JP2023105838A - Hybrid microscope device and microscope system - Google Patents

Abstract

To provide a device capable of performing optical microscope observation and scanning electron microscope observation of a same part of a same sample by the transmitted light in a single microscope device without using a separate microscope.SOLUTION: A hybrid microscope device 100 according to one embodiment of the present invention includes a light source 122 for optical microscope observation on a same side as the side where an observation target sample is irradiated with an electron beam, and an optical image detector 121 for acquiring a transmitted light image of the observation target sample is arranged on a side opposite to the electron beam irradiation side with respect to the observation target sample.SELECTED DRAWING: Figure 1

Description

The present invention relates to a hybrid microscope apparatus and microscope system, and more particularly to a multi-purpose microscope apparatus and microscope system having the functions of an optical microscope and a scanning electron microscope.

In recent years, Correlative Light and Electron Microscopy (CLEM) has come to be used for pathological tissue research and diagnosis.

For example, Patent Literature 1 discloses a scanning electron microscope capable of performing scanning electron microscopic observation using a slide glass used for observation with an optical microscope.
In addition, as a CLEM system that facilitates observation of the same location with an optical microscope and a scanning electron microscope, a system that supports CLEM analysis using a field-emission scanning electron microscope (FE-SEM) has been developed and is commercially available. (Hitachi High-Tech Co., Ltd., MirrorCLEM).

In addition, Patent Document 2 and Non-Patent Document 1 propose a hybrid microscope with a reflective optical system that enables fluorescent microscopic observation from above a specimen observed with a scanning electron microscope.

However, in general, optical microscopic observation of pathological tissue specimens on glass slides is often performed using transmitted light, and CLEM, which combines optical microscopic observation using transmitted light and scanning electron microscopic observation, requires separate microscopes. There was no choice but to use it, and the problem remained that the observation operation was complicated.

In Patent Document 2, in the section of the problem to be solved by the invention, it is stated that the purpose is to provide a microscope that can also perform fluoroscopic image observation. do not describe a mechanism for performing fluoroscopy.

An example of a combination of optical microscope observation using transmitted light and scanning electron microscope observation is shown in Patent Document 3, but this apparatus is a special-purpose microscope called an atmospheric pressure SEM.

JP 2012-109171 A JP 2014-211448 A JP 2018-26197 A

Takaaki Kanamaru et al., "Photon/Electron Hybrid Microscope Development -Observation by 'FL-SEM'-", Microscope Vol. 46, No. 1 (2011), p. 66-70

As described above, although there is a need for simple CLEM observation of specimens on slide glasses for pathological research, etc., the combination of the optical microscope provided so far and the scanning electron microscope, or the proposed In hybrid microscopes, practical problems have not yet been solved.

In response to this, the present inventors conceived of a new hybrid microscope mechanism that enables CLEM observation with a simple structure.

An object of the present invention is to provide an apparatus capable of performing optical microscope observation using transmitted light and scanning electron microscope observation of the same portion of the same sample in a single microscope apparatus without using separate microscopes, and the apparatus. To provide a system comprising

As a result of intensive studies by the present inventors, the above problems can be solved by a hybrid microscope device and a microscope system having the following aspects.

[1] A light source for optical microscope observation is provided on the same side as the electron beam irradiation of the sample to be observed, and an optical image detector for obtaining a transmitted light image of the sample to be observed irradiates the sample with the electron beam. A hybrid microscope apparatus of an optical microscope and a scanning electron microscope, characterized in that it is arranged on the side opposite to the irradiation side.
[2] The hybrid microscope apparatus of [1], wherein the optical image detector is a detector selected from CCD and CMOS, and the detector substantially functions as a sample stage on which the sample to be observed is placed.
[3] The light-receiving area of the optical image detector is larger than the area of the sample to be observed, and a condensing optical system separate from the optical components constituting the optical image detector is provided between the optical image detector and the sample to be observed. The hybrid microscope device of [1] or [2], which does not have.
[4] Coordinate information for the entire light-receiving element of each pixel constituting the optical image detector is configured to be available for alignment during magnified observation of a sample to be observed with a scanning electron microscope, [1] to [ 3].
[5] A hybrid microscope device according to any one of [1] to [4], and an analysis device, wherein the analysis device includes a control unit that controls the operation of the hybrid microscope device, and a scanning type obtained by the hybrid microscope device An image acquisition unit that acquires image information about an electron microscope image and an optical microscope image, a data processing unit that processes data from the control unit and the image acquisition unit, and an output unit that outputs the processing result of the data processing unit, A hybrid microscope system for performing scanning electron microscope observation and optical microscope observation of a sample placed in a sample chamber of a hybrid microscope device.
[6] The analysis device acquires coordinate information for the entire light receiving element of each pixel that constitutes the optical image detector of the hybrid microscope device, and uses the coordinate information to perform magnified observation of the sample to be observed by the scanning electron microscope. The hybrid microscope system of [5], configured to be able to control the operation of the hybrid microscope device for alignment.

