WO2023013325A1 - 顕微ラマン分光装置 - Google Patents

顕微ラマン分光装置 Download PDF

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Publication number
WO2023013325A1
WO2023013325A1 PCT/JP2022/026204 JP2022026204W WO2023013325A1 WO 2023013325 A1 WO2023013325 A1 WO 2023013325A1 JP 2022026204 W JP2022026204 W JP 2022026204W WO 2023013325 A1 WO2023013325 A1 WO 2023013325A1
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Prior art keywords
light
raman
excitation
diffraction grating
scattered light
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PCT/JP2022/026204
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English (en)
French (fr)
Japanese (ja)
Inventor
智生 篠山
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Shimadzu Corp
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Shimadzu Corp
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Application filed by Shimadzu Corp filed Critical Shimadzu Corp
Priority to CN202280053789.XA priority Critical patent/CN117859048A/zh
Priority to JP2023539715A priority patent/JP7582487B2/ja
Priority to EP22852751.1A priority patent/EP4382892A4/en
Priority to US18/294,858 priority patent/US12480880B2/en
Publication of WO2023013325A1 publication Critical patent/WO2023013325A1/ja
Anticipated expiration legal-status Critical
Priority to JP2024190296A priority patent/JP2025016619A/ja
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0208—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/021—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using plane or convex mirrors, parallel phase plates, or particular reflectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0229—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using masks, aperture plates, spatial light modulators or spatial filters, e.g. reflective filters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0294—Multi-channel spectroscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/06—Scanning arrangements arrangements for order-selection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J3/18—Generating the spectrum; Monochromators using diffraction elements, e.g. grating
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2803—Investigating the spectrum using photoelectric array detector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4412—Scattering spectrometry
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/10—Arrangements of light sources specially adapted for spectrometry or colorimetry
    • G01J2003/102—Plural sources

