Micro-Raman spectroscopy apparatus
A simplified micro-Raman spectrometer with shared components and a single imaging lens addresses structural complexity and cost, ensuring high-precision analysis by avoiding fluorescence overlap and positional inaccuracies.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- SHIMADZU SEISAKUSHO LTD
- Filing Date
- 2024-10-30
- Publication Date
- 2026-05-15
AI Technical Summary
Existing micro-Raman spectrometers with multiple excitation laser light sources, apertures, diffraction gratings, and CCD detectors have complex structures, leading to large size, high cost, and potential analysis inaccuracies due to movable parts affecting reproducibility.
A micro-Raman spectrometer with multiple excitation light sources and a simplified optical system using a single imaging lens and shared components to guide and separate Raman scattered light, eliminating the need for physical switching mechanisms.
The spectrometer is miniaturized, cost-effective, and ensures high-precision analysis by preventing fluorescence overlap and reducing positional errors from movable parts.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to a microscopic Raman spectrometer capable of obtaining a high spatial resolution on the order of μm by condensing excitation light using an objective lens of a microscope.
Background Art
[0002] When a substance (sample) is irradiated with light of a specific wavelength (such as monochromatic light or laser light), the irradiated light is scattered, and a part of it becomes weak Raman scattered light having a wavelength different from that of the irradiated light due to molecular vibration. Since the frequency of this Raman scattered light matches the intrinsic vibration frequency of the molecule, the Raman scattered light appears at a certain fixed wave number (Raman shift) based on the vibration and rotation of the molecules constituting the sample. An apparatus for analyzing the molecular-level structure of a sample by detecting a Raman spectrum obtained by spectroscopically analyzing this Raman scattered light is a Raman spectrometer, and this Raman spectrometer is composed of an excitation light source, a spectroscope, and a detector, and various proposals have been made for this so far (see, for example, Patent Document 1).
[0003] In addition, proposals have also been made for a microscopic Raman spectrometer capable of obtaining a high spatial resolution on the order of μm by condensing irradiation light (excitation light) using an objective lens of a microscope in the optical system of the Raman spectrometer (see, for example, Patent Document 2).
[0004] By the way, since the sample has various fluorescence characteristics with respect to ultraviolet rays and visible light, fluorescence corresponding to the irradiation light is emitted from the sample. In Raman spectroscopy, when the autofluorescence of the sample is strong, the wavelengths of the Raman scattered light and the fluorescence overlap, and the very weak Raman peaks in the Raman spectrum are buried in the fluorescence spectrum, resulting in a problem that the analysis of the sample cannot be performed with high precision.
[0005] Therefore, Patent Document 3 proposes a micro-Raman spectrometer that can perform highly accurate analysis of a sample by performing fluorescence observation as a preliminary measurement on the sample, predicting what wavelength of fluorescence the sample will emit in response to laser light of a certain excitation wavelength, and selecting an excitation laser light such that the wavelength of fluorescence and the wavelength of Raman scattered light do not overlap. Specifically, this micro-Raman spectrometer is equipped with multiple excitation laser light sources and an automatic laser switch for switching between these excitation laser light sources. The spectrometer is equipped with aperture switching means for selecting the optimal aperture from among multiple apertures, diffraction grating switching means for switching to an appropriate diffraction grating from among multiple diffraction gratings according to the excitation laser light, and detector switching means for switching to an appropriate CCD detector from among multiple CCD detectors according to the excitation laser light. [Prior art documents] [Patent Documents]
[0006] [Patent Document 1] Japanese Patent Publication No. 2006-113021 [Patent Document 2] Japanese Patent Publication No. 2017-207522 [Patent Document 3] Patent No. 6788298 [Overview of the project] [Problems that the invention aims to solve]
[0007] However, the micro-Raman spectrometer proposed in Patent Document 3 employs a configuration in which the spectrometer is equipped with multiple apertures (the same number as the number of excitation laser light sources), diffraction gratings, and CCD detectors, and these are switched by aperture switching means, diffraction grating switching means, and detector switching means to switch them according to excitation laser light of different wavelengths. This leads to a problem in that the structure of the spectrometer becomes complex, resulting in a large size and high cost.
[0008] Furthermore, each switching mechanism for physically switching between the aperture, diffraction grating, and detector has movable parts, but problems such as the reproducibility of the positions of these movable parts may negatively affect the analysis results of the sample.
