JP2010054368A - Optical microscope and observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical microscope capable of measuring spectra by a spectrograph and imaging a sample speedily with high resolution, and to provide an observation method. <P>SOLUTION: The optical microscope 100 comprises a laser light source 10, a Y scanning device 40 for performing scanning of light beams in a Y direction, an objective lens 23, an X scanning device 20 for performing scanning of light beams in an X direction, the spectrograph 31 for measuring spectra, and a line CCD camera 50. Then, a switching mirror 27 is provided so that it can be inserted and removed, where it switches a first optical path 51 toward the spectrograph 31 and an optical path 52 toward the line CCD camera 50. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an optical microscope and an observation method, and more particularly to an optical microscope and an observation method including a spectroscope for measuring a spectrum.

  Raman spectroscopic measurement is possible regardless of whether the sample is a gas, liquid, crystal, or amorphous solid, and the temperature can be high or low, and there is an advantage that a special measurement atmosphere such as a vacuum is not required for the measurement. Have. Furthermore, there is an advantage that the sample can be measured as it is without requiring any pre-treatment of the sample, and many measurements are made by taking advantage of these advantages. By utilizing Raman spectroscopy, it is possible to observe molecules without staining and to observe impurities in the semiconductor.

  In order to perform such Raman spectroscopic measurement, a Raman microscope using a spectroscope has been disclosed (Patent Documents 1 and 2). In this Raman microscope, a sample is irradiated with focused laser light. A Raman spectrum can be measured by spectroscopically analyzing Raman scattered light from the sample. Furthermore, in these Raman microscopes, the spatial distribution of the Raman scattered light intensity at a specific wavelength can be measured by moving the sample and measuring. In order to shorten the measurement time, a Raman microscope in which a sample is illuminated in a line by a cylindrical lens and detected by a CCD camera is also disclosed (Non-Patent Document 1). Since this Raman microscope illuminates in a line shape, it can irradiate a wide area at a time, and the measurement time can be shortened. However, when the light is formed in a line shape using a cylindrical lens, it is difficult to irradiate the light uniformly, and the peripheral area becomes dark.

Furthermore, an optical microscope that scans by deflecting laser light is disclosed (Patent Document 3). In this document, an X-direction scanning device and a Y-direction scanning device are provided. The scanning period of the Y scanning device is made shorter than the exposure time of the camera provided in the spectroscope (paragraph 0042). Thereby, the Raman spectrum in a linear area | region can be measured with 1 frame of a camera.
JP 2002-14043 A Japanese Patent Laid-Open No. 2003-344776 JP 2007-179002 A CHARLENE A. DRUMM and 1 other "Microscopic Raman Line-Imaging With Principle Component Analysis" APPLIED SPECTROCOPY, 1995, 49, No. 9, p. 1331-1337

  As described above, in Patent Document 3 and Non-Patent Document 1, a spectroscope is used to measure a Raman spectrum. By the way, in the Raman microscope as described above, there is a demand for acquiring information on a sample that is not subjected to spectroscopy together with information on a Raman spectrum. For example, there is a demand for observing a sample with reflected light reflected from the sample. Alternatively, there is a demand for observation using fluorescence generated in a sample. When performing such observation, it is preferable to image the sample at high speed and with high resolution.

  However, in the above-mentioned Raman microscope, the reflected light is imaged by the spectroscope camera through the spectroscope, and there are the following problems. Since the spectroscope camera is required to have low noise, the readout speed is slow. That is, it becomes difficult for the spectroscope camera to capture images at high speed. In addition, aberration is generated by passing through the spectroscope. In particular, the Zernitana spectrometer has a large aberration because it uses a concave mirror. When there is aberration, the image due to reflected light is blurred. In such a case, it becomes difficult to image with high resolution. As described above, an optical microscope using a spectroscope for measuring a spectrum has a problem that a sample cannot be imaged at high speed and with high resolution.

As described above, the conventional optical microscope has a problem that it is difficult to image a sample at high speed and with high resolution in a configuration using a spectroscope for measuring a spectrum.
The present invention has been made in view of the above-described problems, and an optical microscope capable of imaging a sample at high speed and with high resolution, and observation even with a configuration using a spectroscope for measuring a spectrum. It aims to provide a method.

  An optical microscope according to a first aspect of the present invention includes a laser light source, first scanning means for deflecting a light beam from the laser light source and scanning in a first direction, and scanning by the first scanning means. An objective lens that collects the incident light beam and makes it incident on the sample, and a second position that scans in the second direction by moving the relative position of the light beam scanned by the first scanning means and the sample. Scanning means, light emitted from the sample to the objective lens side among the light beams incident on the sample and disposed in the optical path from the first scanning means to the sample, and from the laser light source to the sample A light branching means for separating the incident light incident on the light beam, and an incident light arranged on the incident side where the outgoing light separated by the light branching means is collected and incident along a direction corresponding to the first direction. Having a slit, A spectroscope that spatially disperses the emitted light that has passed through the emission slit, a two-dimensional array photodetector that detects the emitted light dispersed by the spectrometer, and a position conjugate with the sample. And a photodetector having light receiving elements arranged along a direction corresponding to the first direction, and the outgoing light separated by the light branching unit is directed to the spectrometer or to the photodetector. And switching means for switching whether to go. Thereby, even if it is the structure which can measure a spectrum with a spectroscope, the sample can be imaged through a confocal optical system. Further, the sample is imaged by a photodetector different from the two-dimensional array photodetector for the spectroscope. Therefore, the sample can be imaged at high speed and with high resolution.

  An optical microscope according to a second aspect of the present invention is the optical microscope described above, wherein the light branching unit branches the light regardless of the wavelength, and a first beam splitter that branches the light according to the wavelength. A second beam splitter, wherein the first beam splitter and the second beam splitter are used exclusively, and when the emitted light is directed to the spectrometer, the first beam splitter In the case where the incident light and the emitted light are separated and the emitted light is directed to the photodetector, the reflected light of the sample is captured by the photodetector. The outgoing light is separated. As a result, the emitted light can be efficiently propagated to the photodetector or the two-dimensional array photodetector with a simple configuration.

  An optical microscope according to a third aspect of the present invention is the above-described optical microscope, wherein the fluorescence generated in the sample is detected by the photodetector in a state where the first beam splitter is inserted in an optical path. Is. Thereby, the fluorescence image by the fluorescence which generate | occur | produced with the sample can be imaged.

An optical microscope according to a fourth aspect of the present invention is the above-described optical microscope, wherein the first scanning unit scans the light beam at least once with the exposure time of the two-dimensional array photodetector. A scanner and a high-speed scanner that scans the light beam at a higher speed than the low-speed scanner, and the low-speed scanner is used to observe a predetermined position on the sample when the emitted light is directed to the photodetector. The incident light is positioned by a scanner. Thereby, it can position correctly and can observe the time change of the sample in a predetermined position.
An optical microscope according to a fifth aspect of the present invention is the above-described optical microscope, wherein the light branching unit includes an edge filter that transmits or reflects light according to a wavelength, and the light beam includes the edge filter. The incident angle with respect to the edge filter is 12 ° or less. Thereby, outgoing light and incident light can be reliably separated.

