 121 is a circuit that controls electron beam energy and current. The scanning signal control circuit 122 is a circuit that outputs to the scanning coil a signal that irradiates the target position with the electron beam one after another. The electron beam irradiation control circuit 123 is a circuit that controls opening and closing of the shutter.

  The photodetector power supply circuit 124 is a high-voltage power supply for a photomultiplier tube in the case of the device of the present application, and is incorporated in the light emission measuring module 13 in the case of the device of the present application. In the case of the device of the present application, the optical signal shaping circuit 125 includes a preamplifier and a photon counting circuit, and outputs a photon count value. In the case of the present device, the preamplifier is incorporated in the vicinity of the detector in the vacuum chamber. The secondary electron signal shaping circuit 126 is a shaping circuit that amplifies the signal from the secondary electron detector 127. The secondary electron detector 127 is a detector originally provided in the SEM.

<Near-field optical observation device>
Next, a near-field optical observation apparatus that is an embodiment of the present invention will be described.
FIG. 2 is a schematic diagram showing an example of a near-field optical observation apparatus that is an embodiment of the present invention. FIG. 3 is a schematic diagram illustrating an example of a state in which a sample is filled in the environmental cell of the near-field optical observation apparatus according to the embodiment of the present invention.

As shown in FIGS. 2 and 3, the near-field optical observation apparatus 11 according to the embodiment of the present invention has an environment cell 12 and a light emission measurement module 13 and is schematically configured.
The environmental cell 12 includes an environmental cell 12 including a space portion 19 capable of sealing and holding a sample, an incident window 30 provided on one side of the space portion 19, and an exit window 34 provided on the other side of the space portion 19. And the light emission measuring module 13.

The environmental cell 12 includes a frame portion 35, a hole portion 35c communicating with one surface side and the other surface side of the frame portion 35, an incident window 30 provided on one surface side of the frame portion 35 of the hole portion 35c, and a hole portion 35c. And an emission window 34 provided separately on the other surface side of the frame portion 35.
As shown in FIGS. 2 and 3, the exit window 34 may be provided on the other surface side of the frame portion 35 or separated from the other surface side, and is disposed in contact with the other surface of the frame portion 35. May be.

  As shown in FIG. 3, the environmental cell 12 includes a space 19 that can hold the sample 41 hermetically between the entrance window 30 and the exit window 34. The space 19 includes a hole 22c1, a hole 35c, a filling medium discharge hole 22c2, and a hole 38Ac. The space portion 19 is filled with a sample 41 and a filling medium 42.

  As the frame portion 35, any material of Si, stainless steel, copper, molybdenum, platinum, nickel, and gold can be used. Thereby, the size of the hole portion 35c can be accurately controlled, and the sample holding state can be accurately controlled. Moreover, it does not react with the sample or the filling medium.

  It is preferable that a hole 35c that communicates the one surface side and the other surface side of the frame portion 35 is formed so as to expand from the entrance window side toward the exit window side. Thereby, the emitted light can be efficiently transmitted to the light emission measurement module.

  It is preferable that the wall surface of the frame part 35 facing the hole part 35c is covered with a highly light-reflective material. Thereby, the emitted light can be efficiently transmitted to the light emission measurement module. As the material having high light reflectivity, any of Ag, Al, Au, and Pt can be used. It is possible to coat these materials only on the required wall surface by the oblique deposition method.

  The incident window 30 includes two layers: a structural layer 31 made of a high-strength material and a light-emitting layer 32 that is stacked on the structural layer 31 and has a light-emitting material that can emit light by electron beam excitation. However, the present invention is not limited to this, and three or more layers may be used.

The light emitting layer 32 itself is made of a light emitting material capable of emitting light by electron beam excitation, or is formed by adding another light emitting material capable of emitting light by electron beam excitation to the light emitting material. Thereby, the polymer material of the light emitting layer 32 can be utilized for a light source. Thereby, the electron beam irradiation part irradiated to the light emitting layer 32 can be used as a near-field light source.
The light emitting layer 32 itself can be a light emitting material that can emit light by electron beam excitation, from a group of π-conjugated polymers such as PPV derivatives, organic EL materials such as polyimide and Alq, plastic scintillators such as BC400, SiN, and ZnO. Any one selected material can be mentioned. In addition, another light emitting material that is added and can emit light by electron beam excitation is any one selected from the group of laser dyes such as Alq, Bbq, Zn-PBO, rubrene, Ir (ppy) ₃ , coumarin, and DCM. Can be mentioned. Specifically, the light emitting layer (coumarin dispersion PVK) which added coumarin to PVK, the light emitting layer which added Ag to ZnS, etc. can be mentioned. Examples of coumarin include coumarin 6 having a band gap of 2.3 eV.

The structural layer 31 is a membrane made of a high-strength material SiN. The structural layer 31 may be a membrane made of a high-strength material such as SiO ₂ , SiC, Si, polyimide, parylene, carbon, collodion, form bar, or triacetyl cellulose. These membranes can maintain strength even when the film thickness is reduced, and can prevent the incident window from being broken even when the environmental cell is held in the vacuum chamber.

Examples of the combination of the frame portion and the structural layer include a structural layer made of any one membrane selected from the group of SiN, SiO ₂ , SiC, and Si in the Si frame portion.
As another combination, a frame portion made of any one metal selected from the group of SUS, Cu, Mo, Pt, Ni, Au, polyimide, parylene, carbon, collodion, formbar, triacetyl cellulose. The structural layer which consists of a membrane of any one organic material selected from the group of these can be mentioned.

  The frame portion is not limited to one provided with one hole portion, and a frame portion provided with a plurality of hole portions may be used. For example, 3 × 3 = 9 holes may be arranged. Thereby, a mesh-shaped hole can be formed. The mesh-shaped hole can be formed in a Si frame or a frame made of a metal such as SUS, Cu, Mo, Pt, Ni, or Au. A membrane made mainly of an organic material such as polyimide, parylene, carbon, collodion, formbar, or triacetylcellulose can be attached as a structural layer to a frame portion made of a metal mesh.

The film thickness of the entrance window 30 is preferably 5 nm or more and 500 nm or less.
The planar view diameter of the light emitting layer 32 of the entrance window 30 is preferably 5 μm or more and 1 mm or less. Thereby, two-dimensional observation can be performed in this range.

As the exit window 34, any material of glass, polyester, polyimide, SiN, SiO ₂ , SiC, Si, parylene, carbon, collodion, form bar, and triacetyl cellulose can be used. As glass, quartz glass is preferable.
The planar view diameter of the emission window 34 is, for example, 5 μm or more and 10 mm or less.

  The luminescence measurement module 13 is disposed on the exit window 34 side of the environmental cell 12. The luminescence measurement module 13 is a module having a photomultiplier tube. Thereby, the emitted light can be captured efficiently.

  The luminescence measurement module 13 may have a configuration including a photodetector such as a photodiode or an avalanche photodiode. Moreover, it is good also as a structure which has multi-element photodetectors, such as a multi-element photomultiplier tube, a multi-element photodiode, CCD, CMOS.

<Principle of near-field optical observation device>
The principle of the near-field optical observation apparatus according to the embodiment of the present invention will be described.
FIG. 4 is a schematic diagram showing an example of the principle of the near-field optical observation apparatus according to the embodiment of the present invention.

