JP2005121479A - Confocal microscopic spectroscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscopic spectroscope for simultaneously measuring scattered light, such as Raman scattered light and fluorescence, and the optical image (confocal optical microscopic image)of a sample, and for clarifying the position relationship. <P>SOLUTION: Reflection light and scattered light from a sample S are separated by a notch filter 27, the quantity of light in the reflection light is measured by a photodetector 4, and Raman scattered light or a fluorescent spectrum is simultaneously measured by a photomultiplier tube 3 or a CCD 46. A location to be measured in the sample S is moved successively for accumulating the measured data of the quantity of reflection light, Raman scattered light, and fluorescent spectrum, and a two-dimensional or three-dimensional distribution image based on the measured data is displayed. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a confocal microspectroscopic device that measures reflected light and scattered light generated when a sample is irradiated with excitation laser light by a confocal optical microscope.

  Conventionally, there has been proposed an apparatus for detecting Raman scattered light, fluorescence, and the like generated when a sample is irradiated with excitation laser light through a confocal optical microscope. As this confocal optical microscope, what is described in Patent Document 1 is a first collection of excitation laser light emitted from a laser light source 101 and converted into a parallel light beam through a beam splitter 102 as shown in FIG. The laser beam is incident on the optical lens 103, the excitation laser beam is condensed by the first condenser lens 103, and the pinhole 105 of the pinhole mask (spatial filter) 104 is placed on the focal point of the first condenser lens 103. Further, the excitation laser beam that diffuses through the pinhole 105 is guided to the second condenser lens (objective lens) 106, and the excitation laser is applied onto the sample S by the second condenser lens 106. Those configured to condense light have been proposed.

In this confocal optical microscope, reflected light and scattered light of the excitation laser light are generated from the sample S when the excitation laser light condensed on the sample S is irradiated. The reflected light and scattered light return to the pinhole 105 while converging via the second condenser lens 106, and return to the beam splitter 102 via the pinhole 105 and the first condenser lens 103. The beam returned to the beam splitter 102 is branched by the beam splitter 102 from the optical path returning to the laser light source 101 side, and is guided to a photomultiplier tube (PMT) 108 side which is a detection means. In the photomultiplier tube 108, among the light beams reflected or scattered from the sample S, light having a wavelength different from that of the excitation laser beam emitted from the laser light source 101, such as Raman scattered light or fluorescence generated in the sample S, is emitted. Detected.
JP 2000-321200 A

  By the way, in the apparatus for detecting Raman scattered light or fluorescence using a confocal optical microscope as described above, the scattered light such as Raman scattered light or fluorescence, and an optical image of the sample (confocal optical microscope image) The positional relationship was unclear.

  In other words, when specific data on Raman scattered light, fluorescence, etc. is obtained at a certain measurement location on the sample, where the measurement location is in the confocal optical microscope image (optical image). It was difficult to identify whether there was.

  If the chemical state of the measurement site to be analyzed based on Raman scattered light or fluorescence and the optical image (three-dimensional information) of the measurement site can be measured simultaneously, the state of the measurement site can be determined. Although it is possible to measure more accurately, in the conventional apparatus, switching of the optical system and changing the position of the sample between the state of measuring Raman scattered light and fluorescence and the state of measuring the optical image Etc., and these could not be measured at the same time.

  Therefore, the present invention has been proposed in view of the above circumstances, and can simultaneously measure scattered light such as Raman scattered light and fluorescence and an optical image of a sample (confocal optical microscope image). Therefore, an object of the present invention is to provide a confocal microspectroscopic device that can clarify the positional relationship between them.

