WO2011126041A1 - 複合顕微鏡装置 - Google Patents
複合顕微鏡装置 Download PDFInfo
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- WO2011126041A1 WO2011126041A1 PCT/JP2011/058684 JP2011058684W WO2011126041A1 WO 2011126041 A1 WO2011126041 A1 WO 2011126041A1 JP 2011058684 W JP2011058684 W JP 2011058684W WO 2011126041 A1 WO2011126041 A1 WO 2011126041A1
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- optical
- objective lens
- microscope
- sample
- reflecting mirror
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/22—Optical or photographic arrangements associated with the tube
- H01J37/226—Optical arrangements for illuminating the object; optical arrangements for collecting light from the object
- H01J37/228—Optical arrangements for illuminating the object; optical arrangements for collecting light from the object whereby illumination and light collection take place in the same area of the discharge
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/04—Means for controlling the discharge
- H01J2237/045—Diaphragms
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2237/00—Discharge tubes exposing object to beam, e.g. for analysis treatment, etching, imaging
- H01J2237/26—Electron or ion microscopes
- H01J2237/2602—Details
- H01J2237/2605—Details operating at elevated pressures, e.g. atmosphere
- H01J2237/2608—Details operating at elevated pressures, e.g. atmosphere with environmental specimen chamber
Definitions
- the present invention relates to a scientific instrument using an electron beam, and more particularly to a transmission electron microscope and an electron beam analyzer.
- FIG. 8A and 8B show a conventional composite microscope apparatus.
- the optical microscope is placed adjacent to the electron microscope body, and the sample is horizontally moved between the optical microscope and the electron optical axis of the electron microscope, thereby switching between the optical microscope and the electron microscope for observation.
- FIG. 8B an apparatus in which two microscopes are integrated is also known. This apparatus rotates a sample by 90 ° and observes an optical microscope image (J. Struct. Biol. 164 (2008)). 183-189).
- an operation for searching for fluorescently stained viruses in cells is performed with a fluorescence microscope, and an operation for enlarging and fixing (photographing) the searched viruses is performed with an electron microscope. It is impossible to do.
- the conventional apparatus is not suitable for so-called high-throughput operation for quickly detecting and fixing an object to be detected.
- Patent Documents 2 and 3 also disclose an apparatus for observing the same sample by combining an electron microscope and an optical microscope, but these techniques are for a scanning electron microscope, and are a transmission electron microscope. There is still no technology that can simultaneously observe the same sample using a combination of an optical microscope and an optical microscope.
- the main object of the present invention is to provide an apparatus capable of observing the same sample at the same time by combining a transmission electron microscope and an optical microscope.
- the composite microscope apparatus of the present invention includes a transmission electron microscope and an optical microscope.
- the electron microscope includes an electron gun that emits an electron beam toward a sample, an electromagnetic objective lens that forms an image of the electron beam, and a detection unit that receives the electron beam that has passed through the electromagnetic objective lens.
- a reflecting mirror is disposed in the middle of the traveling path, and the optical microscope is provided with an optical objective lens disposed at a position separated from the traveling path.
- the reflecting surface of the reflecting mirror is inclined toward the sample and the optical objective lens.
- An installation center hole penetrating the reflecting mirror is formed at a position where the reflecting mirror intersects the traveling path.
- the diameter of the installation center hole is preferably 0.1 to 1 mm.
- an angle adjusting mechanism for adjusting the tilt angle of the reflecting mirror it is more desirable to provide a lens adjusting mechanism for adjusting the optical objective lens.
- the electromagnetic objective lens one having a cylindrical coil and a yoke covering the coil can be used. A notch is formed in a part of the yoke, and the part in which the notch of the yoke is formed projects into the inside of the coil, so that a gap can be formed inside the electromagnetic objective lens.
- an optical objective lens and a sample holder in which a sample is arranged can be arranged.
- An optical microscope having a light source, a dichroic mirror, and an optical detection unit can be used.
- the dichroic mirror, the optical objective lens, and the optical detection unit can be arranged on a straight line that intersects the traveling path. It is desirable to incline the reflecting surface of the dichroic mirror toward the optical objective lens and the light source.
- a fluorescent microscope lens can be used as the optical objective lens.
- the optical microscope can include an illumination reflector disposed on the traveling path and a light source disposed away from the traveling path. The illumination reflector is opposite to the reflector with the sample interposed therebetween. It is desirable to be located on the side.