As shown in FIG. 4, in the environment cell of the near-field optical observation apparatus according to the embodiment of the present invention, the incident window 30 in which the structural layer 31 and the light emitting layer 32 are laminated in this order from the electron gun side, and the sample 41 The filling medium 42 and the emission window 34 are stacked in this order, and the light emission measurement module 13 is disposed on the surface of the environmental cell 12 opposite to the electron gun.
The filling medium 42 is, for example, PBS as a liquid and air (Air) as a gas.

  Examples of the sample 41 include biological samples such as nanoparticles and cells made of organic compounds or inorganic compounds. It can even be applied to unstained, non-fixed biological samples in liquids that could not be observed with super-resolution optics. As samples other than living organisms, it can be said that particles of various compositions such as inorganic, organic and semiconductor are observed in an arbitrary solution or gas. Even if it is a biological sample, not only unstained and non-fixed but also stained cells and fixed cells can be targeted. Even a biological sample can be observed in air or a gas of any composition, not in a liquid, depending on the type, such as bacteria, viruses, and yeast cells having cell walls.

  An electron beam 91 emitted from the electron gun 102 is irradiated onto an arbitrary point of the light emitting layer 32, and the arbitrary point of the light emitting layer 32 emits light as a light source 92 by CL. The light 93 emitted from the light source 92 irradiates the sample 41 disposed in the near field, and as a result of the near field interaction with the sample 41, the transmitted light 93 is captured by the photocathode 13 a of the light emission measurement module 13. . The captured light is analyzed by a PC and displayed on a monitor.

  The electron beam 91 can obtain the one-dimensional direction information of the sample 41, for example, by scanning in the direction of the arrow 91a, and the information on the two-dimensional surface of the sample 41 can be obtained by sequentially shifting the scanning direction in parallel. Can be obtained.

  The light 93 from the light source 92 is arbitrary light (wavelength 200 to 2000 nm) centered on visible light, which is determined according to the type of fluorescent material, but the size of the light source 92 is nano-order corresponding to the electron beam diffusion region. Since the distance from the light source 92 to the sample 41 is as short as nano-order, the contrast of the transmitted light by the sample 41 is determined by the near-field interaction, and the near-field having a resolution exceeding the diffraction limit of visible light. Optical observation is possible.

  Since the thickness 30t of the incident window 30 is 5 nm or more and 500 nm or less, the distance between the sample 41 and the light source 92 can be shortened, and the electron beam irradiation part of the light emitting layer 32 can be used as a near-field light source. . Further, the intensity of the incident window 30 can be sufficiently maintained. In particular, by making the incident window 30 into a two-layer structure that is functionally separated into a structural layer that retains mechanical strength and a light-emitting layer having a light-emitting function, the light-emitting function is enhanced and the mechanical strength is also increased. it can.

The thickness of the light emitting layer 32 is 5 nm or more and 500 nm or less. Thereby, the distance of a sample and a light source can be shortened, and the electron beam irradiation part of a light emitting layer can be utilized as a near field light source.
The thickness of the exit window 34 is 5 nm or more and 1 mm or less. Thereby, the distance of a sample and the light emission measurement module can be shortened, and the light 93 in a wide angle range can be efficiently captured.

  The thickness of the frame part 35 is 10 μm or more and 1 mm or less. The thickness of the frame portion 35 does not affect the optical path length, but the position of the exit window 34 cannot approach the light emitting layer side from the thickness of the frame portion 35. Moreover, since the wall surface of the frame part 35 acts as a reflector, the hole 35c is formed so as to expand from the incident window side toward the emission window side, thereby increasing the efficiency of the emitted light incident on the light emission measurement module 13. Can do.

<Sample-containing environmental cell preparation method 1: When the sample is nanoparticles>
Next, a sample-containing environmental cell manufacturing method according to an embodiment of the present invention, in which the sample is nanoparticles, will be described.
5 to 12 are process diagrams showing an example of a sample-containing environment cell manufacturing method according to the embodiment of the present invention.
The sample-containing environmental cell manufacturing method according to the embodiment of the present invention includes a sample attachment / filling medium dropping step S1 on the incident window of the first substrate, a filling medium dropping step S2 on the exit window of the second substrate, The first substrate and the second substrate overlap step S3 are schematically configured.

  In addition, as a pre-process of the sample attachment / filling medium dropping step S1, it is preferable to drop and coat a light emitting layer on a structural layer made of a high-strength material to form an incident window. Thereby, even if it is a very small area, it is possible to form the light emitting layer by uniformly dispersing the fluorescent dye without biasing the film, and the resolution can be increased.

(Sample Attachment to Filling Window of First Substrate / Filling Medium Dropping Step S1)
In this step, first, as shown in FIG. 5, a first substrate 21 having an entrance window 30 provided on one surface side of a frame portion 35 having a hole portion 35c communicating one surface side and the other surface side is prepared. The frame portion 35 is bonded and fixed to the hole portion 21c1 of the first substrate 21 on the outer peripheral side. Further, the first substrate 21 is provided with a screw hole 21c2 in the substrate body 21A.
The incident window 30 includes a structural layer 31 adhered to one surface side of the frame substrate 35 and a light emitting layer 32 adhered to the other surface side of the structural layer 31.

Next, as shown in FIG. 5, the sample 41 is attached to the other surface side of the incident window 30.
Next, as shown in FIG. 6, the filling medium 42 is dropped so as to cover the sample 41.

  Next, as shown in FIG. 7, the first substrate 21 is disposed on the third substrate 23. The third substrate 23 is formed by forming a hole 23c1 and a screw hole 23c2 in the substrate body 23A. In a later step, the first substrate 21 and the second substrate 22 are fixed to the third substrate 23 with screws.

(Filling medium dropping step S2 on the exit window of the second substrate)
In this step, the second substrate 22 having the exit window 34 is prepared. The second substrate 22 has an environmental cell hole 22c1, a filling medium discharge hole 22c2, an emitted light hole 22c3, and a screw hole 22c4 formed in the substrate body 22A. Become. A sealing seal 38A provided with a hole 38Ac is attached to the filling medium discharge hole 22c2. An O-ring 37 is fitted in the environmental cell hole 22c1. The O-ring 37 is a rubber packing.
Next, as shown in FIG. 8, the filling medium 42 is dropped so as to cover the emission window 34 and to fill the environmental cell hole 22 c 1.

(First substrate and second substrate superposition step S3)
In this step, as shown in FIG. 9, the first substrate 21 and the second substrate 22 are overlaid so that the filling medium 42 is mixed. The direction in which the arrows overlap. Since the O-ring 37 is arranged, leakage of the filling medium 42 is prevented.

As shown in FIG. 10, when the first substrate 21 and the second substrate 22 are overlapped, the filling medium discharge hole 22c2 and the hole 38Ac of the sealing seal 38A provided in the second substrate are formed. Accordingly, the excess filling medium 42 is discharged.
Next, as shown in FIG. 11, the discharged filling medium 42 is removed with filter paper or the like.

Next, as shown in FIG. 12, another sealing seal 38B is adhered so as to overlap the sealing seal 38A, thereby completely isolating and sealing the inside of the environmental cell from the outside. The material of the sealing seals 38A and 38B is polyimide.
Since there are a wide variety of adhesives that can be bonded with high strength in a short time if they are made of the same material, the sealing seals can be sealed in a short time and reliably by overlapping the sealing seals.