Definitions

  • the present invention relates to a microscopic Raman spectroscopic device that can obtain high spatial resolution on the ⁇ m order by condensing excitation light using a microscope objective lens.
  • a Raman spectrometer is a device that analyzes the structure of a sample at the molecular level by detecting the Raman spectrum obtained by spectroscopy of this Raman scattered light.
  • the sample since the sample has fluorescence properties of various sizes for ultraviolet light and visible light, the sample emits fluorescence according to the irradiation light.
  • Raman spectroscopy when the autofluorescence of a sample is strong, the wavelengths of Raman scattered light and fluorescence overlap, and very weak Raman peaks in the Raman spectrum are buried in the fluorescence spectrum, making sample analysis difficult. can not be performed with high precision.
  • Patent Document 3 fluorescence observation is performed as a preliminary measurement for a sample, and the sample emits fluorescence at what wavelength with respect to which excitation wavelength of laser light.
  • a microscopic Raman spectroscopic apparatus has been proposed that can analyze a sample with high accuracy by selecting an excitation laser beam that does not overlap with .
  • this microscopic Raman spectroscopic device is equipped with a plurality of excitation laser light sources and an automatic laser switching device for switching between these excitation laser light sources, and the spectroscope has a plurality of apertures to select the optimum one.
  • a spectroscope is provided with a plurality of (the same number as the number of excitation laser light sources) apertures (incidence apertures), diffraction gratings, and CCD detectors, and these are arranged at different wavelengths.
  • the structure of the spectrometer is complicated, resulting in an increase in size and cost due to the use of an aperture switching means, a diffraction grating switching means, and a detector switching means for switching according to the excitation laser beam.
  • each switching means for physically switching the aperture, diffraction grating, and detector is provided with a movable part, but problems such as the position reproducibility of these movable parts adversely affect the analysis results of the sample. There is also the possibility of affecting
  • the present invention has been made in view of the above-mentioned problems, and its object is to realize miniaturization and cost reduction by simplifying the structure of the spectroscope, and to always analyze samples with high accuracy.
  • An object of the present invention is to provide a microscopic Raman spectrometer.
  • the spectroscope includes a plurality of excitation light sources that emit excitation light with different wavelengths, and excitation light emitted from a selected excitation light source.
  • an optical system that guides Raman scattered light incident from the diffraction grating to the diffraction grating; and an imaging lens that forms an image of a plurality of Raman scattered light beams separated by the diffraction grating on the detector, wherein the optical system is provided in the same number as the entrance apertures, and the image forming lens receives a plurality of light beams separated by the diffraction grating and forms an image on the detector.
  • the spectrometer corresponds to a plurality of excitation light sources emitting excitation light beams having different wavelengths, the same number of entrance apertures as the excitation light sources, and Raman light incident from one of these entrance apertures. are equipped with a plurality of optical systems (the same number as the number of excitation light sources) to guide the light beams to the diffraction grating, so there is no need for switching means for switching between the entrance aperture (aperture) and the diffraction grating. Since the lens can form an image on the detector, the structure of the spectroscope is simple, and miniaturization and cost reduction of the spectroscope can be realized.
  • an excitation light source that emits excitation light with a wavelength that does not overlap with the wavelength of fluorescence emitted from the sample is selected, and in the spectrometer, an optical system corresponding to the selected excitation light source is used to generate Raman scattered light.
  • an optical system corresponding to the selected excitation light source is used to generate Raman scattered light.
  • the wavelengths of the Raman scattered light and the fluorescence overlap each other, preventing the extremely weak Raman peak of the Raman spectrum from being buried in the fluorescence spectrum. be able to.
  • switching means for physically switching apertures, diffraction gratings, and detectors which were conventionally required, are no longer required, problems such as position reproducibility of the movable parts of the switching means can affect sample analysis results. There are no adverse effects, and this also ensures that sample analysis can always be performed with high accuracy.
  • FIG. 1 is a diagram showing the overall configuration of a microscopic Raman spectrometer according to the present invention
  • FIG. 1 is a cross-sectional view showing an internal configuration of a spectroscope of a microscopic Raman spectrometer according to the present invention
  • FIG. It is a figure which shows the Raman spectrum of cyclohexane, (a) is the figure at the time of excitation by the excitation wavelength of 532 nm, (b) is the figure at the time of excitation at the excitation wavelength of 785 nm.
  • FIG. 1 is a diagram showing the overall configuration of a microscopic Raman spectroscopic device according to the present invention.
  • the molecular structure and physical properties of the sample S are analyzed by the Raman spectrum obtained by spectroscopically dispersing R1 or R2. can be analyzed locally, and is structured as follows.
  • the microscopic Raman spectroscopic device 1 shown in FIG. A spectroscope 10 that disperses the radiated Raman scattered light R1 or R2, a CCD detector 20 that detects and photoelectrically converts the Raman scattered light dispersed by the spectroscope 10 for each wavelength (wave number), and the CCD detection.
  • a personal computer (PC) 30 is basically provided for converting the signal obtained by the device 20 into a Raman shift value (wavenumber shift value) and displaying it as a Raman spectrum.
  • the micro Raman spectrometer 1 irradiates the sample S with excitation laser light L1 or L2 emitted from one of the selected laser oscillators 2 or 3, and irradiates the weak Raman scattered light R1 emitted from the sample S.
  • an optical system X is provided that guides R2 to the spectroscope 10 .
  • the two laser oscillators 2 and 3 emit excitation laser beams L1 and L2 having different wavelengths, respectively.
  • One laser oscillator 2 emits an excitation laser beam L1 with a wavelength of 532 nm and the other laser oscillator 3 emits an excitation laser beam L2 having a wavelength of 785 nm.
  • the optical system X irradiates the sample S with the excitation laser light L1 or L2 emitted from the two laser oscillators 2 and 3, respectively, and guides the Raman scattered light R1 or R2 emitted from the sample S to the spectroscope 10.
  • FIG. 2 is a cross-sectional view showing the internal configuration of the spectroscope.
  • the illustrated spectroscope 10 includes two (the same number as the laser oscillators 2 and 3) diffraction gratings 12 and 12 in a rectangular box-shaped case 11. 13, one imaging lens 14, and two incident apertures 11a and 11b (the same number as the laser oscillators 2 and 3) formed on the top surface of the case 11. It is constructed by housing two optical systems I and II leading to one diffraction grating 12 or 13 .
  • one optical system I includes a reflecting mirror 15, a collimator lens 16, and a folding mirror 17, and the other optical system II includes a reflecting mirror 15 and a collimator lens .
  • one reflecting mirror 15 is shared by the optical systems I and II.
  • the configuration of the spectroscope 10 can be simplified, and the size and cost of the spectroscope 10 can be reduced.