- the reflective surface of the illumination reflector can be inclined from the traveling path toward the sample and the light source, and an installation center hole that penetrates the illumination reflector can be formed at a position where the traveling path of the illumination reflector intersects. desirable.
- Reflector mirror surface, reflecting mirror installation center hole inner wall surface, lighting reflector mirror surface, lighting reflecting mirror installation center hole inner wall surface, lighting reflector mirror surface, lighting reflector mirror installation A transparent conductive material film is formed on one or more of the inner wall surface of the central hole, the surface of the optical condenser lens, the inner wall surface of the central hole of the optical condenser lens, and the surface of the optical objective lens. It is desirable. It is desirable to dispose an electromagnetic objective lens inside the vacuum chamber, and to form an antireflection film in the inner space of the vacuum chamber around the light source.
- an electron microscope image and an optical microscope image can be observed (photographed) simultaneously for the same sample.
- FIG. 1 is a cross-sectional view schematically showing a composite microscope of the present invention.
- FIG. 2 is a schematic cross-sectional view showing an example of an electromagnetic objective lens.
- FIG. 3 is a schematic cross-sectional view showing an example of an optical microscope.
- FIG. 4 is a schematic cross-sectional view showing another example of an optical microscope.
- FIG. 5 is a photograph showing a specific example of the composite microscope apparatus of the present invention.
- FIG. 6 is a photographed image by the composite microscope apparatus of the present invention.
- FIG. 7a is an image taken by a fluorescence microscope
- FIG. 7b is an image taken by an electron microscope at a low magnification
- FIGS. 7c, 7d, and 7e are images taken by an electron microscope at a high magnification.
- FIG. 8A is a schematic diagram for explaining a conventional composite microscope
- FIG. 8B is a side view for explaining a conventional composite microscope.
- the composite microscope apparatus 1 indicates an example of the composite microscope apparatus of the present invention.
- the composite microscope apparatus 1 includes an electron microscope 2 and an optical microscope 4.
- the structure of the electron microscope 2 is not specifically limited, For example, it has the vacuum chamber 20, the electron gun 21, the converging lens 22, the objective lens 25, the projection lens 31, and the detection part 30.
- a vacuum pump (not shown) is connected to the vacuum chamber 20, and a vacuum atmosphere is formed inside the vacuum chamber 20.
- the electron gun 21 has an electron beam (electron beam) emission port directed toward the internal space of the vacuum chamber 20, and the electron beam travels inside the vacuum chamber 20 in which a vacuum atmosphere is formed.
- a symbol C in FIG. 1 indicates a traveling path (electron optical axis) of an electron beam emitted from the electron gun 21.
- the converging lens 22, the objective lens 25, the projection lens 31, and the detection unit 30 are arranged along the electron optical axis C in the order described from the side close to the electron gun 21.
- the electron beam is converged by the convergence lens 22, imaged by the objective lens 25, magnified by the projection lens 31, and then incident on the detection unit 30.
- the electron microscope 2 used in the present invention is a transmission electron microscope (TEM), and the sample to be observed is arranged on the electron optical axis C.
- TEM transmission electron microscope
- the arrangement of the sample and the optical microscope 4 will be described together with a specific example of the objective lens 25.
- FIG. 2 is an enlarged sectional view of the objective lens 25.
- the objective lens 25 is, for example, an electromagnetic lens (magnetic field lens).
- the objective lens 25 of the electron microscope 2 is referred to as an electromagnetic objective lens 25.
- the electromagnetic objective lens 25 includes a coil 24 and a yoke 23 that covers the coil 24.
- the coil 24 has a cylindrical shape (ring shape), and the entire electromagnetic objective lens 25 has a cylindrical shape.
- the electromagnetic objective lens 25 is arranged so that one end opening of the tube is directed to the electron gun 21 and the center axis of the tube is parallel to the electron optical axis C of the electron beam. Therefore, the space inside the cylinder becomes the electron beam passage 29, and the electron beam enters the electromagnetic objective lens 25 from one end (upper end) of the cylinder and is emitted downward from the other end (lower end) of the cylinder.
- a notch 26 is formed in the yoke 23.
- the shape and position of the notch 26 are not particularly limited, but the notch 26 can be formed on the inner side, upper end, or lower end of the cylinder (electromagnetic objective lens 25). In any case, the shape of the notch 26 is preferably a ring shape surrounding the electron beam.