After the superimposing step S3, the first substrate and the second substrate are preferably screwed and fixed to the third substrate. Thereby, the environmental cell 12, especially the space part 19, can be stably held.
Through the above steps, the sample-containing environmental cell can be easily produced in a short time.
With the sample-containing environment cell, particles of various compositions such as inorganic, organic, and semiconductor can be observed in an arbitrary solution or gas.

<Sample-containing environmental cell preparation method 2: When the sample is a living cell>
Next, a sample-containing environmental cell manufacturing method according to an embodiment of the present invention, in which the sample is a living cell, will be described.
13 to 18 are process diagrams showing an example of a sample-containing environment cell manufacturing method according to an embodiment of the present invention.
The sample-containing environmental cell manufacturing method according to the embodiment of the present invention is different in the sample attachment / filling medium dropping step S1 to the entrance window of the first substrate.

  First, the 1st board | substrate 21 which provided the entrance window 30 in the one surface side of the flame | frame part 35 which has the hole 35c which connects the one surface side and the other surface side is prepared. The frame portion 35 is bonded and fixed to the hole portion 21c1 of the first substrate 21 on the outer peripheral side. Further, the first substrate 21 is provided with a screw hole 21c2 in the substrate body 21A. The incident window 30 includes a structural layer 31 adhered to one surface side of the frame substrate 35 and a light emitting layer 32 adhered to the other surface side of the structural layer 31.

Next, as shown in FIG. 13, the silicone ring 45 is disposed on the first substrate 21. The silicone ring 45 adheres to the first substrate 21 with an adhesive force unique to silicone.
Next, the cells 47 cultured in another flask are dropped into the ring of the silicone ring 45 while being suspended in the culture solution 46. As shown in FIG. 14, the cells 47 float in the culture solution 46 dropped into the ring of the silicone ring 45.

As time passes, the cells 47 gradually settle and adhere to the first substrate 21, the other surface side of the entrance window 30, and the frame portion 35 as shown in FIG. 15. The cells 47 start to grow while attached to these cells, and become normal.
Next, as shown in FIG. 16, the culture solution 46 is replaced with the filling liquid 42. In the filling liquid 42, the cells 47 remain alive for several hours.
Next, as shown in FIG. 17, the silicone ring 45 is removed.

In place of the first substrate with the nanoparticle sample attached to the other surface side of the entrance window 30 shown in FIG. 5, a substrate with the cell sample shown in FIG. 17 is used. In the same manner as the environmental cell manufacturing method, a cell sample-containing environmental cell can be easily manufactured in a short time.
FIG. 18 is a diagram showing an example of a sample-containing environment cell of a living cell.

This makes it possible to perform near-field optical observation of an unstained, non-fixed biological sample in the liquid, which has not been possible with super-resolution optical observation until now.
Not only unstained and non-fixed but also stained cells and fixed cells can be targeted. In addition, biological samples such as bacteria, viruses, and yeast cells having cell walls can be observed not in liquid but in air or a gas of any composition.

<How to use a scanning electron optical microscope>
The usage method of the scanning electron optical microscope according to the embodiment of the present invention is a usage method of the scanning electron optical microscope 101.
An incident beam of an environmental cell is irradiated with an electron beam having an energy of 0.8 kV or more and 1.2 kV or less. When the energy is 0.8 kV or more and 1.2 kV or less, the electron beam of this energy cannot penetrate the incident window, and damages or destroys the unstained, unfixed biological sample in the liquid in the space. And high-resolution optical observation.

(Second embodiment of the present invention)
Next, a scanning electron optical microscope that is a second embodiment of the present invention will be described.
FIG. 19 is a schematic diagram showing an example of a scanning electron optical microscope according to the second embodiment of the present invention.
As shown in FIG. 19, the scanning electron optical microscope 1102 according to the second embodiment of the present invention has an optical path system 1201 between the environmental cell 12 and the light emission measurement module 13. Other than the arrangement outside the vacuum chamber, the configuration is the same as that of the scanning electron optical microscope 101 according to the first embodiment of the present invention.
The near-field optical observation device 1211 includes an environment cell 12, a light emission measurement module 13, and an optical path system 1201.

  The environmental cell 12 and the luminescence measurement module 13 are arranged apart from each other, and the luminescence measurement module 13 is arranged outside the vacuum chamber. As a result, not only the PMT but also a complex module such as a spectroscopic measurement system or a multiwavelength light emission measurement module can be used as the light emission measurement module 13.

  An optical path system 1201 that connects the exit window side of the environmental cell 12 and the photocathode of the light emission measurement module 13 is provided. By the optical path system 1201, the emitted light is efficiently transmitted to the light emission measurement module.

The optical path system 1201 is formed by combining two or more optical components selected from the group of a lens, a mirror, and an optical fiber.
Specifically, in the optical path system 1201, the light emitted from the environment cell 12 is once converted into parallel light by the chromatic aberration correction objective lens 1122, changed in direction by the mirror 1119, and again by the chromatic aberration correction lens 1123 by the optical fiber 1125. It is condensed on the end face. The optical fiber 1125 is connected to the outside of the vacuum chamber through a feedthrough, and light is incident on the light emission measurement module 13 placed in the atmosphere.
In the optical path system 1201, the chromatic aberration correction objective lens 1122, the mirror 1119, and the chromatic aberration correction lens 1123 are attached to the holding unit 109.

  When a spectroscopic measurement system is used as the luminescence measurement module 13, in addition to two-dimensional image information, a sample can be analyzed from the viewpoint of spectrum. If the sample shows no absorption, the observed spectrum is simply the CL spectrum of the light emitting layer. If the sample absorbs a specific wavelength, the intensity of the wavelength decreases, so that a near-field optical image including absorption spectrum information of the sample can be acquired.

When a multi-wavelength light emission measurement module is used as the light emission measurement module 13, a color image can be observed.
FIG. 20 is a schematic diagram illustrating an example of a multiwavelength light emission measurement module.
As shown in FIG. 20, light from an optical fiber 1125 is converted into parallel light by a lens 1121, and divided into three optical paths of blue, green, and red by dichroic mirrors 1117 and 1118. Providing the photodetectors 1113, 1114, and 1115 in front of each enables color near-field optical observation.
Although it is difficult to directly incorporate such a complex module into a narrow SEM sample chamber, by taking out an optical signal through the optical fiber 1125 to the outside of the vacuum chamber, there is no restriction on the space of the light emission measurement module 13. Such a degree of freedom can be obtained.

(Third embodiment of the present invention)
Next, a scanning electron optical microscope that is a third embodiment of the present invention will be described.
FIG. 21 is a schematic diagram showing an example of an environmental cell provided in the scanning electron optical microscope according to the third embodiment of the present invention. The scanning electron optical microscope according to the third embodiment of the present invention has the same configuration as that of the scanning electron optical microscope 101 according to the first embodiment of the present invention, except that the environment cell shown in FIG. 21 is provided.

As shown in FIG. 21, the environmental cell 2012 is configured by laminating a mesh-shaped frame portion 2035, an incident window 2030, a spacer 2055, an emission window 2034, and a support substrate 2051 from the electron beam side.
The incident window 2030 includes an antistatic layer 2053 and a light emitting layer 2052.
In the space 2019, the sample 2041 is arranged on the light emitting layer side and is filled with a filling medium 2042.
In FIG. 21, the spacer and the exit window are joined with an adhesive. However, even if the O-ring is sandwiched between the spacer and the exit window, the spacer and the exit window may be fixed by screwing as described above. Good.