  • only one imaging lens 14 is provided, and as will be described later, the Raman scattered light R1 or R2 guided to the diffraction grating 12 or 13 by the optical system I or II and dispersed is combined into a common lens.
  • the structure of the spectroscope 10 can also be simplified by adopting a configuration in which the light is condensed by two imaging lenses 14 and imaged on the detection surface 20a of the CCD detector 20, and the spectroscope 10 can be made smaller, more compact, and at a lower cost. can be improved.
  • this laser oscillator 2 when one of the laser oscillators 2 on the short wavelength side is selected and this laser oscillator 2 is driven (turned on), this laser oscillator 2 emits an excitation laser with a wavelength of 532 nm indicated by a broken line in FIG.
  • Light L1 is emitted.
  • This excitation laser beam L1 is sequentially reflected by reflecting mirrors M1, M2, and M3 and guided to a long-pass filter LPF1, reflected by this long-pass filter LPF1 and guided to a reflecting mirror M4, where After being reflected, the light is reflected by the dichroic mirror DM and the reflecting mirrors M5 and M6, and the sample S is irradiated.
  • the excitation laser beam L1 with a wavelength of 532 nm directed toward the sample S is condensed by the objective lens 6, whereby a high spatial resolution of ⁇ m order can be obtained.
  • the Raman scattered light R1 is condensed by the condensing lens 4, and passes through one of the incident apertures 11a in the case 11 of the spectroscope 10. introduced into.
  • this laser oscillator 3 when the other laser oscillator 3 on the longer wavelength side is selected and this laser oscillator 3 is driven (turned on), this laser oscillator 3 emits an excitation laser beam L2 having a wavelength of 785 nm indicated by a broken line in FIG. do. Then, this excitation laser beam L2 is successively reflected by the reflection mirrors M2 and M3 and guided to the long-pass filter LPF2. After that, the excitation laser beam L2 is reflected by the long-pass filter LPF2, passes through the dichroic mirror DM, reaches the reflection mirror M5, and is sequentially reflected by the reflection mirror M5 and the reflection mirror M6 to irradiate the sample S. .
  • the excitation laser beam L2 with a wavelength of 785 nm directed toward the sample S is condensed by the objective lens 6, whereby a high spatial resolution on the order of ⁇ m (diameter of 5 ⁇ m in this embodiment) can be obtained.
  • the sample S when the sample S is irradiated with the excitation laser light L2 having a wavelength of 785 nm emitted from the other laser oscillator 3, the sample S emits weak Raman scattered light R2, and this Raman scattered light R2 is , and sequentially reflected by the reflecting mirrors M6 and M5 as indicated by the solid line in FIG. Then, this Raman scattered light R2 is reflected by the reflecting mirror M7, then condensed by the condensing lens 5, and introduced into the spectroscope 10 through the other incident aperture 11b opened in the case 11 of the spectroscope 10. .
  • the two laser oscillators 2 and 3 which respectively emit excitation laser beams L1 and L2 having different wavelengths, emit excitation laser beams such that the wavelength of the fluorescence emitted from the sample S does not overlap with the wavelength of the Raman scattering light R1 or R2.
  • One that emits L1 or L2 is selected, and one of the selected laser oscillators 2 or 3 is selectively driven (lighted).
  • the Raman scattered light R1 or R2 When the Raman scattered light R1 or R2 is condensed by the incident aperture 11a or 11b and introduced into the spectroscope 10 as described above, the Raman scattered light R1 or R2 becomes is guided to the diffraction grating 12 or 13 by the optical system I or II shown in FIG.
  • this Raman scattered light R2 constitutes an optical system II. After being reflected by the common reflecting mirror 15, the light is collimated by passing through the collimator lens 18 to form a parallel light beam. Then, this parallel light flux is guided to the diffraction grating 13, and by passing through this diffraction grating 13, the light is split into wavelengths.
  • the wavelength resolution of the spectroscope 10 is selected to be about 0.3 ⁇ m in consideration of the balance between the minimum required value and the energy to be obtained. are doing. Since the diameter of the entrance apertures 11a and 11b corresponding to the wavelength resolution of 0.3 ⁇ m is about 30 ⁇ m in this embodiment, the Raman scattered light from the 5 ⁇ m diameter region of the sample S is incident on the entrance apertures 11a and 11b.
  • the diameter of the Raman scattered light beams R1 and R2 emitted from the sample S should be set to about 6 ⁇ m. It is sufficient to magnify it twice and form an image on the incident apertures 11a and 11b. Therefore, the F values of the Raman scattered light beams R1 and R2 incident on the spectroscope 10 do not have to be so small (there is no need to enter a bright light flux).
  • the Raman scattered light R1 or R2 is split by the diffraction grating 12 or 13
  • the split Raman scattered light R1 or R2 is imaged on the detection surface 20a of the CCD detector 20 by the imaging lens 14.
  • the CCD detector 20 detects and photoelectrically converts the spectroscopic Raman scattered light R1 or R2 for each wavelength (wavenumber), and the signal obtained by the CCD detector 20 is converted to Raman light by a personal computer (PC) 30. It is converted into a shift value (wavenumber shift value) and displayed as a Raman spectrum.
  • PC personal computer
  • the Raman scattered light beams R1 and R2 can be incident on one imaging lens 14 in tandem, and the diameter of the imaging lens 14 can be kept small so that the imaging lens 14 can be made small and compact. , and by extension, the miniaturization and compactness of the spectroscope 10 as a whole can be realized.
  • FIG. 3 shows the Raman spectrum of cyclohexane, where (a) is the case of excitation at an excitation wavelength of 532 nm, and (b) is the case of excitation at an excitation wavelength of 785 nm.
  • the horizontal axis of the Raman spectra shown in FIGS. 3(a) and 3(b) is the Raman shift (cm ⁇ 1 ), and the vertical axis is the Raman intensity.
  • the spectroscope 10 corresponds to the two laser oscillators 2 and 3 that emit excitation laser beams having different wavelengths, Two incident apertures 11a and 11b, which are the same in number as these laser oscillators 2 and 3, and a plurality of (laser (the same number as the oscillators 2 and 3) are provided. is simple, and the size and cost of the spectroscope 10 can be reduced.
  • the laser oscillators 2 and 3 that emit the excitation laser beams L1 and L2 having wavelengths that do not overlap with the wavelength of the fluorescence emitted from the sample S are selected, and in the spectrometer 10, the selected laser oscillator 2 or 3 can direct Raman scattered light R1 or R2 to the corresponding diffraction grating 12 or 13 using optical system I or II. Therefore, the wavelengths of the Raman scattering lights R1 and R2 overlap with the fluorescence wavelengths, preventing the occurrence of a problem that a very weak Raman peak of the Raman spectrum is buried in the fluorescence spectrum. It can be done with high accuracy.
  • the present invention is applied to the microscopic Raman spectroscopic device 1 provided with two laser oscillators 2 and 3 that respectively emit excitation laser beams L1 and L2 having different wavelengths.
  • the present invention can be similarly applied to a microscopic Raman spectrometer equipped with three or more laser oscillators that emit excitation laser beams of different wavelengths.
  • the excitation laser light L1 having a wavelength of 532 nm and the laser light L2 having a wavelength of 785 nm are used as examples of the excitation laser light having different wavelengths, but any other wavelength ( For example, 488 nm, 633 nm, etc.) can be selected.
  • excitation light source in addition to a laser oscillator that emits excitation laser light, any other excitation light source that emits monochromatic light other than laser light can be used.