  In the observation method according to the sixth aspect of the present invention, the light beam from the laser light source is deflected and scanned in the first direction, and the light beam scanned in the first direction is condensed and incident on the sample. And moving the relative position between the light beam scanned in the first direction and the sample to scan in the second direction, and out of the light beam incident on the sample from the sample to the objective lens side. Before the outgoing light emitted from the laser beam is descanned in the first direction, the outgoing light and the incident light incident on the sample from the laser light source are separated, and the outgoing light separated from the incident light is The optical path is switched so as to propagate through one of the first optical path and the second optical path, and when the emitted light propagates through the first optical path, the incident is arranged along a direction corresponding to the first direction. The light exits from the entrance slit and passes through the entrance slit. Light is spatially dispersed according to wavelength, and the dispersed outgoing light is separated by a two-dimensional array photodetector in which light receiving elements are arranged along the direction of the incident slit and the direction in which the outgoing light is dispersed. When detecting and measuring the spectrum, and the emitted light propagates through the second optical path, a light receiving element disposed at a position conjugate with the sample and provided along a direction corresponding to the first direction It detects with the photodetector which has these. Thereby, even if it is the structure which can measure a spectrum with a spectroscope, the sample can be imaged through a confocal optical system. Further, the sample is imaged by a photodetector different from the two-dimensional array photodetector for the spectroscope. Therefore, the sample can be imaged at high speed and with high resolution.

  An observation method according to a seventh aspect of the present invention is the observation method described above, wherein when separating the incident light and the emitted light, a first beam splitter that branches light according to the wavelength, and a wavelength If the second beam splitter that splits the light is used exclusively and the emitted light propagates through the first optical path, the incident light and the emitted light are separated by the first beam splitter. When the outgoing light propagates through the second optical path, the incident light and the outgoing light are separated by the second beam splitter in order to capture a reflected image with the photodetector. It is what. As a result, the emitted light can be efficiently propagated to the photodetector or the two-dimensional array photodetector with a simple configuration.

  An observation method according to an eighth aspect of the present invention is the observation method described above, wherein when the emitted light propagates through a second optical path, the incident light and the emitted light are transmitted by the first beam splitter. And the fluorescence generated in the sample is detected by the photodetector. Thereby, the fluorescence image by the fluorescence which generate | occur | produced with the sample can be imaged.

An observation method according to a ninth aspect of the present invention is the observation method described above, wherein the low-speed scanner scans the light beam at least once with the exposure time of the two-dimensional array photodetector, and the low-speed scanner. A high-speed scanner for scanning the light beam at high speed, and positioning the incident light with the low-speed scanner for observing a predetermined position on the sample when the emitted light is directed to the photodetector. To do. Thereby, it can position correctly and can observe the time change of the sample in a predetermined position.
The observation method according to the tenth aspect of the present invention is such that the light beam and the emitted light are separated by inserting an edge filter that transmits or reflects light in accordance with the wavelength into the optical path. And the incident angle with respect to the said edge filter of the said light beam is 12 degrees or less. Thereby, outgoing light and incident light can be reliably separated.

  According to the present invention, it is possible to provide an optical microscope and an observation method capable of imaging a sample at high speed and with high resolution even with a configuration using a spectroscope for measuring a spectrum.

  Hereinafter, embodiments to which the present invention can be applied will be described. The following description is to describe the embodiment of the present invention, and the present invention is not limited to the following embodiment. For clarity of explanation, the following description is omitted and simplified as appropriate. Further, those skilled in the art will be able to easily change, add, and convert each element of the following embodiments within the scope of the present invention. In addition, what attached | subjected the same code | symbol in each figure has shown the same element, and abbreviate | omits description suitably.

Embodiment 1 of the Invention
An optical microscope according to an embodiment of the present invention will be described with reference to FIG. FIG. 1 is a diagram schematically showing a configuration of an optical system of an optical microscope 100 according to the present embodiment. The optical microscope 100 is configured to observe the sample 24 as a laser light source 10, a beam expander 11, a laser line filter 12, a Y scanning device 40, a relay lens 14, a relay lens 15, a relay lens 17, and a relay lens 18. , Edge filter 19, X scanning device 20, relay lens 21, tube lens 22, objective lens 23, half mirror 25, imaging lens 26, switching mirror 27, spectrometer 31, line CCD camera 50, and stage 60. is doing.

  The optical microscope 100 is a Raman microscope. A light beam from the laser light source 10 is incident on the sample 24, and Raman scattered light from the sample 24 is detected by the spectroscope 31. Since the Raman scattered light is split by the spectroscope 31, the Raman spectrum can be measured. Furthermore, since the optical microscope 100 can scan in the XY direction (horizontal direction) and the Z direction (vertical direction), a three-dimensional Raman spectrum image can be measured. Further, the optical microscope 100 is provided with a line CCD camera 50 in order to capture a reflected image via a confocal optical system. That is, the reflected light reflected from the sample 24 is detected through the line confocal optical system. It is possible to switch between an optical system for measuring the spectrum of Raman scattered light and an optical system for capturing a reflected image. The optical system for measuring the spectrum of Raman scattered light and the optical system for capturing a reflected image share a part of the optical path, and are switched by the switching mirror 27.

  The overall configuration of the optical microscope 100 will be described. First, an illumination optical system for measuring a Raman spectrum will be described. In the illumination optical system, the laser light source 10 emits laser light serving as excitation light. The laser light source 10 emits light having a predetermined laser wavelength. The laser light source 10 emits red or green monochromatic light, for example. For example, Millennia manufactured by Spectra Physics Co., Ltd. can be used as the laser light source 10. The laser light source 10 is an Nd / YVO4 laser having a laser wavelength of 532 nm, a line width of 0.24 nm, and a maximum output of 10 W. The laser light source 10 emits laser light having this laser wavelength.

  The laser light is magnified by the beam expander 11. That is, the beam expander 11 expands the beam diameter so that the laser beam spot has a predetermined size. Thereafter, the laser light enters the laser line filter 12. The laser line filter 12 blocks light other than the laser wavelength. Thereby, the stray light used as noise can be reduced. Then, the laser light passes through the laser line filter 12 and enters the high-speed scanner 13.