- FIG. 3 is an enlarged cross-sectional view of the portion indicated by reference A in FIG.
- the portion of the yoke 23 in which the notch 26 is formed protrudes more inside the cylinder than the coil 24, and the diameter of the passage 29 is reduced.
- the protruding portion (pole piece) of the yoke 23 the side closer to the electron gun 21 is distinguished as the upper pole 27, and the side far from the electron gun 21 is distinguished as the lower pole 28.
- At least a portion constituting the upper pole 27 and the lower pole 28 is made of a high magnetic material such as iron cobalt (FeCo).
- FeCo iron cobalt
- Magnetic field lines formed by the energized coil 24 leak into the passage 29 from the upper pole 27 and the lower pole 28, and the electron beam is rotated and refracted by the leakage magnetic field.
- the leakage magnetic field is controlled by adjusting the shape and / or size of the pole piece, the energization amount of the coil 24, and the like, and the imaging position of the electron beam is changed.
- the gap 26 is formed in the pole piece portion of the electromagnetic objective lens 25 by the portion of the notch 26 protruding inward.
- a sample holder 11 is disposed in the gap. In the sample holder 11, a portion where the sample 10 is installed protrudes from the notch 26 to the passage 29, and the electron beam passing through the passage 29 passes through the sample 10 and travels toward the detection unit 30.
- the focal length As the distance (working distance) between the electromagnetic objective lens 25 and the sample 10 increases, the focal length also increases and the aberration increases. However, as shown in FIG. 3, the sample 10 is placed inside the electromagnetic objective lens 25 (passage 29). If it is placed in the position, the aberration becomes small and the resolution becomes high.
- a reflecting mirror 41 is disposed in the passage 29, and a through hole (installation center hole) 42 is formed in the reflecting mirror 41.
- the installation center hole 42 is located on the electron optical axis C and has a diameter of 0.1 to 1 mm, which is larger than the beam diameter of the electron beam. Therefore, the electron beam passes through the installation center hole 42 without being reflected by the reflecting mirror 41.
- the reflecting mirror 41 may be arranged on either the detection unit 30 side or the electron gun 21 side of the sample 10, and an electron beam transmitted through the sample 10 or an electron beam transmitted through the sample 10 is installed. It passes through the center hole 42.
- the reflecting mirror 41 is located between the upper pole 27 and the lower pole 28.
- a part or all of the optical microscope 4 is disposed in the gap of the electromagnetic objective lens 25.
- the optical microscope 4 has an objective lens 43, a light source 45, and an optical detection unit 46. At least the objective lens 43 is disposed in the gap of the electromagnetic objective lens 25, and the objective lens 43 faces the reflecting mirror 41.
- the objective lens 43 of the optical microscope 4 is referred to as an optical objective lens.
- the light source 45 is, for example, a mercury lamp, and the light emitted from the light source 45 is converted into parallel light parallel to the electron optical axis C by the irradiation lens 47.
- a spectroscopic means is arranged ahead of the traveling direction of the parallel light.
- the spectroscopic means includes, for example, a dichroic mirror 52.
- the reflecting surface of the dichroic mirror 52 is inclined by a predetermined angle (here, 45 °) from the direction parallel to the electron optical axis C toward the optical objective lens 43 and the light source 45.
- excitation light light having a predetermined wavelength
- wavelength light goes straight. Accordingly, the excitation light is incident on the optical objective lens 43. It is more desirable to arrange the excitation filter 51 between the dichroic mirror 52 and the irradiation lens 47 and extract the excitation light in advance.
- the dichroic mirror 52, the optical objective lens 43, and the reflecting mirror 41 are arranged on a straight line orthogonal to the electron optical axis C, that is, on the excitation light path, at a position between the upper pole 27 and the lower pole 28. Yes.
- the reflecting surface of the reflecting mirror 41 is inclined from the electron optical axis C by a predetermined angle (here, 45 °) toward the sample 10 and the optical objective lens 43, and the excitation light passes through the optical objective lens 43 and is then reflected by the reflecting mirror 41. And is incident on the sample 10.
- Specimen 10 is stained with a fluorescent material and emits light when excitation light enters.
- the fluorescent light is reflected by the reflecting mirror 41 and enters the optical objective lens 43.