As a material of the mesh-shaped frame portion 2035, stainless steel can be used. The film thickness is 1 to 100 μm. The opening is, for example, a square and has a side of 100 to 1000 μm.
As a material of the light emitting layer 2052, a polyimide membrane can be given. The film thickness is 100 to 1000 nm. Since polyimide emits fluorescence having a peak wavelength of 550 nm by electron beam irradiation, it can be used as a light source.
An example of the material of the exit window is a PET film. The film thickness is 1 to 10 μm.
An example of the material for the antistatic layer 2053 is carbon. The film thickness is 1 to 100 nm. Thereby, charging of polyimide can be prevented.
The spacer 2055 is a ring-shaped component, and examples of the material of the spacer 2055 include polystyrene, SUS, Al, and Cu. Thereby, the space part of an environmental cell can be maintained mechanically stably, maintaining airtightness.
An example of the filling medium 2042 is air.

  With the above configuration, the incident window is irradiated with an electron beam having a low electron beam energy of 1.0 to 5.0 kV, the light of the light source excited and emitted by the incident window is transmitted through the sample, and the emission window side. The near-field optical observation can be performed by a light emission measuring module such as PMT.

(Fourth to eighth embodiments of the present invention)
Next, scanning electron optical microscopes according to fourth to eighth embodiments of the present invention will be described.
FIGS. 22B to 22E are schematic views showing an example of an environmental cell provided in a scanning electron optical microscope that is the fourth to eighth embodiments of the present invention. A scanning electron optical microscope 101 according to the fourth to eighth embodiments of the present invention is provided with the environmental cell shown in FIGS. 22B to 22E, and the scanning electron optical microscope 101 according to the first embodiment of the present invention. It is set as the same structure. For comparison, FIG. 22A shows an environmental cell of the scanning electron optical microscope 101 according to the first embodiment of the present invention.

  FIG. 22B is a schematic diagram illustrating an example of an environmental cell provided in the scanning electron optical microscope according to the fourth embodiment of the present invention. The environment cell 12D of the scanning electron optical microscope according to the fourth embodiment of the present invention is the environment of the scanning electron optical microscope 101 according to the first embodiment of the present invention except that the stacking order of the structural layer and the light emitting layer is reversed. The configuration is the same as that of the cell 12.

  FIG. 22C is a schematic diagram illustrating an example of an environmental cell provided in a scanning electron optical microscope according to the fifth embodiment of the present invention. The environmental cell 12E of the scanning electron optical microscope according to the fifth embodiment of the present invention is provided with a structural layer that also serves as a light emitting layer instead of the structural layer and the light emitting layer. It is set as the structure similar to the environmental cell 12 of the scanning electron optical microscope 101 which is.

  FIG. 22D is a schematic diagram showing an example of an environmental cell provided in the scanning electron optical microscope according to the sixth embodiment of the present invention. An environmental cell 12F of a scanning electron optical microscope according to the sixth embodiment of the present invention has a scanning electron optical microscope 101 according to the first embodiment of the present invention, except that a frame portion is provided on one surface side of the structural layer. The environmental cell 12 has the same configuration.

  FIG. 22E is a schematic diagram showing an example of an environmental cell provided in the scanning electron optical microscope according to the seventh embodiment of the present invention. The environmental cell 12G of the scanning electron optical microscope according to the seventh embodiment of the present invention is the environmental cell of the scanning electron optical microscope according to the sixth embodiment of the present invention except that the stacking order of the structural layer and the light emitting layer is reversed. The configuration is the same as 12F.

  FIG. 22E is a schematic view showing an example of an environmental cell provided in the scanning electron optical microscope according to the eighth embodiment of the present invention. The environmental cell 12H of the scanning electron optical microscope according to the eighth embodiment of the present invention is provided with a structural layer that also serves as a light emitting layer instead of the structural layer and the light emitting layer, and is the seventh embodiment of the present invention. It is set as the structure similar to the environment cell 12G of the scanning electron optical microscope which is.

(Ninth and Tenth Embodiments of the Present Invention)
Next, scanning electron optical microscopes according to ninth and tenth embodiments of the present invention will be described.
23 (d) to (f) and (g) to (i) are examples of a combination of a structural layer and a frame portion of an environmental cell included in a scanning electron optical microscope according to the ninth and tenth embodiments of the present invention. It is a schematic diagram which shows. The scanning electron optical microscope according to the ninth and tenth embodiments of the present invention includes a combination of the structural layer and the frame portion of the environmental cell shown in FIGS. 23 (d) to (f) and (g) to (i). Is configured similarly to the scanning electron optical microscope 101 according to the first embodiment of the present invention. For comparison, FIGS. 23A to 23C show a combination of the structural layer and the frame portion of the environmental cell of the scanning electron optical microscope 101 according to the first embodiment of the present invention.
23 (a), (d), and (g) are plan views, and FIGS. 23 (b), (e), and (h) are rear views of the plan views (a), (d), and (g). 23 (c), (f), and (i) are cross-sectional views taken along one-dot chain lines in plan views (a), (d), and (g).

  FIGS. 23D to 23F are schematic views showing an example of a combination of a structural layer and a frame portion of an environmental cell provided in a scanning electron optical microscope according to the ninth embodiment of the present invention. The combination of the structural layer and the frame part of the environmental cell of the scanning electron optical microscope according to the ninth embodiment of the present invention is the same as that of the first embodiment of the present invention except that the hole is formed by 3 × 3 in the frame part. The configuration is the same as the combination of the structural layer and the frame portion of the environmental cell of the scanning electron optical microscope.

  FIGS. 23G to 23I are schematic views showing an example of a combination of a structural layer and a frame portion of an environmental cell provided in a scanning electron optical microscope according to the tenth embodiment of the present invention. The scanning electron optical microscope according to the third embodiment of the present invention is the same as the combination of the structural layer and the frame portion of the environmental cell of the scanning electron optical microscope according to the tenth embodiment of the present invention except that the antistatic layer is omitted. The structure is the same as the combination of the structural layer and the frame part of the environmental cell of the microscope.

  A near-field optical observation apparatus 11 according to an embodiment of the present invention includes a space 19 that can hold a sample in a sealed state, an incident window 30 provided in one of the spaces 19, and an exit provided in the other of the spaces 19. Since the environment cell 12 including the window 34 and the light emission measurement module are included, and the incident window has a light emitting material capable of emitting light by electron beam excitation, the vacuum chamber is obtained by holding the environment cell. It is possible to measure non-stained, non-fixed biological samples in the liquid, and use high-resolution optics by using the cathodoluminescence light of the fluorescent material in the window of the environmental cell as a near-field light source. Can be observed.

  The near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the frame portion 35 is provided so as to be in contact with the entrance window 30, so that it is in a closed environmental cell and between the entrance window and the exit window. It is possible to hold an unstained, non-fixed biological sample in the liquid and optically observe with high resolution.

  Since the near-field optical observation apparatus 11 according to the embodiment of the present invention is configured so that the hole 35c is formed in the frame portion 35 so as to communicate the one surface side and the other surface side, An unstained, non-fixed biological sample in the liquid can be held between the entrance window and the exit window, and optical observation can be performed with high resolution.

  Since the near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the incident window 30 is provided on one surface side of the frame portion 35, the near-field optical observation apparatus 11 is in a closed environment cell and between the incident window and the emission window. It is possible to hold an unstained, non-fixed biological sample in the liquid and optically observe with high resolution.