According to the present invention, there is provided a hybrid microscope apparatus that allows easy CLEM observation of an observation target sample such as a pathological tissue specimen on a slide glass.
Further, according to the present invention, there is provided a hybrid microscope system including the above hybrid microscope device.

1 is a schematic diagram showing the configuration of a hybrid microscope apparatus according to an embodiment of the present invention; FIG. FIG. 4 is a schematic diagram showing the configuration of a hybrid microscope apparatus according to another embodiment of the present invention; FIG. 4 is a schematic diagram showing the configuration of a hybrid microscope apparatus according to another embodiment of the present invention; FIG. 4 is a schematic diagram showing the configuration of a hybrid microscope apparatus according to another embodiment of the present invention; 1 is a block diagram showing the configuration of a hybrid microscope system according to one embodiment of the present invention; FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

[Configuration and function of hybrid microscope device]
The hybrid microscope apparatus of the present invention has the function of an optical microscope and the function of a scanning electron microscope, and is capable of performing both optical microscope observation using transmitted light and scanning electron microscope observation with a single microscope apparatus. is. That is, the microscope apparatus of the present invention is a hybrid microscope apparatus of an optical microscope and a scanning electron microscope.

The hybrid microscope apparatus of the present invention includes a light source for optical microscope observation on the same side as the electron beam irradiation of the observation target sample, and an optical image detector that acquires a transmitted light image of the observation target sample. is arranged on the side opposite to the electron beam irradiation side.

<Embodiment 1>
FIG. 1 is a schematic diagram showing the configuration of a hybrid microscope apparatus according to one embodiment of the present invention.

As shown in FIG. 1, a hybrid microscope apparatus 100 (hereinafter also simply referred to as microscope apparatus 100) of this embodiment includes a scanning electron microscope section 110, an optical microscope section 120, a sample chamber 130, and a sample chamber bottom. A material 140 and evacuation means 150a and 150b are provided. Thereby, the microscope apparatus 100 of this embodiment has a scanning electron microscope function and an optical microscope function.

The scanning electron microscope section 110 includes a lens barrel 111 , an objective lens 112 and an electron beam detector 113 .

An electron gun (electron beam generator) is arranged inside the lens barrel 111 . The electron gun can generate an electron beam by any principle, and typically includes thermionic emission type, field emission type, and Schottky type. Of these, the use of thermionic emission type is preferable in terms of size reduction of the entire device. As for the configuration of the electron gun and the electron optical components arranged inside the lens barrel 111, the configuration similar to that of the conventional scanning electron microscope can be adopted, so detailed description thereof will be omitted in this specification. .

An objective lens 112 is arranged at the lower end of the lens barrel 111 . An electron beam emitted from an electron gun in a lens barrel 111 is irradiated onto a sample to be observed while its beam width is controlled by an objective lens 112 .

Although not shown in FIG. 1, the sample to be observed is introduced into the sample chamber 130 from the bottom side of the sample chamber 130 while being placed on top of an optical image detector, which will be described later. In other words, the microscope apparatus 100 shown in FIG. 1 is configured such that the sample to be observed is introduced into the sample chamber 130 from the atmospheric pressure condition outside the apparatus. However, the mode of introducing the observation target sample into the sample chamber 130 is not limited to this.

The electron beam detector 113 detects reflected electrons and secondary electrons generated by irradiating the sample to be observed with the electron beam, and provides a scanning electron microscope observation image of the sample to be observed. Here, the electron beam detector 113 may have a function of detecting fluorescent X-rays.