  The high speed scanner 13 and the low speed scanner 16 constitute a Y scanning device 40. That is, the Y scanning device 40 includes the high speed scanner 13 and the low speed scanner 16. The Y scanning device 40 scans by deflecting the laser light in the Y direction. The Y scanning device 40 deflects the light beam by changing the emission angle of the incident light beam. Thereby, the incident position of the light beam on the sample 24 changes along the Y direction. For example, the high-speed scanner 13 is a resonance type galvanometer mirror, and scans the laser beam at about 8 kHz. Specifically, the direction of the laser beam is changed by changing the angle of the reflection surface of the galvanometer mirror. The low-speed scanner 16 is a servo galvanometer mirror and operates at a lower speed than the high-speed scanner 13. Thereby, the laser beam is scanned. The high speed scanner 13 and the low speed scanner 16 scan the laser beam in the Y direction. The operation of the Y scanning device 40 will be described later.

  Relay lenses 14 and 15 are provided between the high-speed scanner 13 and the low-speed scanner 16. The relay lenses 14 and 15 refract laser light and relay images. A diaphragm may be provided between the relay lens 14 and the relay lens 15. The laser light reflected by the low-speed scanner 16 enters the relay lenses 17 and 18. The relay lenses 17 and 18 refract the laser light and relay the image. The laser light from the relay lens 18 enters the edge filter 19.

  The edge filter 19 reflects or transmits light according to the wavelength. That is, the edge filter 19 has a transmittance and a reflectance according to the wavelength. Specifically, the edge filter 19 reflects light having a laser wavelength and transmits light having a wavelength longer than the laser wavelength. Thereby, Raman scattered light can be measured efficiently. That is, most of the laser light serving as excitation light is reflected by the edge filter 19 and travels in the direction of the sample 24. On the other hand, most of the Raman scattered light having a wavelength longer than that of the laser light passes through the edge filter 19 and travels toward the spectroscope 31. Here, an edge filter 19 manufactured by Semrock is used.

  The laser light reflected by the edge filter 19 enters the X scanning device 20. The X scanning device 20 is a servo galvanometer mirror, for example, and deflects the light beam by changing the angle of the reflecting surface. That is, since the inclination angle of the reflection surface of the X scanning device 20 with respect to the optical axis changes, the emission angle of the light beam can be changed. As a result, the incident position of the light beam on the sample 24 changes along the X direction. A light beam can be scanned in the X direction on the sample 24. The deflection angle in the X scanning device 20 is controlled by an electrical signal. Further, since the X direction and the Y direction are orthogonal to each other, the two-dimensional region can be scanned on the sample 24 by scanning in the XY direction by the X scanning device 20 and the Y scanning device 40.

  The laser light reflected by the X scanning device 20 is refracted by the relay lens 21 and the tube lens 22 and enters the objective lens 23. The objective lens 23 collects the light beam and makes it incident on the sample 24. That is, the objective lens 23 collects the light beam on the sample 24 and illuminates the sample 24. Thereby, the spot-like region of the sample 24 is illuminated. As the objective lens 23, for example, Nikon Apochromate NA 1.2 x60 can be used.

  The objective lens 23 collects the laser beam and makes it incident on the sample 24. At this time, the laser beam is scanned in the XY directions by the X scanning device 20 and the Y scanning device 40. That is, the incident position of the laser beam on the sample 24 changes according to the operations of the X scanning device 20 and the Y scanning device 40. Thereby, a two-dimensional area can be scanned on the sample 24.

  Incident light that has entered the sample 24 from the objective lens 23 is reflected by the sample 24. A part of the incident light incident on the sample 24 is Raman scattered. Out of the incident light incident on the sample 24, light emitted to the objective lens 23 side is defined as emitted light. That is, light traveling from the sample 24 toward the objective lens 23 is referred to as outgoing light. The emitted light includes Rayleigh scattered light having the same wavelength as the laser wavelength and Raman scattered light having a wavelength different from the laser wavelength. Further, when the sample 24 includes a fluorescent material, the emitted light includes fluorescence.

  The Raman scattered outgoing light has a wavelength different from that of the incident light. That is, the outgoing light is scattered by being shifted from the frequency of the incident light by the Raman shift. The spectrum of the emitted light becomes a Raman spectrum. Therefore, by measuring the spectrum of the emitted light, the chemical structure and physical state of the substance contained in the sample 24 can be specified. That is, since the Raman spectrum includes information on the frequency of the substance constituting the sample 24, the substance in the sample 24 can be identified by detecting the emission light by spectroscopically detecting it. Then, by scanning the focal position of the incident light in the XYZ directions and measuring the spectrum of the emitted light from the entire surface or a part of the sample 24, the three-dimensional measurement of the Raman spectrum can be performed. By paying attention to a specific wavelength in the measured Raman spectrum, it is possible to measure the three-dimensional spatial distribution of the specific substance. Specifically, when the sample 24 is a living cell, the spatial distribution of nucleic acids and lipids or the spatial distribution of sucrose and polystyrene spheres can be measured.

  The sample 24 is placed on the stage 60. The stage 60 is, for example, an XYZ stage. This stage 60 is a movable stage and can illuminate an arbitrary position of the sample 24. Further, the distance between the objective lens 23 and the sample 24 can be changed by driving the stage 60 in the Z direction. Therefore, the focal position of the objective lens 23 can be changed along the optical axis direction. Since the optical microscope 100 constitutes a laser confocal microscope as will be described later, scanning in the Z direction is possible by changing the focal position. That is, a tomographic image of the sample 24 can be taken by moving the stage in the Z direction. Raman scattered light from an arbitrary height of the sample 24 can be detected, and a three-dimensional Raman spectrum image can be measured.

  Next, an optical system for detecting light emitted from the sample 24 will be described. Light emitted from the sample 24 placed on the stage 60 propagates on the same optical path as the incident light. That is, the light is refracted by the objective lens 23, refracted by the tube lens 22 and the relay lens 21, and enters the X scanning device 20. The X scanning device 20 reflects incident outgoing light in the direction of the edge filter 19. At this time, the emitted light is descanned by the X scanning device 20. That is, by being reflected by the X scanning device 20, the emitted light propagates in the direction opposite to the traveling direction of the incident light incident on the X scanning device 20 from the laser light source 10. Further, the Rayleigh scattered light from the sample 24 also propagates in the same optical path as the Raman scattered light.

  The outgoing light reflected by the X scanning device 20 enters the edge filter 19. The edge filter 19 branches the light emitted from the sample 24 and the incident light incident on the sample 24 from the laser light source 10 based on the wavelength. That is, the edge filter 19 is provided such that its reflection surface is inclined with respect to the optical axis of the incident light. When the light emitted from the sample 24 passes through the edge filter 19, the optical axis of the light emitted from the sample 24 is different from the optical axis of the incident light incident on the sample 24 from the laser light source 10. Therefore, the light emitted from the sample 24 can be separated from the incident light incident on the sample 24 from the laser light source 10. Thus, the edge filter 19 constitutes a light branching unit that branches incident light and outgoing light.