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  • Spectroscopy & Molecular Physics (AREA)
  • General Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Spectrometry And Color Measurement (AREA)
PCT/JP2022/026204 2021-08-04 2022-06-30 顕微ラマン分光装置 Ceased WO2023013325A1 (ja)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CN202280053789.XA CN117859048A (zh) 2021-08-04 2022-06-30 显微拉曼分光装置
JP2023539715A JP7582487B2 (ja) 2021-08-04 2022-06-30 顕微ラマン分光装置
EP22852751.1A EP4382892A4 (en) 2021-08-04 2022-06-30 RAMAN MICROSPECTROMETER
US18/294,858 US12480880B2 (en) 2021-08-04 2022-06-30 Microscopic raman spectroscopy device
JP2024190296A JP2025016619A (ja) 2021-08-04 2024-10-30 顕微ラマン分光装置

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Application Number Priority Date Filing Date Title
JP2021-127928 2021-08-04
JP2021127928 2021-08-04

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US (1) US12480880B2 (https=)
EP (1) EP4382892A4 (https=)
JP (2) JP7582487B2 (https=)
CN (1) CN117859048A (https=)
WO (1) WO2023013325A1 (https=)

Cited By (1)

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CN116625960A (zh) * 2023-05-06 2023-08-22 深圳综合粒子设施研究院 一种基于双针孔的原位光谱测试装置

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CN120314282B (zh) * 2025-05-09 2025-10-21 奥谱天成(厦门)光电股份有限公司 一种libs与拉曼光谱联用系统

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