That is, in the microscope apparatus 100 of the present embodiment, any mode of scanning electron microscopic observation of a sample to be observed may be used, and the general modes of electron microscopic observation, such as backscattered electron mode, secondary electron mode, or fluorescence X-ray mode, may be used. A line detection mode may also be used. Of these, in the present invention, the detection mode by the backscattered electron mode or the secondary electron mode is preferable.

The optical microscope section 120 includes an optical image detector 121 and a light source 122 .

The optical image detector 121 is for acquiring a transmitted light image of a sample to be observed, and is arranged on the opposite side of the sample to be observed from the electron beam irradiation side.

As the optical image detector 121, CMOS and CCD are generally used, and both can be preferably used in the present invention. Here, in the microscope apparatus 100 of this embodiment, it is preferable that the optical image detector 121 substantially has the function of a sample stage on which the sample to be observed is placed. For example, in FIG. 1, the optical image detector 121 constitutes a part of the sample chamber bottom member 140, and the sample chamber bottom member 140 places the sample to be observed on top of the portion where the optical image detector 121 is located. configured as possible.

Moreover, in the microscope apparatus 100 of the present embodiment, the light receiving area of the optical image detector 121 is preferably larger than the area of the sample to be observed. For example, in a mode in which the specimen to be observed is a general histopathological specimen, the light receiving area (length x width size) of the optical image detector 121 is preferably 3.5 mm in length x 4.5 mm in width or more, more preferably It is 4.5 mm×5.5 mm or more, more preferably 5.5 mm×7 mm or more.

Furthermore, in the microscope apparatus 100 shown in FIG. 1, the sample to be observed can be placed directly on the optical image detector 121 . As described above, in the microscope apparatus 100 of the present embodiment, there is no condensing optical system separate from the optical components constituting the optical image detector 121 between the optical image detector 121 and the sample to be observed. preferable. As the configuration of such an optical image detector, for example, the one described in JP-A-2018-42283 can be adopted.

The light source 122 is for performing optical microscope observation of the observation target sample by the optical image detector 121, and is arranged on the same side as the electron beam irradiation of the observation target sample. Specifically, in the microscope apparatus 100 shown in FIG. 1, the light source 122 is arranged at the lower end of the objective lens 112 .

Any light source 122 may be used, for example, an LED (light emitting diode), a mercury lamp, a xenon lamp, or the like can be used, but an LED is mentioned as a preferable light source. In this case, the LED is preferably a white LED in which blue light and yellow light are mixed, or a three-color mixed white LED in which blue light, green light and red light are mixed, and more preferably a three-color mixed type. Moreover, it is preferable that the light source is configured to irradiate not only white light but also blue light, green light, and red light independently. Further, it is also preferable to use a configuration in which a UV-LED for irradiating ultraviolet light is provided as the light source 122 in addition to the LED.

FIG. 1 shows an example in which the light source 122 is arranged on both sides of the lower part of the objective lens 112, but it is well known that the above-described LED can be a light source of any shape via a light guide plate. As for the shape of the light source 122, it is preferable to have a doughnut-like shape surrounding the objective lens 112 from the viewpoint of reducing non-uniformity of light when obtaining an optical microscope observation image of the observation target sample. .

Evacuation means 150a and 150b are attached to the lens barrel 111 and the sample chamber 130, respectively. It can be adjusted to a predetermined degree of vacuum. The evacuation means 150a and 150b are composed of vacuum pumps.

In a preferred aspect of the microscope apparatus 100 of the present embodiment, the scanning electron microscope observation is not performed under atmospheric pressure as described in Patent Document 3, but the inside of the lens barrel 111 and the inside of the sample chamber 130 are predetermined. is performed under vacuum conditions adjusted to a degree of vacuum of The degree of vacuum is preferably 100 Pascal or less, more preferably 10 Pascal or less, and most preferably 10 ⁻² Pascal or less. Here, from the viewpoint of efficiently performing scanning electron microscope observation under vacuum conditions in a short time, the electron gun in the lens barrel 111 is arranged in a vacuum tube preliminarily adjusted to a predetermined degree of vacuum. may be adopted.