  The edge filter 19 used as the light branching unit transmits or reflects light according to the wavelength. It is preferable to reduce the incident angle of the light beam with respect to the edge filter 19. Here, the incident angle of the light beam is an angle between the principal ray of the light beam and the normal of the reflection surface of the edge filter 19. The incident angle of the light beam from the relay lens 18 with respect to the reflection surface of the edge filter 19 is preferably set to 12 ° or less, for example, and more preferably set to 10 °. In this way, the light beam is incident on the edge filter 19 from a direction close to the vertical direction. In addition, the incident angle is preferably set to 8 ° or more so that the optical path of the relay lens 18 does not overlap, but may be set to 8 ° or less depending on the design. Thereby, the characteristics of the edge filter 19 can be improved, and the Rayleigh scattered light and the Raman scattered light can be reliably separated. That is, the reflectance of the edge filter 19 with respect to Rayleigh scattered light and the transmittance of the edge filter 19 with respect to Raman scattered light can be improved. Therefore, the Rayleigh scattered light and the Raman scattered light can be reliably separated based on the wavelength difference between the Rayleigh scattered light and the Raman scattered light. Of course, in the optical microscope according to the present invention, the incident angle of the light beam with respect to the edge filter 19 is not limited to the above value.

  Furthermore, the edge filter 19 has a characteristic that reflects light having a laser wavelength and transmits Raman scattered light. Accordingly, the Rayleigh scattered light from the sample 24 is reflected by the edge filter 19, and the Raman scattered light passes through the edge filter 19. That is, by using the edge filter 19, it is possible to remove the Rayleigh scattered light based on the difference between the wavelengths of the Rayleigh scattered light and the Raman scattered light. Further, most of the laser light from the laser light source 10 is reflected by the edge filter 19 and travels toward the sample 24. Thereby, the loss of a laser beam can be reduced and only Raman scattered light can be detected efficiently. The reflection characteristic of the edge filter 19 may be determined according to the spectrum range to be measured. Here, the edge filter 19 is disposed between the Y scanning device 40 and the sample 24. Therefore, the edge filter 19 separates the outgoing light before being descanned by the Y scanning device 40 and the light beam from the laser light source 10.

  The outgoing light that has passed through the edge filter 19 enters the imaging lens 26. The imaging lens 26 forms an image of the sample 24 on the entrance slit 30 or the line CCD camera 50. Further, the outgoing light refracted by the imaging lens 26 is reflected by the switching mirror 27 and enters the incident slit 30 provided on the incident side of the spectroscope 31. At this time, the imaging lens 26 condenses outgoing light on the entrance slit 30. That is, the imaging lens 26 forms an enlarged image of the area illuminated by the sample 24 on the entrance slit 30. The entrance slit 30 is provided with a line-shaped opening. The opening is provided along a direction corresponding to the Y direction. That is, the opening of the entrance slit 30 is provided along a direction corresponding to the scanning direction (Y direction) of the Y scanning device 40 on the sample 24.

  The imaging lens 26 refracts the emitted light and forms an image on the entrance slit 30. Here, since the incident light is imaged in a spot shape on the surface of the sample 24, the emitted light is collected in a spot shape on the incident slit 30. The direction of the opening of the entrance slit 30 and the scanning direction of the Y scanning device 20 are matched. The emitted light is incident on the edge filter 19 without being descanned by the Y scanning device 20. For this reason, when scanning with the Y scanning device 20, the spot position of the light beam moves on the entrance slit 30 in the direction of the line-shaped opening of the entrance slit 30. It arrange | positions so that the light scanned in the Y direction on the sample 24 may form an image in the opening part of the entrance slit 30. FIG. In other words, the entrance slit 30 and the sample 24 are arranged so as to have a conjugate relationship with each other.

  Therefore, the Raman microscope is configured as a confocal optical system. For example, the stop (not shown) and the surface of the sample 24 are arranged so as to be conjugated with each other, and the surface of the sample 24 and the entrance slit 30 are arranged so as to be conjugated with each other. Incident light is collected in a spot shape on the XY plane on which the diaphragm is provided and on the surface of the sample 24. Then, the emitted light scattered and emitted from the sample 24 is collected in a spot shape on the entrance slit 30. The incident slit 30 has an opening along the Y direction, and only the outgoing light incident on the opening is transmitted to the two-dimensional detector of the spectroscope 31. By using such an imaging optical system for the illumination optical system from the laser light source 10 to the sample 24 and the observation optical system from the sample 24 to the array photodetector 36, a confocal Raman microscope can be obtained. Thereby, measurement with high resolution in the Z direction can be performed. Then, by moving the stage 60 in the Z direction, Raman scattered light from an arbitrary height of the sample 24 can be detected separately from Raman scattered light from other heights.

  The outgoing light that has passed through the entrance slit 30 enters the main body of the spectroscope 31. The spectroscope 31 will be described with reference to FIG. FIG. 2 is a diagram showing the configuration of the spectroscope 31. Here, a Zellnitana type spectrometer 31 is shown. The spectroscope 31 is provided with an entrance slit 30, a mirror 32, a concave mirror 33, a grating 34, a concave mirror 35, and an array photodetector 36.

  The incident slit 30 is disposed on the incident side of the spectroscope 31. Moreover, the entrance slit is arrange | positioned along the Y direction. The outgoing light passing through the entrance slit 30 is reflected by the mirror 32 and the concave mirror 33 and enters the grating 34. The grating 34 spatially disperses the light incident from the incident slit 30 according to the wavelength. Here, a reflection type diffraction grating is used as the grating 34. The grating 34 disperses the emitted light in a direction perpendicular to the direction of the entrance slit 30. The light dispersed by the grating 34 is reflected by the concave mirror 35 and enters the array photodetector 36.

  Of course, a spectroscope 31 having a configuration other than the above may be used. The outgoing light is dispersed by the spectroscope 31 in a direction perpendicular to the direction of the entrance slit 30. That is, the spectroscope 31 wavelength-disperses the emitted light in a direction perpendicular to the line-shaped opening of the entrance slit 30. The outgoing light separated by the spectroscope 31 enters the array photodetector 36. The array photodetector 36 is an area sensor in which light receiving elements are arranged in a matrix. Specifically, the array photodetector 36 is a two-dimensional array photodetector such as a CCD camera in which pixels are arranged in an array.

  For the array photodetector 36, for example, a cooled CCD can be used. Specifically, an electronically cooled CCD (−25 ° C.) of 1024 × 256 pixels manufactured by Princeton Instruments can be used as the array photodetector 36. It is also possible to attach an image intensifier to the array photodetector 36. The pixels of the array photodetector 36 are arranged along the direction corresponding to the entrance slit 30. Therefore, one arrangement direction of the pixels of the array photodetector 36 coincides with the direction of the entrance slit 30, and the other arrangement direction coincides with the dispersion direction of the spectrometer 31. The direction corresponding to the direction of the entrance slit 30 of the array photodetector 36 is the Y direction, and the direction perpendicular to the entrance slit 30, that is, the direction in which the emitted light is dispersed by the spectroscope 31 is the X direction.