  The near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the hole portion 35 is a part of the space portion 19, so that it is in a closed environmental cell and is in a liquid between the entrance window and the exit window. Thus, an unstained, non-fixed biological sample can be held and optically observed with high resolution.

  Since the near-field optical observation apparatus according to the embodiment of the present invention has a configuration in which the incident window 2030 is provided on the other surface side of the frame portion 2035, it is in a closed environment cell and between the incident window and the emission window. It is possible to hold an unstained, non-fixed biological sample in the liquid and optically observe with high resolution.

  The near-field optical observation device 11 according to an embodiment of the present invention is configured to have an exit window provided on the other surface side of the frame portion or spaced apart from the other surface side. The unstained, non-fixed biological sample in the liquid can be held between the entrance window and the exit window, and optical observation can be performed with high resolution.

  The near-field optical observation device 11 according to the embodiment of the present invention has a configuration in which the hole portion that communicates the one surface side and the other surface side of the frame portion 12 is formed so as to spread from the entrance window side toward the exit window side. By efficiently capturing light emitted from the near-field light source through the sample, an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  The near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the wall surface of the frame portion facing the hole portion 35c is covered with a highly light-reflective material, and thus affects the sample in the environmental cell. First, an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  In the near-field optical observation apparatus 11 according to an embodiment of the present invention, the entrance window 30 is formed of two or more layers, and is laminated on the structural layer 31 made of a high-strength material and excited by electron beam. And a light emitting layer 32 having a light emitting material capable of emitting light, so that the strength of the environmental cell is secured by the structural layer, while a near-field light source is formed in the light emitting layer, An unstained, non-fixed biological sample can be optically observed with high resolution.

  In the near-field optical observation apparatus according to the embodiment of the present invention, the light-emitting layer 2052 itself is made of a light-emitting material that can emit light by electron beam excitation. Stained, non-fixed biological samples can be optically observed with high resolution.

  The near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the light emitting layer 32 is formed by adding another light emitting material capable of emitting light by electron beam excitation to the light emitting material. An unstained, non-fixed biological sample in the liquid can be optically observed with high resolution without affecting the sample.

In the near-field optical observation apparatus 11 according to the embodiment of the present invention, the structural layer 31 is a high-strength material of any one of SiN, SiO ₂ , SiC, Si, polyimide, parylene, carbon, collodion, formval, and triacetylcellulose. Therefore, the strength of the environmental cell can be secured, and a stable, unstained, non-fixed biological sample can be optically observed with high resolution without rupturing the environmental cell even in a vacuum.

  The near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the film thickness 30t of the incident window 39 is 5 nm or more and 500 nm or less, so the distance from the near-field light source in the environmental cell to the luminescence measurement module is shortened. Thus, an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

In the near-field optical observation apparatus 11 according to the embodiment of the present invention, the exit window 34 is any one of glass, polyester, polyimide, SiN, SiO ₂ , SiC, Si, parylene, carbon, collodion, formbar, and triacetyl cellulose. Therefore, the emitted light from the near-field light source can be efficiently transmitted to the luminescence measurement module, and an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  Since the near-field optical observation apparatus according to the embodiment of the present invention has a configuration in which a plurality of entrance windows are provided, the light emitted from the near-field light source is efficiently transmitted to the luminescence measurement module, and the non-stained, non-stained liquid is obtained. A fixed biological sample can be optically observed with high resolution.

  The near-field optical observation apparatus 11 according to the embodiment of the present invention has a configuration in which the luminescence measurement module 13 is disposed on the exit window side of the environmental cell, so that an unstained, non-fixed biological sample in the liquid is high. Optical observation with resolution is possible.

  In the near-field optical observation device 1211 according to the embodiment of the present invention, the luminescence measurement module 13 is disposed away from the environment cell 12 and connects the exit window side of the environment cell and the photocathode of the luminescence measurement module. Since the optical path system 1202 is provided, an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  In the near-field optical observation apparatus 1211 according to the embodiment of the present invention, the optical path system 1201 is formed by combining two or more optical components selected from the group of lenses 1122 and 1123, a mirror 1119, and an optical fiber 1125. Therefore, an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  The near-field optical observation apparatuses 11 and 1211 according to the embodiments of the present invention have a configuration in which the light emission measurement module 13 is any one of a module having a photomultiplier tube, a spectroscopic measurement system, and a multiwavelength light emission measurement module. In the case of a module having a light source, the light emitted from the near-field light source can be efficiently captured by the light emission measurement module, and an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution. In addition to typical image information, the sample can be analyzed from the viewpoint of spectrum, and color near-field optical observation becomes possible.

  In the sample-containing environmental cell manufacturing method according to the embodiment of the present invention, a first substrate provided with an entrance window on one side or the other side of a frame portion having a hole communicating with one side and the other side is prepared. A step S1 of dropping a filling medium so as to cover the sample after the sample is attached to the other surface side of the entrance window and a second substrate having an exit window are prepared, and the exit window is covered. Since the configuration includes the step S2 of dropping the filling medium and the step S3 of superimposing the first substrate and the second substrate so that the filling medium is mixed, the sample is attached to the incident window and the environment is filled with the filling medium. An environmental cell containing an unstained, non-fixed biological sample in a liquid can be easily prepared as a system in which the inside of the cell is filled and the inside is closed.

  In the sample-containing environmental cell manufacturing method according to the embodiment of the present invention, as a pre-process of the step S1 of dropping the filling medium, a light emitting layer is dropped and coated on a structural layer made of a high-strength material to form an incident window. Since it is configured, a smooth and uniform light emitting layer can be formed, and an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  In the sample-containing environmental cell manufacturing method according to the embodiment of the present invention, the excess filling medium is discharged / removed from the filling medium discharging hole provided in the first substrate or the second substrate in the overlapping step S3. Then, another sealing seal is overlapped and adhered to the sealing seal provided on the filling medium discharge side of the filling medium discharge hole, so that the environment cell is completely filled with the filling medium. An environmental cell containing an unstained, non-fixed biological sample can be easily produced.

  Since the sample-containing environmental cell manufacturing method according to the embodiment of the present invention is configured to screw and fix the first substrate and the second substrate to the third substrate after the superimposing step S3, An environmental cell containing an unstained, non-fixed biological sample in a liquid that is not ruptured in a vacuum chamber can be easily produced.

  Since the scanning electron optical microscopes 101 and 1102 according to the embodiment of the present invention include the near-field optical observation apparatuses 11 and 1211 and the electron microscope, they contain an unstained, non-fixed biological sample in the liquid. An environmental cell is placed in a vacuum chamber of an electron microscope, and an unstained, non-fixed biological sample in the liquid can be optically observed with high resolution.

  Since the scanning electron optical microscope 101 which is an embodiment of the present invention has a configuration in which the near-field optical observation device 11 is disposed in a vacuum chamber of an electron microscope, an unstained, non-fixed biological sample in liquid is used. Optical observation can be performed with high resolution.

  In the scanning electron optical microscope 101 according to the embodiment of the present invention, the environment cell 12 of the near-field optical observation apparatus 11 is held by the environment cell holding unit 110, and the light emission measurement module 13 of the near-field optical observation apparatus 11 is Since it is held by the holding unit 109 and is provided with a mechanism for aligning and moving the environmental cell holding unit with respect to the holding unit, the environmental cell holding unit can be replaced to easily change the sample.