Further, in a preferred aspect of the microscope apparatus 100 of the present embodiment, the coordinate information of the entire light-receiving element of each pixel constituting the optical image detector 121 is used for alignment during enlarged observation of a sample to be observed using a scanning electron microscope. preferably configured to be available. As a result, for example, when a pathological tissue specimen on a slide glass is observed with an optical microscope and a desired observation site or observation area is found, the accuracy of the enlargement operation for observation with a scanning electron microscope is improved, and the time can be shortened. can be observed efficiently. In addition, by recording the coordinate information acquired for each pathological tissue sample, even when the sample is taken out of the apparatus and then subjected to observation again, the target observation site can be determined based on the recorded coordinate information. Alternatively, since it can be aligned with the observation area, the observation operation is further simplified.

<Embodiment 2>
FIG. 2 is a schematic diagram showing the configuration of a hybrid microscope device according to another embodiment of the present invention. In FIG. 2, the same reference numerals are assigned to the same components as those of the microscope apparatus 100 shown in FIG. 1, and detailed description thereof will be omitted below.

As shown in FIG. 2, in a hybrid microscope device 200 (hereinafter also simply referred to as a microscope device 200) of this embodiment, the electron beam detector 113 and the light source 122 are switched from the microscope device 100 shown in FIG. That is, in the microscope apparatus 200 shown in FIG. 2, the electron detector 113 is arranged at the lower end of the objective lens 112 .

Here, FIG. 2 shows an example in which the light source 122 is arranged on the lower side of the electron beam detector 113, but the above-described LED can be a light source of any shape via a light guide plate. is well known, the shape of the light source 122 should be a doughnut-like shape so as to surround the electron beam detector 113, to reduce the non-uniformity of the light when obtaining an optical microscope observation image of the sample to be observed. is preferable from the viewpoint of reducing

<Embodiment 3>
FIG. 3 is a schematic diagram showing the configuration of a hybrid microscope apparatus according to another embodiment of the invention. 3, the same components as those of the microscope apparatus 100 shown in FIG. 1 or the microscope apparatus 200 shown in FIG. 2 are denoted by the same reference numerals, and detailed description thereof will be omitted below.

As shown in FIG. 3, in a hybrid microscope device 300 (hereinafter also simply referred to as a microscope device 300) of this embodiment, an optical microscope section 120 includes an optical image detector 121, a light source 122, and a movable mirror 123. .

In the microscope device 300 of this embodiment, the light source 122 is arranged inside the lens barrel 111 . Inside the lens barrel 111, a movable mirror 123 is arranged in the direction in which the light emitted from the light source 122 is emitted. , and passes through the objective lens 112 to irradiate the sample to be observed.

The material, size, and the like of the movable mirror 123 are not particularly limited as long as the functions described above are exhibited. The movable mirror 123 can be retracted to a position that does not interfere with the electron beam emitted from the electron gun in the lens barrel 111 during scanning electron microscope observation by the microscope apparatus 300. As for the mechanism, a conventionally known configuration can be adopted.

<Embodiment 4>
FIG. 4 is a schematic diagram showing the configuration of a hybrid microscope device according to another embodiment of the present invention. In FIG. 4, the same components as those of the microscope apparatus 100 shown in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted below.

As shown in FIG. 4, in a hybrid microscope device 400 (hereinafter also simply referred to as a microscope device 400) of this embodiment, an optical microscope section 120 includes an optical image detector 121, a light source 122, and a light-transmissive sample stage. 124 and an optical lens 125 .

A light transmissive sample stage 124 and an optical lens 125 are positioned within the sample chamber 130 . In the microscope apparatus 400 of this embodiment, the observation target sample is placed on the upper part of the light-transmissive sample stage 124 . In other words, in the microscope apparatus 400 of the present embodiment, the light source 122 is arranged on the same side as the electron beam irradiation side with respect to the sample to be observed, and the light transmissive sample table 124, the optical lens 125, and the optical image detector are arranged. 121 is arranged on the side opposite to the electron beam irradiation side with respect to the sample to be observed. The optical lens 125 converges the light emitted from the light source 122 and transmitted through the sample to be observed and the light-transmissive sample table 124, and the optical image detector 121 arranged below receives the light.