  The array photodetector 36 outputs a detection signal corresponding to the light intensity of the emitted light received by each pixel to the processing device 71. The processing device 71 is an information processing device such as a personal computer (PC), for example, and stores a detection signal from the array photodetector 36 in a memory or the like. Then, the detection result is subjected to a predetermined process and displayed on the monitor. Further, the processing device 71 controls scanning of the Y scanning device 40 and the X scanning device 20 and driving of the stage 60. Here, the X direction of the array photodetector 36 corresponds to the wavelength (frequency) of the emitted light. That is, in the pixel array arranged in the X direction, one pixel detects outgoing light having a long wavelength (low frequency), and the other pixel detects outgoing light having a short wavelength (high frequency). Thus, the distribution of light intensity in the X direction of the array photodetector 36 shows the distribution of Raman spectrum.

  The relationship between the scanning by the Y scanning device 40 and the light receiving pixels of the array photodetector 36 will be described in detail with reference to FIG. FIG. 3A is a diagram schematically illustrating the spot shape of the light beam on the incident surface of the incident slit 30. FIG. 3B is a diagram schematically showing a light receiving surface in the array photodetector 36. As shown in FIG. 3A, the entrance slit 30 is provided with an opening 30a along the Y direction. The opening 30a has a length corresponding to the light receiving surface of the array photodetector 36 in the Y direction.

  Since the emitted light is imaged on the entrance slit 30 by the imaging lens 26, a light beam spot 38 is formed in the opening 30a. Here, when the Y scanning device 40 is driven to scan the light beam, the light beam spot 38 moves along the opening 30 a of the entrance slit 30 on the entrance surface of the entrance slit 30. That is, the incident position of the light beam on the incident surface of the incident slit 30 sequentially moves from the spot 38a, the spot 38b,.

  FIG. 3B shows a light receiving pixel 37 provided on the light receiving surface of the array photodetector 36. Each light receiving pixel 37 is formed with a light receiving element such as a photodiode. Further, when the array photodetector 36 is a CCD camera, a charge coupled device (CCD) is formed in each pixel. The light receiving pixels 37 are arranged in a matrix, that is, in the vertical direction and the horizontal direction. For example, 1024 pixels are provided in the horizontal direction and 256 pixels are provided in the vertical direction. As shown in FIG. 3B, the light receiving pixel 37 in the uppermost column among the pixels arranged in a matrix is defined as a light receiving pixel 37a. Further, the light receiving pixels 37 next to the light receiving pixels 37a, that is, the second row from the top are the light receiving pixels 37b, and the light receiving pixels 37 in the bottom row are the light receiving pixels 37n.

  Here, the vertical direction (Y direction) in FIG. 3B is a direction corresponding to the opening 30 a of the entrance slit 30. Therefore, when the light beam is incident on the spot 38a, the emitted light is incident on the uppermost light receiving pixel 37a. At this time, since the emitted light is split by the spectroscope 31, the horizontal direction of the uppermost light receiving pixel 37a corresponds to the wavelength (λ) of the emitted light. That is, the spectroscope 31 disperses outgoing light that has passed through the entrance slit 30 in a direction perpendicular to the opening 30 a of the entrance slit 30. Therefore, the long-wavelength outgoing light is incident on one end of the uppermost light receiving pixel 37a, and the short-wavelength outgoing light is incident on the other end. That is, outgoing light with different wavelengths is incident on each light receiving pixel 37a in the uppermost row. Thus, the spectral information of the Raman scattered light can be developed in a direction orthogonal to the Y direction of the array photodetector 36 in which pixels are arranged in a two-dimensional array.

  The Y scanning device 40 scans the incident light in the Y direction so that the light that has passed through the opening 30a of the incident slit 30 enters the light receiving pixels 37b in the second column from the top of the array photodetector 36. At this time, the incident position of the light beam in the incident slit 30 is 35b. In addition, the illumination position on the sample is scanned in the Y direction. Furthermore, since the outgoing light transmitted through the entrance slit 30 is split by the spectroscope 31, the horizontal direction of the light receiving pixels 37b in the second column corresponds to the wavelength (λ) of the outgoing light. Thus, the vertical direction of the light-receiving pixels arranged in an array corresponds to the scanning direction of the Y scanning device 40, and the horizontal direction corresponds to the wavelength of the Raman scattered light.

  Here, while the array photodetector 36 captures one frame, the light beam is scanned in the Y direction at least once. That is, the scanning period of the Y scanning device 40 is made shorter than the exposure time, and scanning is performed once or more in the Y direction within the exposure time of one frame of the array photodetector 36. As a result, the Raman spectrum of the linear region corresponding to the scanning range can be measured with one frame of the array photodetector 36. That is, the entire scanning area of the Y scanning device 40 is scanned within the exposure time. Thereby, the incident position of the light beam moves from the spot 38a to the spot 38n on the entrance slit 30 within the exposure time. Therefore, on the sample 24, the spectrum of Raman scattered light can be measured for the entire region corresponding to the opening 30a. That is, it is possible to image a linear region having a length corresponding to the opening 30 a of the entrance slit 30 in one frame. Raman spectra from a plurality of points on the sample 24 can be measured by one exposure. Accordingly, it is possible to reduce the number of charges transferred in the CCD of the array photodetector 36 and the number of data transfers from the CCD, thereby shortening the measurement time. Therefore, the Raman spectrum of a plurality of points can be measured with one frame of data transfer. It is not necessary to transfer data for each point, and the measurement time can be shortened. In this case, since there are the light receiving pixels 37 of the array photodetector 36 in rows a to n, the Raman spectrum at n points of the sample 24 can be measured by one exposure. Therefore, the measurement time can be shortened. Therefore, for example, even when a Raman spectrum is measured for a three-dimensional wide region, it is possible to prevent the measurement time from being long and improve the practicality.

  In this way, the spectral information of the Raman scattered light is developed in a direction orthogonal to the Y direction of the array photodetector 36 in which the light receiving pixels are arranged in a two-dimensional array. And the spectral information of the linear area | region in the sample 24 is acquired at once. Therefore, the Raman spectrum can be measured at high speed. Moreover, since it illuminates with spot light, it can illuminate uniformly and can perform a measurement accurately. That is, since the sample 24 is illuminated with the spot light, speckle noise can be prevented. Furthermore, since the spot light is scanned, the fluctuation of the illumination light luminance according to the position on the sample 24 can be reduced. Therefore, accurate measurement can be performed in a short time.