  In order to solve the above-described problems, a confocal microspectroscopic device according to the present invention includes at least two or more excitation laser light sources that continuously or pulse oscillate, and transmit or reflect light of a predetermined wavelength and reflect light of other wavelengths. Or, a plurality of optical filters that pass through and a plurality of shutters that block the optical path, and a pump laser beam that selects one of the pump laser beams from each pump laser light source by switching these optical filters and shutters A selection means, an irradiation means for irradiating the sample with the excitation laser light selected by the excitation laser light selection means, a confocal optical microscope as a means for condensing reflected light or scattered light from the sample, and generated from the sample Spectroscopic means for spectroscopically measuring the scattered light and measuring the spectroscopic spectrum of the sample to be measured, and moving the position of the sample in three dimensions in advance. Spectral distribution image forming means for accumulating spectral intensity data within the set spectral window and displaying the spectral distribution image three-dimensionally based on the accumulated spectral intensity data. The spectroscopic means is configured to share excitation laser light. The reflected light and the scattered light of the excitation laser light generated from the measurement location are separated by a first optical filter that performs wavelength discrimination by being focused and irradiated on the measurement location on the sample by a focusing optical microscope. The intensity of the separated reflected light is detected by a photodetector, and the separated scattered light is light having a wavelength longer than that of the excitation laser light selected in synchronization with the selection of the excitation laser light by the excitation laser light selection means. Scattered light and fluorescence having different excitation wavelengths for the same measurement location on the sample through the second optical filter having the characteristic of reflecting or transmitting the light. The spectrum is dispersed by the spectroscopic device and measured simultaneously. The spectral distribution image forming means repeats the accumulation of measurement data measured in the spectroscopic means while sequentially moving the measurement points in the sample, and the accumulated measurement is performed. A confocal optical microscope image corresponding to reflected light measurement of excitation laser light and a two-dimensional or three-dimensional distribution image corresponding to spectrum measurement are displayed based on the data.

  Further, the present invention includes a beam expander having a function of shaping the beam shape by expanding or reducing the beam diameter of the incident excitation laser light to a preset beam diameter in the above-described confocal microspectroscopic device, By making the excitation laser light that has passed through this beam expander enter a confocal optical microscope, the beam diameter of the excitation laser light is made to coincide with the pupil diameter of the objective lens of the confocal optical microscope. is there.

  Furthermore, the present invention provides a spectroscopic device that calculates the light intensity of the excitation laser light by taking out a part of the excitation laser light through a semi-transmission mirror and measuring the light intensity in the confocal microspectroscopic device described above. By dividing the intensity of the Raman scattered light and the fluorescence spectrum measured after being wavelength-dispersed by the light intensity of the excitation laser light, the fluctuation of the intensity of the Raman scattered light and the fluorescence spectrum caused by the fluctuation of the light intensity of the excitation laser light It is characterized by correcting.

  According to the present invention, in the above-described confocal microspectroscopic device, the pinhole provided in the confocal optical microscope has a first slit whose opening width can be continuously changed, and an opening width which can be continuously changed. The second slit, which is arranged with the opening width direction being substantially perpendicular to the slit, is overlapped with the other slit.

  Further, the present invention is characterized in that, in the above-described confocal microspectroscopic device, the diameter of the pinhole is adjusted according to the required spatial resolution and the amount of light throughput.

  In the confocal microspectroscopic device according to the present invention, the spectroscopic means simultaneously measures the intensity of the reflected light, the Raman scattered light and the fluorescence spectrum having different excitation wavelengths at the same measurement location on the sample, and the spectral distribution. Since the image forming means displays the confocal optical microscope image corresponding to the reflected light measurement of the excitation laser beam and the two-dimensional or three-dimensional distribution image corresponding to the spectrum measurement, the confocal point at the same measurement location on the sample is displayed. The positional relationship between the optical microscope image, the Raman scattered light, and the fluorescence spectrum is clarified.

  That is, the present invention can simultaneously measure scattered light such as Raman scattered light and fluorescence, and an optical image (confocal optical microscope image) of the sample, and can clarify the positional relationship between them. A microspectroscopic device can be provided.

  Further, the present invention provides a beam expander having a function of shaping the beam shape by expanding or reducing the beam diameter of the excitation laser light to a preset beam diameter in the confocal microspectroscopic device described above. By making the beam diameter of the excitation laser light coincide with the pupil diameter of the objective lens of the confocal optical microscope by making it incident on the confocal optical microscope through the excitation laser, the emission energy of the excitation laser light source can be excited with high efficiency. Can be used as light.

  Furthermore, the present invention provides the above-described confocal microspectroscopic device by dividing the intensity of Raman scattered light and fluorescence spectrum measured by wavelength dispersion by a spectroscopic device by the light intensity of the excitation laser light. Because the fluctuation of the intensity of the Raman scattered light and the fluorescence spectrum caused by the fluctuation of the light intensity of the light source is corrected, even if the emission intensity of the excitation laser light in the excitation laser light source fluctuates, Raman scattered light and fluorescence spectrum can be measured.

  According to the present invention, in the above-described confocal microspectroscopic device, the pinhole provided in the confocal optical microscope has a first slit whose opening width can be continuously changed, and an opening width which can be continuously changed. In this case, the diameter of the pinhole is required in a confocal optical microscope, for example. It can be freely set according to the spatial resolution and the light throughput.