  The scanning electron optical microscope 101 according to the embodiment of the present invention removes the environmental cell holding unit from the holding unit by the positioning movement mechanism and puts it in another vacuum chamber 105 connected to the vacuum chamber 103 of the electron microscope. Since the structure is movable, the sample can be easily exchanged by exchanging the environmental cell holder.

  The method of using the scanning electron optical microscope according to the embodiment of the present invention is the method of using the scanning electron optical microscope described above, and electrons having an energy such that the electron beam transmittance of the incident window of the environmental cell is 1% or less. Since the configuration is such that the line is irradiated to the entrance window of the environmental cell, the environmental cell containing an unstained, non-fixed biological sample in the liquid is placed in the vacuum chamber of the electron microscope, and in the liquid, the unstained, Optical observation can be performed with high resolution without damaging or destroying a non-fixed biological sample.

  The method for using a scanning electron optical microscope according to an embodiment of the present invention is the above-described method for using a scanning electron optical microscope, in which an electron beam having an energy of 0.8 kV to 1.2 kV is applied to an incident window of an environmental cell. The environment cell containing the unstained, non-fixed biological sample in the liquid is placed in the vacuum chamber of the electron microscope, and the non-stained, non-fixed biological sample in the liquid is damaged. Or can be optically observed with high resolution without destruction.

  The near-field optical observation apparatus, the sample-containing environment cell manufacturing method, the scanning electron optical microscope, and the method of using the scanning electron optical microscope, which are embodiments of the present invention, are not limited to the above-described embodiments, but are technical in the present invention. Various modifications can be made within the scope of the idea. Specific examples of this embodiment are shown in the following examples. However, the present invention is not limited to these examples.

(Test Example 1)
In order to increase the light extraction efficiency of the near-field optical observation device, the light reflection effect of the inclined surface of the frame portion that defines the space portion was simulated.
FIG. 24 is a diagram illustrating an example of an evaluation result of a light reflection effect at an inclination angle.
Light emitted from the light source in the entrance window is reflected by the inclined surface of the frame portion in the space portion and is emitted to the exit window.
In the exit window, part of the light is reflected a plurality of times at the one-side interface and the other-side interface, and is emitted from the side wall direction.
Most of the light is emitted in the PMT direction from the other surface side, passes through the PMT ground window, and is taken into the PMT.
By setting the angle of the inclined surface to 35.3 degrees from the vertical direction, the ratio of the light emitted to the side wall portion was reduced, the ratio of the light taken into the PMT was increased, and the sensitivity could be increased.

(Test Example 2)
FIG. 25 is a diagram illustrating an example of a simulation result of the electron beam diffusion region in the incident window.
tm is the thickness of the structural layer, and te is the thickness of the light emitting layer.
In FIG. 25, when the thickness tm of the structural layer is 20 nm and the thickness te of the light emitting layer is 0 nm, 30 nm, and 60 nm, the electron beam energy is 0.6 kV, 0.9 kV, and 1.0 kV, respectively. The electron beam diffusion region is shown. This area becomes the light source 92. The result of FIG. 25 shows that if an electron beam with an appropriate energy is incident according to the thickness of the incident window, the electron beam stops inside without passing through the incident window to form a near-field light source having a diameter of several tens of nm. It shows that

(Test Example 3)
A method for manufacturing a light-emitting layer composed of a uniform and smooth film was studied.
First, a solution of coumarin 6 (1 wt%)-dispersed PVK was prepared as a dispersion of organic fluorescent material. As the solvent, a mixture of chloroform and 0.1 to 1% o-dichlorobenzene was used, and coumarin 6 (1 wt%)-dispersed PVK was adjusted to a concentration of 10 to 100 μg / ml.
Next, using this dispersion, a light emitting layer was formed on the membrane by a drop coating method. The microinjection amount was about 0.05 μl.
FIG. 26 is an appearance photograph (a) of the drop coating and an enlarged photograph (b) of the tip of the pipette used for the drop coating.
FIG. 27 is an operation photograph (a) of drop coating and a smoothness evaluation photograph (b) of the drop coating surface.
It was confirmed that a uniform and smooth film was obtained by measuring the film thickness distribution by FTIR.
When the concentration of coumarin 6 was in the range of 0.1 to 10 wt%, the 1 wt% film had the highest emission intensity.

(Test Example 4)
When the thickness tm of the structural layer was 20 nm and the thickness te of the light emitting layer was 0 nm, 30 nm, and 60 nm, the relationship between the electron beam energy and the electron beam transmittance was examined.
FIG. 28 is a graph showing the results.
As the thickness te of the light emitting layer increased, the transmission initiation energy value increased. That is, it was found that the electron beam does not penetrate as the thickness te of the light emitting layer is increased. Thereby, it became clear that the damage of the sample by electron beam irradiation can be avoided by controlling the electron beam energy value and the thickness te of the light emitting layer. More specifically, since the damage to the sample becomes significant when the electron beam transmittance exceeds 1%, the electron beam energy may be set to a value smaller than that.
FIG. 28 suggests that the electron beam energy should be 1.2 kV or less in the range of te up to 60 nm. On the other hand, from the result of te = 0 nm (only the structural layer), when the electron beam energy is 0.7 kV, the electron beam hardly transmits through the structural layer and does not reach the light emitting layer. It is calculated | required that it is a practical area | region where light emission is obtained at the time of -1.2kV, and an electron beam exceeding 1% is not irradiated to a sample.

(Test Example 5)
Using the results of Test Examples 1 to 4, a near-field optical observation apparatus having the configuration shown in the embodiment was manufactured under the design conditions shown in Table 1. The first and second substrate bodies were made of stainless steel, and the third substrate body was made of aluminum.

FIG. 29 is a photograph (a) of the produced first to third substrates and a silicone ring for cell culture, a combined photograph (b) of the first to third substrates, and an explanatory photograph of the first to third substrates. (C).

FIG. 30 is a photograph (a) of the electric / optical feedthrough terminal of the electron microscope and a photograph (b) of the inside of the vacuum chamber.
FIG. 31 is a photograph (a) of the environmental cell holding unit and an enlarged photograph (b) thereof.
FIG. 32 is a photograph (a) and an enlarged photograph (b) of the extracted environmental cell holding unit.
FIG. 33 is a photograph of the holding unit and the environmental cell holding unit.

(Test Example 6)
Next, a living cell sample-containing environment cell was prepared under the conditions shown in Table 2 by using the living cell sample-containing environment cell preparation method described in the embodiment.

  FIG. 34 is a photograph in which a cell culture silicone ring is attached to a first substrate and placed in a petri dish. The silicone ring was filled with cells and a culture solution, and the cells were cultured. Table 3 shows the samples and culture solutions used.

  Through the above steps, a sample-containing environmental cell in which cells were cultured on the light emitting layer could be produced.

Next, the light emission measurement module of the near-field observation apparatus of Test Example 5 was installed at a predetermined position of the electron microscope.
Next, the movable mechanism capable of alignment was operated, and the environmental cell holder was pulled out from the holder into the sample exchange chamber.
Next, after holding this sample-containing environmental cell in the environmental cell holding part, the environmental cell holding part was accurately moved to a predetermined position inside the vacuum chamber of the electron microscope.
Next, emission measurement was performed by irradiating with an electron beam under the conditions shown in Table 4.