  In this way, it is possible to measure the Raman spectrum of the linear region. Then, when the imaging of one frame is finished, the X scanning device 20 shifts the illumination position by one illumination area in the X direction. Then, similarly, one frame is imaged, and the Raman spectrum of the linear region is measured. By repeating this, the Raman spectrum of the two-dimensional region on the sample 24 can be measured. At this time, since a Raman spectrum can be measured for each illumination region of the objective lens, a two-dimensional Raman spectrum image can be measured. That is, since scanning is performed in the X direction by the X scanning device 20, Raman spectroscopic measurement at each point of the sample can be performed. That is, a Raman spectrum in a two-dimensional region on the sample 24 can be measured. Furthermore, by moving the stage 60 in the XY directions, a Raman spectrum in a wider area can be measured. Further, the three-dimensional measurement can be performed by driving the stage 60 in the Z direction and moving the focal position along the optical axis. That is, when the spectrum measurement of the two-dimensional region is completed, the focal position is shifted in the Z direction, and the Raman spectrum of the two-dimensional region is similarly measured. Thereby, the three-dimensional measurement of the Raman spectrum becomes possible. Thus, by scanning in the Y direction, the light becomes a line shape within the exposure time of the array photodetector 36. In other words, considering the exposure time of the array photodetector 36, the sample 24 is illuminated in a line. Then, Raman scattered light is detected via a line confocal (slit confocal) optical system.

  Note that scanning during the exposure time is preferably performed an integer number of times. That is, it is preferable that the scanning cycle is an integral multiple of the exposure time. Here, one scanning is performed from one end to the other end of the scanning range. For example, the scanning in the Y direction is reciprocated n times (n is a natural number) within the exposure time, and the position where the illumination light is incident on the sample 24 is the same at the start and end of the exposure. Alternatively, the exposure of the array light detector 36 and the scan of the Y scanning device 40 are synchronized, and scanning is started from one end of the scanning range simultaneously with the start of exposure, and the exposure ends when the other end or one end of the scanning range is reached. To do. Thereby, the light quantity of the illumination light irradiated to a linear area | region becomes uniform within exposure time. Therefore, the illumination amount in the illumination area corresponding to each of the vertical pixel columns of the array photodetector 36 becomes uniform, and the Raman spectrum can be measured more accurately. Specifically, scanning is performed 100 times or more in one exposure time. Furthermore, by increasing the number of scans in one exposure time, the influence of fluctuations in the light intensity of the light source can be reduced and illumination can be performed more uniformly.

  Next, an optical system for capturing a reflected image by reflected light will be described with reference to FIG. When capturing a reflection image, the edge filter 19 is removed from the optical path. Then, the half mirror 25 is inserted at the position where the edge filter 19 has been inserted. That is, the edge filter 19 and the half mirror 25 are detachably provided, and only one of them is inserted into the optical path. The edge filter 19 and the half mirror 25 are used exclusively. The half mirror 25 is used when detecting reflected light having the same wavelength as the laser wavelength, and the edge filter 19 is used when detecting Raman scattered light. Further, when detecting reflected light having the same wavelength as the laser wavelength, the switching mirror 27 is removed from the optical path. The configuration other than these is the same as the case of detecting Raman scattered light, and thus the description thereof is omitted.

  Laser light from the relay lens 18 enters the half mirror 25. The half mirror 25 reflects a part of the incident light and transmits a part thereof. Here, half of the laser light incident on the half mirror 25 is reflected in the direction of the X scanning device 20. As in the case of detecting Raman scattered light, the laser light is incident on the sample 24. Similarly, the emitted light from the sample 24 is descanned by the X scanning device 20 and enters the half mirror 25. The half mirror 25 transmits half of the emitted light in the direction of the imaging lens 26. The half mirror 25 has constant transmittance and reflectance regardless of the wavelength. For this reason, part of the Raman scattered light also passes through the half mirror 25. However, since the Raman scattered light is sufficiently weaker than the reflected light (Rayleigh scattered light), there is no problem in capturing the reflected image.

  The outgoing light that has passed through the half mirror 25 is refracted by the imaging lens 26. It goes in the direction of the line CCD camera 50. At this time, the switching mirror 27 is separated from the optical path. For this reason, the emitted light is not reflected in the direction of the spectroscope 31 but goes in the direction of the line CCD camera 50. The imaging lens 26 images the emitted light on the light receiving surface of the line CCD camera 50. The line CCD camera 50 has light receiving pixels arranged along a direction corresponding to the Y direction.

  Further, the Y scanning device 40 scans the light beam one or more times in the Y direction while imaging one frame. That is, the scanning cycle of the Y scanning device 40 is made shorter than the exposure time, and scanning is performed once or more in the Y direction within the exposure time of one frame of the line CCD camera 50. Thereby, the Raman spectrum of the line-shaped region corresponding to the scanning range can be measured with one frame of the line CCD camera 50. That is, the entire scanning area of the Y scanning device 40 is scanned within the exposure time. Therefore, the reflected light is detected through a line confocal (slit confocal) optical system. When the imaging of one frame is completed, the illumination position is shifted by one illumination area in the X direction by the X scanning device 20. Similarly, one frame is imaged, and the reflected light of the line-shaped region is detected. By repeating this, a reflected image of a two-dimensional region on the sample 24 can be taken. A detection signal from the line CCD camera 50 is output to the processing device 71. Then, processing is performed by the processing device 71 to form a reflected image.

  The exposure time of the line CCD camera 50 is shorter than that of the array photodetector 36. Therefore, a reflected image can be taken at high speed. That is, the imaging time can be shortened. Further, the outgoing light enters the line CCD camera 50 without going through the spectroscope 31. Thus, the image is not blurred and high-resolution imaging is possible. Therefore, even with the optical microscope 100 capable of measuring a Raman spectrum, it is possible to capture a reflected image at high speed and with high resolution. Further, by scanning at high speed in the Y direction, the light becomes a line shape within the exposure time of the line CCD camera 50. That is, considering the exposure time of the line CCD camera 50, the sample 24 is illuminated in a line.

  In the present embodiment, a switching mirror 27 is provided as switching means for switching the optical path of the emitted light. The switching mirror 27 is detachably provided in the optical path of the emitted light. As the switching mirror 27 is inserted into the optical path, the emitted light is reflected and travels toward the spectroscope 31. On the other hand, when the switching mirror 27 is removed from the optical path, the emitted light goes straight through the insertion position of the switching mirror 27 and travels toward the line CCD camera 50. Thus, by inserting / removing the switching mirror 27, it is possible to switch between Raman spectrum measurement and reflection image capturing. Of course, a reflection image may be taken when the switching mirror 27 is inserted. In this case, the switching mirror 27 is removed from the optical path during spectrum measurement. By operating the switching mirror 27, it is possible to select between measurement by the spectroscope 31 and measurement by the line CCD camera 50. The switching mirror 27 switches the optical path so that the outgoing light separated from the incident light propagates through the optical path 51 toward the spectroscope 31 or the optical path 52 toward the line CCD camera 50. Note that the switching means for switching the optical path is not limited to the switching mirror 27. For example, a prism that refracts light may be used.