As described above, in the hybrid microscope apparatus of the present invention, a condensing optical system separate from the optical components constituting the optical image detector 121 may be provided between the optical image detector 121 and the sample to be observed. .

The material, size, etc. of the light-transmitting sample table 124 and the optical lens 125 are not particularly limited as long as the functions described above are exhibited, and conventionally known structures can be adopted.

In addition, as a method of placing the observation target sample on the light-transmitting sample table 124, for example, a part of the member constituting the side wall of the sample chamber 130 can be opened so that the observation target can be viewed from the side of the sample chamber 130. A configuration can be employed in which the sample is introduced into the sample chamber 130 and placed on the upper portion of the light-transmissive sample stage 124 . Such a configuration is also employed in conventional scanning electron microscopes. However, in the microscope apparatus 400 of the present embodiment, the manner of introducing the observation target sample into the specimen chamber 130 and the manner of placing the observation target sample on the light transmissive sample stage 124 are not limited to this.

[Method of Observing Sample Using Hybrid Microscope]
Next, as a preferred embodiment of the sample observation method using the hybrid microscope apparatus of the present invention, a mode in which the sample to be observed is a pathological tissue specimen on a slide glass will be described.

Conventionally, in studies of pathological diagnosis and pathological diagnostic methods, the collected pathological tissue specimens are stained with a staining agent (hematoxin/eosin staining (HE staining)) or other immunostaining methods (specific antibodies are used to stain desired antigens). method) and observed with an optical microscope are often performed. At this time, the pathological tissue specimen is often fixed on a slide glass using a paraffin embedding agent. In recent years, such paraffin-fixed and stained histopathological specimens have been observed in combination with an optical microscope and a scanning electron microscope, and a methodology has been adopted in which a specific position is enlarged to observe the histological shape. However, when observing such pathological tissue specimens in the conventional scanning electron microscope observation technique, it was necessary to perform metal vapor deposition in advance in order to avoid electrostatic charge-up to the specimen. Once a pathological tissue specimen has been subjected to metal vapor deposition, it becomes impossible to observe the stained image again with an optical microscope.

However, for example, according to the technique described in International Publication No. 2013/035866 (NanoSuit (registered trademark) technique), the above-described metal deposition is performed to scan a pathological tissue specimen without impairing the optical transparency of the sample. observation became possible. If this technique is used, there is no problem in observing the pathological tissue sample to be observed once again with an optical microscope after subjecting it to scanning electron microscope observation. However, since the optical microscope and the scanning electron microscope are separate entities, CLEM observation with precisely matching the target observation position still requires complicated operations.

That is, the hybrid microscope apparatus of the present invention provides great convenience to the user in observing pathological tissue specimens on slide glasses in combination with NanoSuit technology.

For CLEM observation using the hybrid microscope apparatus of the present invention, it is preferable to use an observation auxiliary liquid (hereinafter referred to as NanoSuit solution) as described in WO2013/035866.

The NanoSuit solution improves the sharpness of observed images in observation by the scanning electron microscope function of the hybrid microscope device of the present invention. The NanoSuit solution contains, as an essential component, at least one interface selected from glycerin and glycerin substitutes; It contains an active compound and optionally at least one compound selected from monosaccharides, disaccharides, salts and buffers.

Glycerin is a trihydric alcohol (so-called polyhydric alcohol), has a hydroxyl group in its molecule, and is a low vapor pressure substance. Also, glycerin is viscous. Substances with these characteristics can be included in NanoSuit solutions as replacement ingredients for glycerin. Specifically, glycerin substitutes include, for example, polyethylene glycol, polyvinyl alcohol, triglycerides, polyresorcinol, polyphenols, tannic acid, urushiol, saponin, and the like. Glycerin and glycerin substitutes may be used alone or in combination of two or more.

As used herein, the term "polysorbates" means those produced by reacting sorbitan fatty acid esters (nonionic surfactants) with ethylene oxide. Currently commonly available polysorbates include polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65 (Tween 65), polysorbate 80 (Tween 80), polysorbate 85 (Tween 85). ), but are not limited to polysorbates that can be included in the NanoSuit solution. Substances classified as nonionic surfactants, like polysorbates, can be included in the NanoSuit solution as substitute components for polysorbates. Specifically, polysorbate substitutes include, for example, polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene mono fatty acid ester, sucrose fatty acid ester, polyglycerin fatty acid ester, alkyl polyglycoside, N-methyl Alkyl glucamides and the like can be mentioned. Polysorbates and polysorbate substitutes may be used singly or in combination of two or more.