The result shown in FIG. 35 was obtained.
FIG. 35 (a) is an image obtained by observing an unstained, non-fixed human lung epidermal cell in a liquid in an environmental cell arranged in a vacuum chamber, and (b) is an explanatory diagram thereof. It is. The scale bar in FIG. 35 is 10 μm.
A region in close contact with the light emitting layer surface of the cells was observed brighter than the surroundings. This is considered to be due to the dipole radiation enhancement phenomenon, because the object in the vicinity of the minute light source of the light-emitting film, that is, the cell, has a higher refractive index than the surrounding medium, the light emission intensity from the light source increases, and it is considered that the light was observed brightly. In addition, the intracellular brightness was not uniform, but showed a slight distribution of brightness.

FIG. 35B shows the result of image filtering and extraction of main structures. The outline of the nucleus indicated by the broken line, fine granules, and a fibrous structure running vertically and horizontally inside the cell were observed. This fibrous structure is presumed to have a cytoskeleton observed.
Since the dipole radiation enhancement phenomenon occurs from the surface to about one-tenth of the wavelength, that is, about 50 nm, it is considered that such a skeletal structure exists in the vicinity of the adhesion interface with the light emitting layer of the cell.

  The cytoskeleton has a diameter of several tens of nanometers, and there are many cases that have been stained and observed with a fluorescence microscope. However, because of the fine structure compared to the diffraction limit, a phase contrast microscope can be used without staining. It was not observed. Also, SNOM using an optical fiber probe was not observed without staining.

  After this observation, the environmental cell was removed from the vacuum chamber, disassembled, and subjected to double staining to confirm that the cell remained alive.

  FIG. 36A shows the results demonstrating high-resolution observation. FIG. 36A is an image obtained with this microscope, and FIG. 36B is an image obtained with a conventional phase contrast microscope. A conventional phase contrast microscope image has a high signal-to-noise ratio and is clear because high-intensity illumination is possible. However, the resolution is not always high due to the influence of the diffraction limit.

  In FIG. 36 (a), cytoplasmic granules were observed as bright spots. In FIG. 36 (b), cytoplasmic granules were observed as dark-looking granules. These corresponded well.

FIG. 36 (c) is a luminance profile of a particularly small granule (A in the figure) among the granules of FIGS. 36 (a) and (b). The scale bar in FIGS. 36A and 36B is 10 μm, the vertical axis of the graph in FIG. 36C is the luminance of the image, and the horizontal axis is the position.
As shown in FIG. 36 (c), in the phase contrast image of the conventional microscope, the granule was observed to be blurred to several μm, and the profile was so gentle that it was not understood where to evaluate as the half-value width.
On the other hand, in the scanning electron optical microscope of the present invention, it was possible to observe as a clear bright spot having a half width of 205 nm exceeding the diffraction limit.

  In this way, an unstained, non-fixed biological sample in the liquid could be held in the environmental cell placed in the vacuum chamber and optically observed with high resolution. Thereby, it was clarified that it can be used as a scanning electron optical microscope (SEOM).

(Test Example 7)
An optical path system was arranged between the environmental cell and the luminescence measurement module (spectral measurement system) to produce a scanning electron optical microscope shown in the second embodiment of the present invention. PVK in which coumarin was dispersed was used as the material of the light emitting layer.

FIG. 37 shows a spectrum measured by this system. The spectrum was measured by changing the electron beam energy from 0.6 to 1.5 kV in a place where the sample showed no absorption.
When the electron beam energy was 0.6 kV, a weak CL of SiN and a wide wavelength range were observed. This is probably because the electron beam stopped inside the structural layer.
When the acceleration voltage was increased, the rise of coumarin emission with a peak at a wavelength of 520 nm was observed. Thereby, it was found that the electrons diffused to the light emitting layer.

In addition to the two-dimensional image information, the sample could be analyzed from a spectral point of view.
When the sample showed no absorption at all, the observed spectrum was simply the CL spectrum of the light emitting layer. If the sample absorbed a particular wavelength, the intensity at that wavelength decreased.

(Test Examples 8 to 15)
A scanning electron optical microscope shown in the second embodiment of the present invention was used in the same manner as in Test Example 7 except that a sample made of an inorganic phosphor and other organic phosphors shown in Table 5 was used as the material for the entrance window. Spectrum measurement was performed.

  FIG. 38 is a spectrum showing the result, which is an entire spectrum (a) and expanded spectra (b) and (c). The result of Test Example 7 is also displayed. The spectral intensity of Test Example 7 was the highest, and the spectral intensity of Test Example 14 was the lowest.

(Test Example 16)
The environmental cell shown in FIG. 21 of the third embodiment of the present invention was produced.
As the material of the mesh 2055, stainless steel was used, and the film thickness was 25 μm. The opening was a square and 340 μm per side.
As a material for the light emitting layer 2052, a polyimide membrane was used, and the film thickness was 200 nm.
As the material of the exit window, a PET film was used, and the film thickness was 1.5 μm.
As a material for the antistatic layer 2053, carbon was used, and the film thickness was 10 nm.
Air was used as the filling medium 2042.
As the sample 2041, gold nanoparticles having a diameter of 60 nm were used.

  With the above configuration, an electron beam with a low electron beam energy of 3.0 kV is irradiated on the incident window, and light from a light source having a peak wavelength of 550 nm that is excited by an electron beam with polyimide constituting the incident window is transmitted through the sample. Thus, near-field optical observation can be performed from the exit window side using a light emission measurement module such as PMT.

FIG. 39 is an image obtained with this microscope. The scale bar in FIG. 39 is 1 μm.
Since gold is a material having a large absorption in the blue to green region, in this case, the quenching of CL due to energy transfer was caused instead of the dipole radiation enhancement phenomenon.
Therefore, it looked darker than the surroundings.
In this image, it is visible at about 200 nm. Since individual particles are 60 nm, this appears to be a cluster of several particles rather than a single particle. There was not enough resolution to identify individual gold nanoparticles. This is presumably because the film thickness of the polyimide membrane used as the light emitting layer is 200 nm and the size of the light emitting region is about the same, so the resolution is limited to about 200 nm. However, the size of 200 nm exceeds the diffraction limit, and FIG. 39 is also a result demonstrating that this microscope can obtain a resolution exceeding that of a conventional optical microscope.

From the above, it is clear that not only biological samples in liquid but also various samples in various environments can be optically observed with high resolution without being affected by the diffraction limit, and can be used as a scanning electron optical microscope. did.
Table 6 shows the relationship between the names of materials used in this specification and their abbreviations.

  The near-field optical observation apparatus, the sample-containing environmental cell preparation method and the scanning electron optical microscope of the present invention are a near-field optical observation apparatus capable of optically observing an unstained, non-fixed biological sample in a liquid with high resolution, The present invention relates to a sample-containing environmental cell preparation method and a scanning electron optical microscope, and may be used in a high-resolution optical microscope industry and an optical microscope industry in which a biological sample can be observed with high resolution as it is.