  Further, according to the present invention, in the confocal microspectroscopic device described above, the pinhole is adjusted in aperture according to the required spatial resolution and light throughput, so in the confocal optical microscope The required spatial resolution and light throughput are quickly realized.

  Embodiments of the present invention will be described below with reference to the drawings.

  As shown in FIG. 1, the confocal microspectroscopic device according to the present invention irradiates a sample S installed in the objective lens block unit 1 with a laser beam emitted from an excitation laser light source 2, and this irradiation. The components such as Raman scattering and fluorescence generated from the sample S are detected by a photomultiplier tube (PMT) 3 through a confocal optical microscope and a spectroscopic device, and the intensity of the reflected light from the sample S is detected. This is a device for detecting by the device 4.

  1A is a front view, and FIG. 1B is a side view.

  In the objective lens block unit 1, the sample S is supported by a three-dimensional stage (XYZ stage) (not shown) and can be moved in any three-dimensional direction.

  In this confocal microspectroscopic device, as the excitation laser light source 2, as shown in FIG. 2, at least two or more excitation laser light sources 2a, 2b, 2c,. Is provided. Each of the laser beams emitted from the respective excitation laser light sources 2a, 2b, 2c,... Is selected by the excitation laser beam selection means and guided to the sample S as the excitation laser beam. Become. As shown in FIG. 2, the excitation laser light selecting means includes a plurality of edge filters that are optical filters that transmit or reflect light of a predetermined wavelength and reflect or transmit light of other wavelengths, and a plurality of shutters that block an optical path. It is comprised. Each edge filter can be switched so that any one is positioned on the optical path, and each shutter can be switched so that any one is in an open state.

  That is, in this excitation laser light selection means, the laser light emitted from the first excitation laser light source 2a passes through the first beam combiner (synthesizer) 6 via the opened first shutter 5. Then, the first to third edge filters 10, 11, and 12 are passed through the first to third slow-phase plates 7, 8, and 9 through the slow-phase plate adapted to the first excitation laser light source 2 a. Are emitted through an edge filter adapted to the first excitation laser light source 2a.

  The laser light emitted from the second excitation laser light source 2b passes through the second shutter 13 that is opened, passes through the second beam combiner (synthesizer) 14, and the first beam combiner 6. Of the first to third edge filters 10, 11, 12, the first to third retardation plates 7, 8, 9 pass through the retardation plate adapted to the second excitation laser light source 2 b, and The light is emitted through an edge filter adapted to the second excitation laser light source 2b.

  Further, the laser light emitted from the third excitation laser light source 2c passes through the opened third shutter 15 and the second beam combiner (synthesizer) 14 and the first beam combiner 6, Among the first to third edge filters 10, 11, and 12, the first to third edge filters 10, 11, and 12 pass through the retardation plate that is suitable for the third excitation laser light source 2 c. The light is emitted through an edge filter adapted to the third excitation laser light source 2c.

  In this excitation laser beam selecting means, any one shutter corresponding to the selected excitation laser light source is opened, and the other shutters are closed. Further, in this excitation laser light selection means, one of the first to third retardation plates 7, 8, 9 and the first to third corresponding to the selected excitation laser light source. Any one of the edge filters 10, 11, and 12 is selected and arranged on the optical path.

  As shown in FIG. 1, the excitation laser light emitted from the excitation laser light selection means is reflected by the first mirror 16 and the second mirror 17 to change the optical path, and passes through the plasma line 18. The light is incident on a polarizing beam splitter 19 serving as a polarizer (polarizer). The second mirror 17 is a semi-transmissive mirror, and the second mirror 17 transmits a certain amount of light of the excitation laser light. The laser light that has passed through the second mirror 17 passes through a first ND (Neutral Density) filter 20 and enters a photomultiplier tube (PMT) 21 for calibrating the amount of excitation laser light.

  The excitation laser light incident on the polarization beam splitter 19 passes through the second ND filter 22 and is incident on the beam expander 23. Each of the first and second ND filters 20 and 22 includes a plurality of turret-type filters having different concentrations, and a corresponding filter is selected by driving a stepping motor in accordance with the selected excitation laser light source. It has come to be.

  As shown in FIG. 3, the beam expander 23 includes first to third convex lenses L1, L2, and L3. The distance X1 between the first and second convex lenses L1 and L2, the second and second convex lenses L1, L2, and the second convex lenses L1, L2, and L3. The function of shaping the beam shape by expanding or reducing the beam diameter (beam diameter) of the incident excitation laser light to a preset beam diameter by varying the distance X2 between the three convex lenses L2 and L3, respectively. have.