Monosaccharides include, for example, glucose and fructose.
Disaccharides include, for example, sucrose, trehalose and the like.
Examples of salts include imidazolium salts, pyridinium salts, piperidinium salts, pyrrolidinium salts, quaternary ammonium salts and the like.
Examples of buffers include acetate buffer (acetic acid/sodium acetate buffer), phosphate buffer (phosphate/sodium phosphate buffer), citrate buffer (citric acid/sodium citrate buffer), citric acid phosphate buffer (citric acid/sodium phosphate buffer), borate buffer, tartrate buffer, Tris buffer and the like.
These monosaccharides, disaccharides, salts and buffers may be used singly or in combination of two or more.

An essential component consisting of at least one compound selected from glycerin, glycerin substitutes, polysorbates, and polysorbate substitutes preferably comprises 0.01% by weight to 10% by weight in the NanoSuit solution, and 0.1 More preferred is an embodiment containing from weight percent to 2 weight percent.

Here, a procedure for CLEM observation of a pathological tissue sample on a slide glass using the hybrid microscope apparatus of the present invention will be described. In addition, the concrete aspect of this invention is not restricted to the procedure described below.

(1) A sample for observation is prepared by preliminarily deparaffinizing a pathological tissue sample fixed with paraffin on a slide glass and then subjecting it to hydrophilization.
(2) A NanoSuit solution is applied to the sample to be observed in (1) to form a liquid film of the NanoSuit solution on the surface of the pathological tissue specimen.
(3) The sample to be observed of (2) is introduced into the sample chamber of the hybrid microscope apparatus of the present invention. (For example, in the microscope apparatus 100 shown in FIG. 1, the sample to be observed is placed above the portion of the sample chamber bottom member 140 where the optical image detector 121 is located.)
(4) Using the optical microscope function of the microscope device, the sample to be observed is observed with an optical microscope to obtain an optical microscope image.
(5) After the optical microscope observation in (4), a position (range) for high-magnification observation with a scanning electron microscope is determined. At this time, it is preferable that the microscope apparatus is configured to be able to select a position (range) for the high-magnification observation using coordinate information for the entire light receiving element of each pixel constituting the optical image detector.
(6) Operate the evacuation means (vacuum pump) of the microscope apparatus to adjust the inside of the lens barrel and the inside of the sample chamber to a predetermined degree of vacuum so that scanning electron microscope observation is possible.
(7) Switch the function of the microscope device to the scanning electron microscope function, observe the position (range) determined in (5) with a scanning electron microscope at a predetermined high magnification, and acquire a scanning electron microscope observation image.
(8) If the electron beam detector in the scanning electron microscope section has a function of detecting fluorescent X-rays, obtain a fluorescent X-ray image as necessary.

[Hybrid microscope system]
A hybrid microscope system of the present invention suitable for CLEM observation of pathological tissue samples on glass slides using the hybrid microscope apparatus of the present invention described above comprises a hybrid microscope apparatus and an analysis apparatus. Here, the analysis device acquires coordinate information for the entire light receiving element of each pixel constituting the optical image detector of the hybrid microscope device, and utilizes the coordinate information for magnified observation of the sample to be observed by the scanning electron microscope. It is preferable that the operation of the hybrid microscope apparatus be controllable for the alignment.

FIG. 5 is a block diagram showing the configuration of a hybrid microscope system according to one embodiment of the present invention.

As shown in FIG. 5, a hybrid microscope system 700 (hereinafter simply referred to as microscope system 700) according to the present embodiment includes hybrid microscope apparatus 500 (hereinafter simply referred to as microscope apparatus 500) and analysis apparatus 600. Prepare.

As the microscope device 500, the microscope devices 100, 200, 300, and 400 according to the first to fourth embodiments described with reference to FIGS. 1 to 4 can be used.

The analysis device 600 includes a control section 610 , an image acquisition section 620 , a data processing section 630 and an output section 640 .