- the optical detection unit 46 is disposed at a position opposite to the reflecting mirror 41 with the optical objective lens 43 interposed therebetween. Although there is a dichroic mirror 52 between the optical objective lens 43 and the optical detection unit 46, the fluorescent light has a wavelength different from that of the excitation light, and therefore passes without being reflected by the dichroic mirror 52.
- An absorption filter 53 and an imaging lens 54 may be disposed between the optical detection unit 46 and the dichroic mirror 52.
- the excitation light and scattered light are removed from the fluorescent light by the absorption filter 53, and the fluorescent light is imaged by the imaging lens 54 and enters the optical detection unit 46.
- the optical detection unit 46 is, for example, a CCD camera or the like, and is connected to a processing device such as a computer.
- the optical detection unit 46 performs an arithmetic process on the fluorescent light captured by the optical detection unit 46 and outputs it to the output device (image display, printing, etc.).
- the optical microscope 4 in FIG. 3 is a so-called epi-illumination type fluorescence microscope that can separate the excitation light and the fluorescence light by the dichroic mirror 52 and observe and / or photograph the sample 10.
- the diameter of the electron beam passing through the electromagnetic objective lens 25 is small, and the diameter of the installation center hole 42 is reduced to about 0.1 to 1.0 mm so that a sufficient amount of excitation light and fluorescent light are reflected by the reflecting mirror 41. Even so, it can pass through the installation center hole 42. Therefore, the composite microscope apparatus 1 can perform electron beam irradiation and light irradiation on the sample 10 on the same axis, and the same sample 10 can be observed and photographed by the transmission electron microscope 2. Observation and photographing with the optical microscope 4 can be performed simultaneously.
- the optical microscope 4 used in the present invention is not limited to a fluorescence microscope.
- Reference numeral 8 in FIG. 4 is a bright-field optical microscope, and members having the same configuration as in FIG.
- the optical detection unit 46 and the optical objective lens 43 can have the same structure and the same arrangement as the optical microscope 4 in FIG. 3, but the arrangement of the light source 85 is different.
- the light source 85 is disposed above the upper pole 27, for example.
- the light source 85 is directed to the electronic optical axis C, and an illumination reflecting mirror 81 is disposed at a position facing the light source 85 on the electronic optical axis C.
- the sample 10 can be disposed in the passage 29 inside the electromagnetic objective lens 25 as in FIG.
- the reflecting surface of the illumination reflecting mirror 81 is inclined from the electron optical axis C by a predetermined angle (here, 45 °) toward the sample 10 and the light source 85.
- An irradiation lens 87 is disposed between the light source 85 and the illumination reflecting mirror 81, and light from the light source 85 is condensed on the reflection surface of the illumination reflecting mirror 81 by the irradiation lens 87 and travels toward the sample 10 on the reflection surface. And reflected.
- the illumination reflector 81 is disposed away from the upper pole 27 and has a long distance to the sample 10.
- an optical condenser lens (condenser lens) 83 is disposed between the illumination reflecting mirror 81 and the sample 10.
- the optical condenser lens 83 is attached to the upper pole 27, and the light is collected by the optical condenser lens 83 and then enters the sample 10.
- a daylighting reflector 88 is disposed at a position opposite to the illumination reflector 81 with the sample 10 in between.
- the reflecting mirror 88 for daylighting is inclined at a predetermined angle (here, 45 °) from the electron optical axis C toward the sample 10 and the optical objective lens 43. Therefore, the transmitted light that has passed through the sample 10 is reflected by the reflecting reflector 88 and enters the optical objective lens 43.
- the optical detection unit 46 is disposed at the tip of the traveling direction of the transmitted light, as in the first example (FIG. 3), and the transmitted light is transmitted directly or after passing through the absorption filter 53 and the imaging lens 54. 46 is incident.
- the illumination reflecting mirror 81, the optical condenser lens 83, and the daylighting reflecting mirror 88 are each arranged on the electron optical axis C of the electron beam, and a through hole (installation center hole) 82 is provided in a portion corresponding to the traveling path of the electron beam.
- 84 and 89 are formed, respectively. Similar to the reflecting mirror 41 in FIG. 3, the diameters of the installation center holes 82, 84, and 89 are set to such an extent that they do not hinder the passage of electron beams (0.1 to 1 mm). Therefore, also in the optical microscope 8 of FIG. 4, the same sample 10 can be observed and photographed by the transmission electron microscope 2 and observed and photographed by the optical microscope 8 at the same time.