  In FIG. 3, the distance X1 between the first and second convex lenses L1, L2 is made smaller in the state shown in (B) than in the state shown in (A), and the second and third convex lenses L2, L3. By increasing the distance X2 between them, the beam diameter of the emitted light is increased.

  The beam diameter of the excitation laser light is adjusted to coincide with the pupil diameter of an objective lens of a confocal optical microscope described later. In this confocal microspectroscopic device, the emission energy of the excitation laser light source 2 can be used as the excitation laser light with high efficiency because the beam diameter of the excitation laser light matches the pupil diameter of the objective lens.

  The movement of each convex lens L1, L2, L3 in the beam expander 23 is performed by driving a stepping motor. The beam diameter of the excitation laser light that has passed through the beam expander 23 is adjusted to a value set in advance according to the excitation laser light source to be selected and the objective lens to be used.

  As shown in FIG. 1, the light emitted from the beam expander 23 is reflected by the third mirror 24 and the fourth mirror 25 to change the optical path, passes through the half-wave plate 26, The light is incident on a notch filter (or edge filter) 27 serving as an optical filter 1. The notch filter 27 is a filter having a characteristic of reflecting only a light beam in a specific narrow wavelength range, and here has a characteristic of reflecting the central wavelength band of the light beam emitted from the excitation laser light source 2. . The excitation laser light reflected by the notch filter 27 is incident on the objective lens block unit 1, reflected by the fifth mirror 28, and incident on the objective lens 29 constituting the confocal optical microscope. The confocal optical microscope serves as an irradiating unit that irradiates the sample S with the excitation laser beam selected by the excitation laser beam selecting unit, and a condensing unit that collects reflected light and scattered light from the sample S. It will be. That is, the excitation laser light incident on the objective lens 29 is focused and irradiated on the measurement site on the sample S.

  The excitation laser beam condensed on the sample S is reflected by the sample S including scattered light and returns to the notch filter 27. In the notch filter 27, the reflected light and scattered light of the excitation laser light generated from the measurement site on the sample S are separated into reflected light and transmitted light of the notch filter 27. That is, the notch filter 27 performs wavelength discrimination by reflecting the excitation laser light and transmitting other wavelength light.

  Here, the excitation laser light reflected by the notch filter 27 is reflected light from the sample S, returns to the third mirror 24 through the half-wave plate 26 and the fourth mirror 25. A part of the reflected light from the sample S is transmitted through the third mirror 24, reflected by the sixth mirror 30, and collected by the condenser lens 31. A pinhole 32 of a pinhole mask (spatial filter) is located on a condensing point by the condensing lens 31. The light beam that diffuses through the pinhole 32 passes through the third ND filter 33 and enters the photodetector 4. The photodetector 4 is a photomultiplier tube (PMT) and measures the amount of reflected light.

  On the other hand, the other wavelength light that has passed through the notch filter 27 is scattered light in the sample S, and enters the spectroscopic means for measuring the spectral spectrum of the measured portion of the sample by dispersing the scattered light. In this spectroscopic means, the scattered light passes through an iris 34 and a polarizing beam splitter 35 that serves as a polarizer (polarizer), and further passes through a band-pass filter 36 and iris 37 that serve as a second optical filter, and then enters a condenser lens 38. Incident. The band pass filter 36 has a characteristic of transmitting light having a longer wavelength than the excitation laser light. The band pass filter 36 is selected in synchronization with the selection of the excitation laser light source in the excitation laser light selection means.

  The scattered light incident on the condenser lens 38 is reflected by the seventh mirror 39 while being condensed. A pinhole 40 of a pinhole mask (spatial filter) is located on a condensing point by the condensing lens 38. The light beam that diffuses through the pinhole 40 is incident on the spectroscopic block 41.

  In the confocal microspectroscopic device, in the confocal optical microscope from the objective lens 29 to the pinholes 32 and 40, each pinhole 32 and 40 has a first variable opening width as shown in FIG. The first slit 50 and the second slit 51 that are arranged so that the opening width can be continuously changed and the opening width direction is substantially perpendicular to the first slit 50 are overlapped with each other. . That is, the opening area of the pinholes 32 and 40 is indicated by the product of the opening width of the first slit 50 and the opening width of the second slit 51.