DESCRIPTION OF SYMBOLS 11 ... Near-field optical observation apparatus, 12 ... Environmental cell, 13 ... Luminescence measuring module, 13a ... Photocathode, 19 ... Space part, 21 ... 1st board | substrate, 21A ... 1st board | substrate body, 21c1 ... Hole part, 21c2 ... Hole , 22 ... second substrate, 22A ... second substrate body, 22c1 ... hole, 22c2 ... hole for discharging filling medium, 22c3 ... hole, 22c4 ... hole, 23A ... third substrate body, 23c1 ... hole , 23c2 ... hole, 23c3 ... hole, 23c4 ... hole, 23 ... third substrate, 23A ... third substrate body, 23c1 ... hole, 23c2 ... hole, 30 ... incident window, 30t ... incident window thickness 31 ... Structural layer 32 ... Light emitting layer 34 ... Exit window 35 ... Frame portion 35c ... Hole portion 36 ... Screw 37 ... O-ring 38 ... Sealing seal 38A ... Sealing seal 38Ac ... Hole Part, 38B ... sealing seal, 41 ... sample, 42 Filling medium, 45 ... silicone ring, 46 ... culture solution, 47 ... cell, 91 ... electron beam, 91a ... electron beam scanning direction, 92 ... light source (near-field light source), 93 ... light, 101 ... scanning electron optical microscope (SEOM) ), 102 ... Electron source (electron gun), 103 ... Vacuum chamber, 104 ... Scanning coil, 105 ... Vacuum chamber, 108 ... Shutter, 109 ... Holding unit, 110 ... Environmental cell holding unit, 121 ... Electron gun control circuit, 122 DESCRIPTION OF SYMBOLS ... Scanning signal control circuit, 123 ... Electron beam irradiation control circuit, 124 ... Photo detector power supply circuit, 125 ... Optical signal shaping circuit, 126 ... Secondary electron signal shaping circuit, 127 ... Secondary electron detector, 128 ... Control PC DESCRIPTION OF SYMBOLS 129 ... Monitor, 1102 ... Scanning electron optical microscope (SEOM), 1201 ... Optical path system, 1113, 1114, 1115 ... Photo detector, 1117, 1118 ... Dichro 1119 ... Chromatic aberration correction objective lens, 1123 ... Chromatic aberration correction lens, 1125 ... Optical fiber, 2012 ... Environmental cell, 2019 ... Space part, 2030 ... Incoming window, 2035 ... Frame part, 2034 ... Output window, 2041 ... sample, 2042 ... filling medium, 2051 ... support substrate, 2052 ... light emitting layer, 2053 ... antistatic layer, 2055 ... spacer.

Claims (30)

A space part capable of hermetically holding a sample, an incident window provided in one of the space parts, an environmental cell provided with an exit window provided in the other of the space part, and a luminescence measurement module,
The near-field optical observation apparatus, wherein the incident window has a light emitting material capable of emitting light by electron beam excitation.   The near-field optical observation apparatus according to claim 1, wherein a frame portion is provided so as to be in contact with the incident window. The near-field optical observation apparatus according to claim 2, wherein a hole is formed in the frame portion so as to communicate one surface side and the other surface side.   The near-field optical observation apparatus according to claim 3, wherein the incident window is provided on one surface side of the frame portion. The near-field optical observation apparatus according to claim 3, wherein the hole is a part of the space.   The near-field optical observation apparatus according to claim 3, wherein the incident window is provided on the other surface side of the frame portion. The near-field optical observation apparatus according to claim 1, wherein an exit window is provided on the other surface side of the frame portion or spaced apart from the other surface side.   The near-field optical observation apparatus according to claim 3, wherein the hole portion that communicates the one surface side and the other surface side of the frame portion is formed so as to expand from the entrance window side toward the exit window side. The near-field optical observation apparatus according to claim 8, wherein a wall surface of the frame portion facing the hole is covered with a highly light-reflective material.   The incident window is formed of two or more layers, and includes a structural layer made of a high-strength material, and a light-emitting layer that is stacked on the structural layer and has a light-emitting material that can emit light by electron beam excitation. The near-field optical observation apparatus according to claim 1, wherein the near-field optical observation apparatus is provided. The near-field optical observation apparatus according to claim 10, wherein the light-emitting layer itself is made of a light-emitting material capable of emitting light by electron beam excitation. The near-field optical observation apparatus according to claim 11, wherein the light emitting layer is formed by adding another light emitting material capable of emitting light by electron beam excitation to the light emitting material. Said structural layer, _SiN, claim, characterized in that a SiO 2, SiC, Si, consists of polyimide, parylene, carbon, collodion, Formvar, from any of the high strength material of triacetyl cellulose membrane 10-12 The near-field optical observation apparatus according to any one of the above.   The near-field optical observation apparatus according to claim 1, wherein the incident window has a thickness of 5 nm to 500 nm. The exit window, glass, polyester, polyimide, _SiN, SiO 2, SiC, Si, parylene, carbon, collodion, Formvar, of claims 1 to 14, characterized in that it consists of any material of triacetyl cellulose The near-field optical observation apparatus according to any one of the above.   The near-field optical observation apparatus according to claim 1, wherein a plurality of the incident windows are provided. The near-field optical observation apparatus according to claim 1, wherein the light emission measurement module is disposed on an exit window side of the environmental cell. The light emission measurement module is disposed apart from the environmental cell, and an optical path system that connects an emission window side of the environmental cell and a photoelectric surface of the light emission measurement module is provided. The near-field optical observation apparatus according to any one of -16. 19. The near-field optical observation apparatus according to claim 18, wherein the optical path system is formed by combining two or more optical components selected from a group of a lens, a mirror, and an optical fiber.   The near-field optical observation apparatus according to claim 1, wherein the light emission measurement module is any one of a module having a photomultiplier tube, a spectroscopic measurement system, and a multiwavelength light emission measurement module. After preparing a first substrate having an entrance window on one side or the other side of a frame part having a hole communicating with one side and the other side, and attaching a sample to the other side of the entrance window Dropping a filling medium so as to cover the sample;
Preparing a second substrate having an exit window and dropping a filling medium so as to cover the exit window;
A method for producing a sample-containing environment cell, comprising: stacking the first substrate and the second substrate so that a filling medium is mixed.   23. The sample-containing environment cell according to claim 21, wherein, as a pre-step of the step of dropping the filling medium, the light emitting layer is dropped and coated on the structural layer made of a high-strength material to form an incident window. Method. In the overlapping step, after the excess filling medium is discharged / removed from the filling medium discharge hole provided in the first substrate or the second substrate, the filling medium discharge side of the filling medium discharge hole The sample-containing environment cell manufacturing method according to claim 21 or 22, wherein another sealing seal is overlapped and adhered to the sealing seal provided on the sample. The sample-containing environment cell manufacturing method according to any one of claims 21 to 23, wherein the first substrate and the second substrate are screwed and fixed to the third substrate after the overlapping step. A scanning electron optical microscope comprising the near-field optical observation apparatus according to claim 1 and an electron microscope. 26. The scanning electron optical microscope according to claim 25, wherein the near-field optical observation device is disposed in a vacuum chamber of an electron microscope.   An environmental cell of the near-field optical observation apparatus is held in an environmental cell holding part, and a light emission measurement module of the near-field optical observation apparatus is held in a holding part, and the environmental cell is in relation to the holding part. 27. The scanning electron optical microscope according to claim 25 or 26, further comprising an alignment moving mechanism for the holding portion. 28. The scanning according to claim 27, wherein the environmental cell holder can be removed from the holder and moved to another vacuum chamber connected to a vacuum chamber of an electron microscope by the alignment moving mechanism. Electron optical microscope. 29. The method of using a scanning electron optical microscope according to claim 24, wherein an electron beam having an energy at which an electron beam transmittance of an incident window of the environmental cell is 1% or less is used as an incident window of the environmental cell. A method of using a scanning electron optical microscope characterized by irradiating a laser beam. 30. The method of using a scanning electron optical microscope according to claim 29, wherein an electron beam having an energy of 0.8 kV or more and 1.2 kV or less is irradiated to an incident window of an environmental cell.