- the light source 85 and the illumination reflecting mirror 81 may be disposed below the lower pole 28 (that is, on the detection unit 30 side).
- the reflecting mirror 88 for daylighting is arranged on the upper pole 27 side with respect to the sample 10, and the light transmitted through the sample 10 is sampled from below.
- a spectroscopic means (a dichroic mirror, an excitation filter, etc.) is arranged between the light source 85 and the illumination reflector 81, and the excitation light is extracted, so that it can be used as a fluorescence microscope. .
- the fluorescent light emitted from the sample 10 to the daylighting reflector 88 side is detected.
- the optical objective lens 43 used in the present invention is not particularly limited, and an equivalent product to a commercially available long working distance type objective lens can be used. It is desirable to use one having a working distance that allows the reflecting mirrors 41 and 88 to be installed between the sample 10 and the optical objective lens 43.
- the lens case body holding the optical objective lens 43 is changed from brass to non-magnetic one (for example, phosphor bronze), and a small hole is provided in the lens case body so that the space of the glass lens and the surrounding vacuum part can be communicated. Is desirable.
- the converging lens 22 and the projection lens 31 of the electron microscope 2 are not particularly limited.
- a magnetic lens having a structure similar to that of the electromagnetic objective lens 25 can be used.
- the surface of the optical condenser lens 83 and / or the inner wall surface of the installation center hole 84, and the surface of the optical objective lens 43 It is desirable to apply a conductive coating to prevent charging by an electron beam.
- the material of the conductive coating is any one selected from the group consisting of materials having high light transmittance, for example, indium tin oxide (ITO), zinc oxide (IZO), and indium-gallium-zinc (IGZO).
- ITO indium tin oxide
- IZO zinc oxide
- IGZO indium-gallium-zinc
- the composite microscope apparatus 1 of the present invention emits light for optical observation into the electron beam passage, a member of the electron microscope 2 where light (excitation light, fluorescent light, reflected light, etc.) is emitted. That is, it is desirable to apply an antireflection treatment (antireflection film) to the surface of the member around the sample 10.
- the antireflection treatment is, for example, a black treatment.
- the black processing is performed by, for example, the sample holder 10, the pole piece portion of the electromagnetic objective lens 25, the sample contamination prevention cooling fan of the electron microscope 2, the tip of the aperture device (particularly for the electromagnetic objective lens 25) of the electron microscope 25, the electromagnetic objective lens It is effective from the viewpoint of antireflection to apply to the wall surface of the vacuum chamber 20 and the like around the portion where the 25 diaphragm is set and the electromagnetic objective lens 25.
- the material used for the black treatment is not particularly limited as long as it is a low-reflectivity nonmagnetic material, and various materials such as titanium black can be used.
- the angles of the reflecting mirrors 41, 81, and 88 can be finely adjusted according to the type and location of the sample 10, the type of fluorescent material, excitation light, and the like, so that various samples 10 can be observed and photographed. It can be performed.
- the optical objective lens 43 it is desirable to connect the optical objective lens 43 to the lens adjusting mechanism 92 so that the distance from the optical objective lens 43 to the reflecting mirrors 41 and 88 can be changed by an operation from the outside of the vacuum chamber 20.
- the focus of the optical objective lens 43 can be finely adjusted in accordance with the type of the sample 10, and various samples 10 can be observed and photographed.
- the composite microscope apparatus 1 of the present invention When observing a biological sample in which a specific organ or cell is fluorescently labeled, in order to prevent the sample 10 from being damaged by the electron beam, the electron beam is not irradiated to the sample 10 at first, and the field of view is searched only with the optical microscopes 4 and 8. . Since the sample 10 is fluorescently labeled, it is possible to search for a visual field more efficiently than when the electron microscope 2 is used.
- the electron beam is shifted to a position off the sample 10 by the deflection coil, and when an object to be observed with high resolution is found, the electron beam is returned to the sample 10 and high resolution by the electron microscope 2 is obtained.
- the deflection coil is installed between the converging lens 22 and the electromagnetic objective lens 25, for example.
- FIG. 5 is a photograph of an external appearance of an example of the composite microscope apparatus 1 of the present invention, and a portion surrounded by an ellipse is a portion in which the optical microscopes 4 and 8 are incorporated in the electromagnetic objective lens 25.