  Next, the Y scanning device 40 will be described. In the present embodiment, two high-speed scanners 13 and low-speed scanners 16 are provided as Y scanning devices 40 that scan in the Y direction. Then, one of the scanners is driven according to the application. For example, when the spectroscope 31 performs spectrum measurement, the high-speed scanner 13 is used. The high-speed scanner 13 is a resonant galvanometer mirror, for example, and operates at a frequency of 8 kHz, for example.

  The low-speed scanner 16 is a servo galvanometer mirror and operates at a frequency lower than that of the high-speed scanner 13. The high speed scanner 13 scans the laser beam at a higher speed than the low speed scanner 16. If any one of the scanners is sufficient, the Y scanning device 40 may be configured by only one scanner.

  For example, in the Raman mode for detecting Raman scattered light, the high speed scanner 13 is fixed and the low speed scanner 16 is operated. That is, since the exposure time of the array photodetector 36 is longer than the exposure time of the line CCD camera 50, a low-speed scan by the low-speed scanner 16 is sufficient. At this time, the array photodetector 36 and the low-speed scanner 16 are synchronized. In the Raman mode, the X scanning device 20 is also operated. At this time, the half mirror 25 is removed from the optical path, and the edge filter 19 is inserted in the optical path. A switching mirror 27 is also inserted in the optical path. Thereby, the spectroscope 31 can perform spectroscopic measurement. In the Raman mode, the high speed scanner 13 may be operated and the low speed scanner 16 may be fixed.

  On the other hand, the exposure time of the line CCD camera 50 is shorter than the exposure time of the array photodetector 36. Therefore, in the reflection mode in which the reflected light having the same wavelength as the laser light is detected, the high speed scanner 13 is operated and the low speed scanner 16 is fixed. Thereby, the laser beam is scanned once or more in the Y direction in the exposure time of the line CCD camera 50. In the case of the reflection mode, the X scanning device 20 is also operated. At this time, the edge filter 19 is removed from the optical path, and the half mirror 25 is inserted in the optical path. Further, the switching mirror 27 is removed from the optical path. Thereby, the reflected light can be detected by the line CCD camera 50. The line CCD camera 50 is preferably scanned by the high-speed scanner 13 because the exposure time is shorter than that of the array photodetector 36.

  Further, when observing a specific position of the sample 24, the low-speed scanner 16 irradiates the specific position with laser light. Then, the laser beam scanning is stopped, and the irradiation position is fixed at that position. At this time, the operation of the high-speed scanner 13 is stopped. Thereby, the change of the image at a specific position can be observed. Thus, when positioning accuracy is required, the irradiation position of the laser beam is moved using only the low-speed scanner 16. That is, in order to illuminate a specific position, the low-speed scanner 16 performs positioning. Thereby, the irradiation position of the laser beam is fixed at a specific position. On the other hand, the high-speed scanner 13 is used when detecting reflected light. Of course, if a single scanner is sufficient, scanning may be performed in the Y direction with only one scanner. Thus, the low speed scanner 16 and the high speed scanner 13 are provided as scanning means for scanning in the Y direction. The two scanners are used properly according to the application.

  Furthermore, in this configuration, fluorescence can be detected. In the case of the fluorescence mode for detecting fluorescence, the high speed scanner 13 is operated and the low speed scanner 16 is fixed. In the case of the fluorescence mode, the X scanning device 20 is also operated. At this time, the half mirror 25 is removed from the optical path, and the edge filter 19 is inserted in the optical path. Thereby, excitation light can be branched from fluorescence. That is, fluorescence having a wavelength different from the laser wavelength passes through the edge filter 19, and the laser light is reflected by the edge filter 19. Further, the switching mirror 27 is removed from the optical path. Thereby, a fluorescent image can be acquired by the line CCD camera 50. In this way, switching between the fluorescence mode, the reflection mode, and the Raman mode can be easily performed. Furthermore, by using the edge filter 19 and the half mirror 25 exclusively, it is not necessary to provide a separate band filter or the like. Therefore, the apparatus configuration can be simplified.

  In the present embodiment, incident light is scanned at high speed, and a Raman spectrum at an arbitrary point on the XY plane of the sample 24 is measured. Therefore, the spectrum measurement time can be shortened. That is, the measurement of the Raman spectrum in a wide region of the sample 24 can be performed in a short time. Therefore, even when the observation area of the sample 24 is widened, a long measurement time is not required. Thereby, even when the wavelength resolution of the Raman spectrum is improved or when a spectrum in a wider range is measured, the measurement time does not take a long time, and practical observation can be performed. For example, observation of a two-dimensional image focusing on Raman scattered light having an arbitrary wavelength (frequency) and measurement of a Raman spectrum at a specific point of the sample 24 can be performed with high resolution.

  Further, in the present embodiment, a slit confocal optical system that collects Raman scattered light is configured. Thereby, since the resolution in the Z direction can be improved, three-dimensional measurement of the Raman spectrum becomes possible. In other words, the focal position on the sample 24 can be scanned in the Z direction by changing the distance between the objective lens 23 and the sample 24. As a result, scanning in the XYZ directions becomes possible, and the Raman spectrum can be measured for a three-dimensional spatial distribution. In other words, the Raman spectrum can be measured at an arbitrary point of the three-dimensional sample 24. Even when such a three-dimensional spatial distribution of the Raman spectrum is measured, the measurement time can be shortened by measuring the linear region at a time. Therefore, measurement can be performed in a practical time. Thus, in the above configuration, four-dimensional measurement of XYZ-λ can be performed. Various spectrum analysis can be performed by performing the four-dimensional measurement. Then, by displaying the analysis result on the display of the processing apparatus, more advanced analysis is possible.

  Furthermore, the optical system for detecting reflected light and fluorescence is a slit confocal optical system. Therefore, by moving the stage 60 in the Z direction, a reflection image and a fluorescence image at an arbitrary height can be acquired. Thereby, a tomographic image can be acquired at high speed and with high resolution. Therefore, three-dimensional observation can be easily performed.

  In the above description, the array photodetector 36 and the line CCD camera 50 have been described as CCD cameras, but these are not limited to CCD cameras. That is, a two-dimensional photodetector having light receiving pixels arranged in a matrix can be used for spectroscopic measurement. In addition, a one-dimensional photodetector in which light receiving pixels are arranged in a row can be used for reflected light detection. Further, the detection signal is integrated for a certain exposure time, and an integration type photodetector is output.