  The opening widths of the slits 50 and 51 are variably adjusted by driving the stepping motor. The apertures of the pinholes 32 and 40 can be freely set according to, for example, the spatial resolution and light throughput required in the confocal optical microscope. That is, if the apertures of the pinholes 32 and 40 are reduced, the spatial resolution in the confocal optical microscope is improved, but the throughput amount is reduced, and if the apertures of the pinholes 32 and 40 are increased, the spatial resolution in the confocal optical microscope is reduced. However, the throughput amount increases.

  As described above, in this confocal microspectroscopic device, the spatial resolution and the light throughput necessary for the confocal optical microscope can be quickly set by adjusting the diameters of the pinholes 32 and 40.

  As shown in FIG. 1, the scattered light incident on the spectroscopic block 41 is reflected by the mirror 42, passes through the reflective diffraction grating 43 serving as a spectroscopic device, and further reflected by the mirror 44. After 45, it is detected by the photomultiplier tube 3. Alternatively, the scattered light incident by switching the position of the mirror is incident on a charge-coupled device (CCD) 46.

  When the optical system of the confocal optical microscope is simplified, as shown in FIG. 5, reflected light and scattered light from the sample S are condensed by the objective lens 29, and a pinhole mask is formed at the condensing point. The pinhole 40 is arranged, and the light beam that has passed through the pinhole 40 is detected by the photomultiplier tube 3.

  Of the scattered light from the sample S, Raman scattered light and fluorescence spectrum having different excitation wavelengths are wavelength-dispersed by the reflective diffraction grating 43 and incident on the photomultiplier tube 3 or the charge coupled device 46 by switching the mirror 45. Is done. The photomultiplier tube 3 is mainly used for detecting a fluorescence spectrum having a wide wavelength range. The charge coupled device 46 is mainly used for detection of Raman scattered light having a large number of fine structures.

  The confocal microspectroscopic device is provided with an electronic circuit unit that controls the operation of each part in the device and serves as a spectral distribution image forming means for processing the measured data.

  The electronic circuit unit controls the three-dimensional stage that supports the sample S, accumulates spectral intensity data within a preset spectral window while moving the position of the sample S three-dimensionally, and stores the accumulated spectral intensity. Based on the data, the spectrum distribution image is displayed three-dimensionally through a display device (not shown). In addition, the electronic circuit unit accumulates data on the amount of reflected light from the sample S while moving the position of the sample S three-dimensionally, and based on the accumulated measurement data, through a display device (not shown), A confocal optical microscope image corresponding to the reflected light measurement of the excitation laser light is displayed.

  That is, the electronic circuit unit repeats the accumulation of measurement data measured by the spectroscopic means a predetermined number of times while sequentially moving the measurement site in the sample S, and then, based on the accumulated measurement data, A confocal optical microscope image can be displayed.

  Therefore, in this confocal microspectroscopic device, as described above, the spectroscopic means simultaneously measures the intensity of the reflected light, the Raman scattered light and the fluorescence spectrum having different excitation wavelengths at the same measurement location on the sample S. Further, since the spectral distribution image forming means displays the confocal optical microscope image corresponding to the reflected light measurement of the excitation laser light and the two-dimensional or three-dimensional distribution image corresponding to the spectral measurement, the same object on the sample S is displayed. The positional relationship between the confocal optical microscope image at the measurement location and the Raman scattered light or the fluorescence spectrum is clarified.

  Further, this electronic circuit section takes out a part of the excitation laser light through a second mirror 17 which is a semi-transparent mirror by a photomultiplier tube (PMT) 21 for calibrating the light amount of the excitation laser light, and converts the light intensity. By measuring, the light intensity of the excitation laser beam is calculated. Thus, when the optical system for extracting a part of the excitation laser beam is shown in a simplified manner, as shown in FIG. 6, the second mirror 17 converts a part of the excitation laser beam before entering the confocal optical microscope. As a result, an optical system incident on the photomultiplier tube 21 is obtained.

  The electronic circuit unit calibrates the intensity of Raman scattered light or fluorescence spectrum measured by wavelength dispersion by a spectroscopic device by dividing the intensity by the light intensity of the excitation laser light, thereby adjusting the light intensity of the excitation laser light. Corrections are made for variations in the intensity of the Raman scattered light or the fluorescence spectrum caused by the variations.

  Therefore, in this confocal microspectroscopic device, even if the emission intensity of the excitation laser light in the excitation laser light source 2 fluctuates, the Raman scattered light and the fluorescence spectrum can be stably measured without being affected by the fluctuation. Can do.