- FIG. 6 shows an example in which inorganic Q dots, which are fluorescent agents, are simultaneously observed with the optical microscope 4 and the electron microscope 2 in the composite microscope apparatus 1.
- FIG. 7 shows an example in which cultured cells expressing fluorescent protein-fused actin are simultaneously observed with the optical microscope 4 and the electron microscope 2 of the composite electron microscope apparatus 1. 6 and 7, it is clear that the electron microscope 2 has a higher resolution than the optical microscope 4, but in the case of FIG. 6, the optical microscope 4 is more efficient in searching the field of view for identifying the position of the Q dot. Since it can be performed well, the composite microscope apparatus 1 of the present invention having both the electron microscope 2 and the optical microscopes 4 and 8 enables high-throughput electron microscope observation.
- a fluorescent portion is identified with a fluorescence microscope.
- FIG. 7a if the portion is enlarged by an electron microscope at low magnification (FIG. 7b) and high magnification (FIG. 7c, FIG. 7d, FIG. 7e), only the region of the fluorescent molecule can be observed with high resolution, and It is possible to avoid damage to the sample 10 when searching for the visual field.
- FIG. 7 the actin fibers are clearly visible in the high-magnification electron microscopic image in the presence of the fluorescent protein indicated by the fluorescence microscope together with the microtubules and ribosomes.
- phase contrast electron microscope can be used to observe the same visual field only at a specific moment while observing a living biological sample.
- the method can be observed with high resolution and high contrast, and the function and structure of the living body can be observed and correlated in real time.
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Abstract
Description
反射鏡の、進行経路と交差する位置には、反射鏡を貫通する設置中心孔を形成する。設置中心孔の直径は0.1~1mmにすることが望ましい。
反射鏡の傾斜角度を調整する角度調整機構を具備させることが望ましく、また、光学対物レンズを調整するレンズ調整機構を具備させることがより望ましい。
電磁対物レンズは、筒状のコイルと、前記コイルを覆うヨークとを具備するものを用いることができる。ヨークの一部には切り欠きを形成し、ヨークの切り欠きが形成された部分は、コイルの内側に突き出す形状とし、電磁対物レンズ内部に間隙が形成することができる。その間隙には、光学対物レンズと、試料が配置される試料ホルダとを配置できる。
光学顕微鏡は、光源と、ダイクロイックミラーと、光学検出部とを有するものを用いることができる。ダイクロイックミラーと、光学対物レンズと、光学検出部とは、進行経路と交差する直線上に並べることができる。ダイクロイックミラーの反射面を、光学対物レンズと、光源に向かって傾斜させることが望ましい。光学対物レンズには、蛍光顕微鏡用レンズを用いることができる。