  Further, the beam splitter that branches the Raman scattered light and the laser light is not limited to the edge filter 19. For example, a dichroic mirror that branches light according to the wavelength may be used as the beam splitter. Further, the X scanning device 20 is not limited to the one that deflects the beam, and may be one that drives the stage 60. Note that a slit confocal optical system may be configured using a cylindrical lens or the like instead of the Y scanning device 40. That is, a light converting means for making light into a line shape is used instead of the Y scanning device 40. If a line-shaped light spot is formed on the sample 24, a slit confocal optical system can be configured. Furthermore, it is preferable to exclusively use the half mirror 25 and the edge filter 19 as the light branching means.

Embodiment 2 of the Invention
An optical microscope 100 according to the present embodiment will be described with reference to FIG. FIG. 4 is a diagram illustrating an optical system of the optical microscope according to the present embodiment. In the present embodiment, the relay lenses 14 and 15 are not provided between the high-speed scanner 13 and the low-speed scanner 16. Since the configuration other than this is the same as that of the first embodiment, the description of the overlapping portions is omitted. With such a configuration, the same effect as in the first embodiment can be obtained.

Embodiment 3 of the Invention
An optical microscope 100 according to the present embodiment will be described with reference to FIG. FIG. 5 is a diagram showing an optical system of the optical microscope according to the present embodiment. In the present embodiment, the low speed scanner 16 is provided between the edge filter 19 and the X scanning device 20. That is, incident light from the half mirror 25 or the edge filter 19 enters the low-speed scanner 16. Since the configuration other than this is the same as that of the fourth embodiment, the description thereof is omitted. Even if it is such a structure, the effect similar to Embodiment 1, 2 can be acquired.

It is a figure which shows the structure of the optical microscope concerning Embodiment 1 of this invention. It is a perspective view which shows typically the structure of the spectrometer used for an optical microscope. It is a figure which shows typically the spot shape of the light beam in the entrance plane of an entrance slit, and the light-receiving surface of an array photodetector. FIG. 4 is a diagram illustrating a configuration of an optical microscope according to a second embodiment. FIG. 6 is a diagram illustrating a configuration of an optical microscope according to a third embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Laser light source 11 Beam expander 12 Laser line filter 13 High speed scanner 14 Relay lens 15 Relay lens 16 Low speed scanner 17 Relay lens 18 Relay lens 19 Edge filter 20 X scanning device 21 Relay lens 22 Tube lens 23 Objective lens 24 Sample 25 Half mirror 26 imaging lens 27 switching mirror 30 entrance slit 31 spectroscope 32 mirror 33 concave mirror 34 grating 35 concave mirror 36 two-dimensional CCD camera 37 light receiving pixel 40 Y scanning device 50 line CCD camera 60 stage 71 processing device

Claims (10)

A laser light source;
First scanning means for deflecting a light beam from the laser light source and scanning in a first direction;
An objective lens for condensing the light beam scanned by the first scanning means and entering the sample;
Second scanning means for scanning in a second direction by moving a relative position between the light beam scanned by the first scanning means and the sample;
Outgoing light that is arranged in the optical path from the first scanning means to the sample and is emitted from the sample to the objective lens side among the light beams incident on the sample, and incident that is incident on the sample from the laser light source A light branching means for separating light;
The outgoing light having an incident slit arranged along a direction corresponding to the first direction on the incident side where the outgoing light separated by the light branching unit is collected and incident, and has passed through the incident slit A spectrometer that spatially disperses the light according to the wavelength;
A two-dimensional array photodetector for detecting the emitted light dispersed by the spectrometer;
A photodetector having a light receiving element arranged at a position conjugate with the sample and arranged along a direction corresponding to the first direction;
An optical microscope comprising: switching means for switching whether the emitted light separated by the light branching means is directed to the spectroscope or to the photodetector. The light branching means includes a first beam splitter that branches light according to a wavelength, and a second beam splitter that branches light regardless of the wavelength,
The first and second beam splitters are used exclusively;
When the outgoing light goes to the spectroscope, the incident light and the outgoing light are separated by the first beam splitter,
When the emitted light is directed to the photodetector, the second beam splitter separates the incident light and the emitted light in order to capture a reflected image of the sample with the photodetector. The optical microscope according to claim 1.   The optical microscope according to claim 2, wherein fluorescence generated in the sample is detected by the photodetector in a state where the first beam splitter is inserted in an optical path. A low-speed scanner, wherein the first scanning means scans the light beam at least once with an exposure time of the two-dimensional array photodetector;
A high-speed scanner that scans the light beam at a higher speed than the low-speed scanner, and
The optical microscope according to claim 1, wherein the incident light is positioned by the low-speed scanner in order to observe a predetermined position on the sample when the emitted light is directed to the photodetector. The light branching means has an edge filter that transmits or reflects light according to wavelength,
The optical microscope according to claim 1, wherein an incident angle of the light beam with respect to the edge filter is 12 ° or less. Deflecting the light beam from the laser light source and scanning in the first direction;
The light beam scanned in the first direction is collected and incident on the sample;
Moving the relative position of the light beam scanned in the first direction and the sample to scan in the second direction;
Out of the light beam incident on the sample, the emitted light emitted from the sample to the objective lens side enters the sample from the emitted light and the laser light source before being descanned in the first direction. Separating incident light,
Switching the optical path so that the outgoing light separated from the incident light propagates in one of the first optical path and the second optical path;
When the emitted light propagates through the first optical path, it is incident on an entrance slit arranged along a direction corresponding to the first direction,
The outgoing light that has passed through the entrance slit is spatially dispersed according to the wavelength,
The dispersed outgoing light is detected by a two-dimensional array photodetector in which light receiving elements are arranged along the direction of the incident slit and the direction in which the outgoing light is dispersed, and a spectrum is measured.
When the emitted light propagates through the second optical path, it is detected by a photodetector having a light receiving element disposed at a position conjugate with the sample and provided along a direction corresponding to the first direction. Observation method. When separating the incident light and the emitted light, the first beam splitter that branches the light according to the wavelength and the second beam splitter that branches the light regardless of the wavelength are exclusively used,
When the emitted light propagates through the first optical path, the incident light and the emitted light are separated by the first beam splitter,
The incident light and the emitted light are separated by the second beam splitter in order to capture a reflected image by the photodetector when the emitted light propagates through a second optical path. Item 7. The observation method according to Item 6.   When the outgoing light propagates through a second optical path, the incident light and the outgoing light are separated by the first beam splitter, and the fluorescence generated in the sample is detected by the photodetector. 8. The observation method according to 7. A low-speed scanner that scans the light beam at least once with an exposure time of the two-dimensional array photodetector, and a high-speed scanner that scans the light beam at a higher speed than the low-speed scanner,
The observation method according to claim 6, 7, or 8, wherein the incident light is positioned by the low-speed scanner in order to observe a predetermined position on the sample when the emitted light is directed to the photodetector. By inserting an edge filter that transmits or reflects light according to wavelength into the optical path, the light beam and the emitted light are separated,
The observation method according to claim 6, wherein an incident angle of the light beam with respect to the edge filter is 12 ° or less.

Images