  Note that the confocal microspectroscopic device according to the present invention can be used with the main body portion inverted as shown in FIG.

  7A is a side view, and FIG. 7B is a front view.

  In this case, the sample S is installed below the objective lens 29, and the objective lens 29 irradiates the sample S with excitation laser light from above.

It is the front view (A) and side view (B) which show the structure of the confocal microspectroscopic device which concerns on this invention. It is a side view which shows the excitation laser beam selection means which comprises the said confocal microspectroscopic device. It is a side view which shows the collimator which comprises the said confocal microspectroscopic device. It is a front view which shows the pinhole mask which comprises the said confocal microspectroscopic device. It is a side view which simplifies and shows the optical system of the confocal optical microscope in the said confocal microspectroscopic device. It is a side view which simplifies and shows the optical system which measures the optical intensity of excitation laser beam in the said confocal microspectroscopic device. It is the side view (A) and front view (B) which show the state which is using the said confocal microspectroscopic device in an inverted state. It is a side view which shows the structure of the conventional confocal optical microscope.

Explanation of symbols

  2 laser light source, 3 photomultiplier tube, 31 first condenser lens, 32 pinhole, 38 second condenser lens, 40 pinhole

Claims (5)

At least two or more excitation laser light sources that oscillate continuously or pulse; and
It has a plurality of optical filters that transmit or reflect light of a predetermined wavelength and reflect or transmit light of other wavelengths and a plurality of shutters that block the optical path. An excitation laser beam selecting means for selecting one excitation laser beam from among the excitation laser beams;
An irradiating means for irradiating the sample of the excitation laser light selected by the excitation laser light selecting means, and a confocal optical microscope as a condensing means for reflected light or scattered light from the sample;
A spectroscopic means for spectroscopically measuring the scattered light generated from the sample and measuring the spectroscopic spectrum of the measured portion of the sample;
Spectral distribution image that three-dimensionally moves the position of the sample, accumulates spectral intensity data within a preset spectral window, and displays the spectral distribution image three-dimensionally based on the accumulated spectral intensity data Forming means, and
The spectroscopic means is configured such that the excitation laser light is condensed and irradiated on the measurement site on the sample by the confocal optical microscope, and reflected and scattered light of the excitation laser beam generated from the measurement site. Are separated by a first optical filter that performs wavelength discrimination, the intensity of the separated reflected light is detected by a photodetector, and the separated scattered light is separated from the excitation laser light by the excitation laser light selection means. Raman scattering with different excitation wavelengths for the same measurement location on the sample through a second optical filter having a characteristic of reflecting or transmitting light having a wavelength longer than that of the excitation laser light selected in synchronization with the selection Simultaneously measure the wavelength of light and fluorescence spectrum by spectral dispersion using a spectroscope,
The spectral distribution image forming means repeats accumulation of measurement data measured by the spectroscopic means for a predetermined number of times while sequentially moving the measurement site in the sample, and then based on the accumulated measurement data, A confocal microspectroscopic device, which displays a confocal optical microscope image corresponding to reflected light measurement and a two-dimensional or three-dimensional distribution image corresponding to spectrum measurement. A beam expander having a function of expanding or reducing the beam diameter of the incident excitation laser beam to a preset beam diameter and shaping the beam shape,
The excitation laser light that has passed through the beam expander is incident on the confocal optical microscope so that the beam diameter of the excitation laser light matches the pupil diameter of the objective lens of the confocal optical microscope. Item 2. The confocal microspectroscopic device according to Item 1. By extracting a part of the excitation laser light through a semi-transmission mirror and measuring the light intensity, the light intensity of the excitation laser light is calculated,
By dividing the intensity of the Raman scattered light and the fluorescence spectrum measured by wavelength dispersion by the spectroscopic device by the light intensity of the excitation laser light, the Raman scattered light and the fluorescence generated by the fluctuation of the light intensity of the excitation laser light. 2. The confocal microspectroscopic device according to claim 1, wherein fluctuations in spectrum intensity are corrected.   The pinhole provided in the confocal optical microscope has a first slit whose opening width can be continuously changed, and an opening width which can be continuously changed, and the opening width direction is substantially perpendicular to the first slit. The confocal microspectroscopic device according to claim 1, wherein the confocal microspectroscopic device is configured by overlapping with the second slit arranged and arranged.   5. The confocal microspectroscopic device according to claim 4, wherein the diameter of the pinhole is adjusted in accordance with a required spatial resolution and light throughput.

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