A control unit 610 controls the operation of the microscope apparatus 500 . Specifically, the control unit 610 is configured to be able to control operations of at least the scanning electron microscope unit 110, the optical microscope unit 120, the sample chamber 130, and the sample chamber bottom member 140 of the microscope device 500. Information (data) regarding control of the microscope apparatus 500 by the control unit 610 can be transmitted to the data processing unit 630 .

The image acquisition unit 620 acquires image information regarding the scanning electron microscope image and the optical microscope image obtained by the microscope device 500 . In other words, the image acquisition section 620 acquires image information obtained by the scanning electron microscope section 110 and the optical microscope section 120 of the microscope device 500 . Image information (image data) acquired by the image acquiring section 620 can be transmitted to the data processing section 630 .

The data processing section 630 can process data from the control section 610 and the image acquisition section 620 and transmit the processing result to the output section 640 .

The output unit 640 outputs the processing result of the data processing unit 630 . As the output unit 640, for example, a liquid crystal display device, a printer, or the like is used. A recording device that records the processing result of the data processing unit 630 on a recording medium such as a USB memory may be used as the output unit 640 .

Scanning electron microscope observation and optical microscope observation of the observation target sample are performed by the microscope system 700 having such a configuration. Here, the image acquisition unit 620 stores a computer program (CLEM observation program for an observation target sample) for executing CLEM observation of a pathological tissue specimen on a slide glass as described above, and executes the program. A storage area is reserved for various analysis data stored and referenced therein. The analysis data includes coordinate information and the like for the entire light receiving element of each pixel that constitutes the optical image detector 121 of the microscope device 500 .

As described above, with the microscope system 700 of the present embodiment, CLEM observation of an observation target sample, that is, scanning electron microscope observation and optical microscope observation, can be easily performed with a simple device configuration.

Reference Signs List 100, 200, 300, 400, 500 hybrid microscope device 110 scanning electron microscope section 111 lens barrel 112 objective lens 113 electron beam detector 120 optical microscope section 121 optical image detector 122 light source 123 movable mirror 124 optically transparent sample stage 125 optics Lens 130 Sample Chamber 140 Sample Chamber Bottom Member 150a, 150b Evacuation Means 600 Analyzer 610 Control Unit 620 Image Acquisition Unit 630 Data Processing Unit 640 Output Unit 700 Hybrid Microscope System

Claims (6)

A light source for optical microscope observation is provided on the same side as the electron beam irradiation side of the observation target sample, and an optical image detector that acquires a transmitted light image of the observation target sample is on the electron beam irradiation side of the observation target sample. A hybrid microscope apparatus of an optical microscope and a scanning electron microscope, characterized in that they are arranged on opposite sides. 2. A hybrid microscope apparatus according to claim 1, characterized in that the optical image detector is a detector selected from CCD and CMOS, and that the detector has substantially the function of a sample stage on which the sample to be observed is placed. The light-receiving area of the optical image detector is larger than the area of the sample to be observed, and there is no focusing optical system separate from the optical components constituting the optical image detector between the optical image detector and the sample to be observed. A hybrid microscope device according to claim 1 or 2. 4. The optical image detector according to any one of claims 1 to 3, wherein the coordinate information of the entire light receiving element of each pixel constituting the optical image detector can be used for alignment during magnified observation of a sample to be observed by a scanning electron microscope. 10. The hybrid microscopy device of claim 1. A hybrid microscope device according to any one of claims 1 to 4 and an analysis device,
The analyzer is
a control unit that controls the operation of the hybrid microscope device;
an image acquisition unit that acquires image information about a scanning electron microscope image and an optical microscope image obtained by the hybrid microscope;
a data processing unit that processes data from the control unit and the image acquisition unit;
An output unit for outputting the processing result of the data processing unit,
Scanning electron microscope observation and optical microscope observation of the sample placed in the sample chamber of the hybrid microscope device,
Hybrid microscope system. The analysis device acquires coordinate information for the entire light receiving element of each pixel that constitutes the optical image detector of the hybrid microscope device, and uses the coordinate information to perform position alignment during magnified observation of the observation target sample by the scanning electron microscope. 6. The hybrid microscope system according to claim 5, configured to be able to control the operation of the hybrid microscope device for the purpose.