光学顕微鏡には、進行経路上に配置された照明用反射鏡と、進行経路から離間して配置された光源とを具備させることもでき、照明用反射鏡は、試料を挟んで反射鏡と反対側に位置させることが望ましい。照明用反射鏡の反射面を、試料と光源に向かって、進行経路から傾斜させ、照明用反射鏡の進行経路が交差する位置に、照明用反射鏡を貫通する設置中心孔を形成することが望ましい。
進行経路上の、照明用反射鏡と試料との間の位置には、光学コンデンサレンズを配置することが望ましく、光学コンデンサレンズの進行経路が交差する位置には、光学コンデンサレンズを貫通する設置中心孔を形成することが望ましい。
反射鏡の表面と、反射鏡の設置中心孔内壁面と、照明用反射鏡の表面と、照明用反射鏡の設置中心孔内壁面と、採光用反射鏡の表面と、採光用反射鏡の設置中心孔内壁面と、光学コンデンサレンズの表面と、光学コンデンサレンズの設置中心孔内壁面と、光学対物レンズの表面のうち、いずれか一箇所以上の面には、透明導電材料の膜を形成することが望ましい。
電磁対物レンズを真空槽内部に配置し、真空槽の内部空間のうち、光源の周囲の部分には、反射防止膜を形成することが望ましい。
特定の器官や細胞を蛍光標識した生体試料を観察する場合、電子線による試料10の損傷を防ぐため、最初は電子線を試料10に照射せず、光学顕微鏡4、8のみで視野探しを行う。試料10が蛍光標識されているため、電子顕微鏡2を使用する場合と比較して効率的な視野探しが可能である。
2 電子顕微鏡
4、8 光学顕微鏡
10 試料
11 試料ホルダ
20 真空槽
21 電子銃
23 ヨーク
24 コイル
25 電磁対物レンズ
30 検出部
41、81、88 反射鏡
42、82、89 設置中心孔(貫通孔)
43 光学対物レンズ
45 光源
46 光学検出部
52 ダイクロイックミラー
83 光学コンデンサレンズ
91 角度調整機構
92 レンズ調整機構
C 電子光軸(進行経路)
Claims (11)
- 透過型の電子顕微鏡と、光学顕微鏡と、を有し、
前記電子顕微鏡は、試料に向かって電子線を放出する電子銃と、前記電子線を結像する電磁対物レンズと、前記電磁対物レンズを通過した前記電子線が入射する検出部とを有し、
前記電子線の進行経路の途中には反射鏡が配置され、
前記光学顕微鏡は前記進行経路から離間した位置に配置された光学対物レンズを有し、
前記反射鏡の反射面は、前記試料と前記光学対物レンズに向かって傾斜し、
前記反射鏡の、前記進行経路と交差する位置には、前記反射鏡を貫通する設置中心孔が形成された複合顕微鏡装置。 - 前記設置中心孔の直径は0.1~1mmであることを特徴とする請求項1記載の複合顕微鏡装置。
- 前記反射鏡の傾斜角度を調整する角度調整機構を有する請求項1又は請求項2のいずれか1項記載の複合顕微鏡装置。
- 前記光学対物レンズを調整するレンズ調整機構を有する請求項1乃至請求項3のいずれか1項記載の複合顕微鏡装置。
- 前記電磁対物レンズは、筒状のコイルと、前記コイルを覆うヨークとを有し、
前記ヨークの一部に切り欠きが形成され、
前記ヨークの前記切り欠きが形成された部分は、前記コイルの内側に突き出され、前記電磁対物レンズ内部に間隙が形成され、
前記間隙には、前記光学対物レンズと、前記試料が配置される試料ホルダとが配置された請求項1乃至請求項4のいずれか1項記載の複合顕微鏡装置。 - 前記光学顕微鏡は、光源と、ダイクロイックミラーと、光学検出部とを有し、
前記ダイクロイックミラーと、前記光学対物レンズと、前記光学検出部とは、前記進行経路と交差する直線上に並べられ、
前記ダイクロイックミラーの反射面は、前記光学対物レンズと、前記光源に向かって傾斜する請求項1乃至請求項5のいずれか1項記載の複合顕微鏡装置。 - 前記光学対物レンズは、蛍光顕微鏡用レンズである請求項6記載の複合顕微鏡装置。
- 前記光学顕微鏡は、前記進行経路上に配置された照明用反射鏡と、前記進行経路から離間して配置された光源とを有し、
前記照明用反射鏡は、前記試料を挟んで前記反射鏡と反対側に位置し、
前記照明用反射鏡の反射面は、前記試料と前記光源に向かって、前記進行経路から傾斜し、
前記照明用反射鏡の、前記進行経路と交差する位置には、前記照明用反射鏡を貫通する設置中心孔が形成された請求項1乃至請求項5のいずれか1項記載の複合顕微鏡装置。 - 前記進行経路上の、前記照明用反射鏡と前記試料との間の位置には、光学コンデンサレンズが配置され、
前記光学コンデンサレンズの、前記進行経路と交差する位置には、前記光学コンデンサレンズを貫通する設置中心孔が形成された請求項8記載の複合顕微鏡装置。 - 前記反射鏡の表面と、前記反射鏡の前記設置中心孔内壁面と、前記照明用反射鏡の表面と、前記照明用反射鏡の前記設置中心孔内壁面と、前記採光用反射鏡の表面と、前記採光用反射鏡の前記設置中心孔内壁面と、前記光学コンデンサレンズの表面と、前記光学コンデンサレンズの前記設置中心孔内壁面と、前記光学対物レンズの表面のうち、いずれか一箇所以上の面には、透明導電材料の膜が形成された請求項9記載の複合顕微鏡装置。
- 前記電磁対物レンズは真空槽内部に配置され、
前記真空槽の内部空間のうち、前記光源の周囲の部分には、反射防止膜が形成された請求項1乃至請求項10のいずれか1項記載の複合顕微鏡装置。
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KR101693539B1 (ko) * | 2015-11-12 | 2017-01-06 | 한국표준과학연구원 | 고분해능 광-전자 융합현미경 |
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