[0009] The present invention has been made in view of the above problems, and its objective is to provide a micro-Raman spectrometer that can be miniaturized and cost-effective by simplifying the structure of the spectrometer, and that can always analyze samples with high precision. [Means for solving the problem]
[0010] The micro-Raman spectrometer according to the present invention is Multiple excitation light sources, each emitting excitation light of different wavelengths, and a single unit that focuses the excitation light and irradiates the sample with it. Tsu The objective lens and A spectrometer that spectrally separates Raman scattered light emitted from a sample by irradiation with excitation light from one selected excitation light source through one objective lens using a diffraction grating, A detector that detects the Raman scattered light spectrally separated by the spectrometer and performs photoelectric conversion, A micro-Raman spectrometer equipped with, The aforementioned spectrometer is, Multiple entrance apertures, An optical system that directs the Raman scattered light incident from the aforementioned incident aperture into a parallel beam and guides it to the diffraction grating, The system comprises an imaging lens that frames the beams of multiple Raman scattered light spectrally separated by the diffraction grating onto the detector, The optical system is provided in the same number as the number of the entrance apertures, A micro-Raman spectrometer characterized in that one imaging lens receives multiple light beams spectrally separated by the diffraction grating and forms an image on the detector. [Effects of the Invention]
[0011] According to the present invention, the spectrometer has, corresponding to a plurality of excitation light sources that emit excitation light with different wavelengths, the same number of incident apertures as these excitation light sources, and a plurality (the same number as the excitation light sources) of optical systems that guide Raman light incident from one of these incident apertures to a diffraction grating. Therefore, switching means for switching the incident aperture (aperture) and the diffraction grating are not required. Also, since a plurality of light beams can be imaged on a detector by one imaging lens, the structure of the spectrometer is simplified, and miniaturization and cost reduction of the spectrometer can be achieved.
[0012] Also, an excitation light source that emits excitation light with a wavelength that does not overlap with the wavelength of the fluorescence emitted from the sample is selected, and in the spectrometer, the Raman scattered light can be guided to the corresponding diffraction grating using the optical system corresponding to the selected excitation light source. For this reason, the problem that the wavelengths of the Raman scattered light and the fluorescence overlap with each other and the very weak Raman peaks in the Raman spectrum are buried in the fluorescence spectrum is prevented, and the analysis of the sample can always be performed with high accuracy.
[0013] Furthermore, since each switching means for physically switching the aperture, the diffraction grating, and the detector, which were conventionally necessary, is not required, the analysis result of the sample is not affected by problems such as the position reproducibility of the movable parts of the switching means, and also by this, the analysis of the sample can always be performed with high accuracy.
Brief Description of the Drawings
[0014] [Figure 1] It is a diagram showing the overall configuration of a microscopic Raman spectrometer according to the present invention. [Figure 2] It is a cross-sectional view showing the internal configuration of the spectrometer of the microscopic Raman spectrometer according to the present invention. [Figure 3] It is a diagram showing the Raman spectrum of cyclohexane, where (a) is a diagram when excited with an excitation wavelength of 532 nm, and (b) is a diagram when excited with an excitation wavelength of 785 nm.
Embodiments for Carrying Out the Invention
[0015] Embodiments of the present invention will be described below based on the accompanying drawings.
[0016] FIG. 1 is a diagram showing the overall configuration of a microscopic Raman spectrometer according to the present invention. The illustrated microscopic Raman spectrometer 1 analyzes the molecular structure and physical properties of a sample S based on a Raman spectrum obtained by spectroscopically analyzing weak Raman scattered light R1 or R2 emitted from the sample S irradiated with excitation laser light L1 or L2. In particular, by condensing the excitation laser light L1 or L2 with the objective lens 6 of the microscope, local analysis on the order of several micrometers is possible, and it is configured as follows.
[0017] That is, the microscopic Raman spectrometer 1 shown in FIG. 1 basically includes two laser oscillators 2 and 3 as excitation light sources, a spectroscope 10 that spectroscopically analyzes Raman scattered light R1 or R2 emitted from the sample S by irradiation with excitation laser light L1 or L2 from one of the selected laser oscillators 2 or 3, a CCD detector 20 that detects and photoelectrically converts the Raman scattered light spectroscopically analyzed by the spectroscope 10 for each wavelength (wave number), and a personal computer (PC) 30 that converts the signal obtained by the CCD detector 20 into a Raman shift value (wave number shift value) and displays it as a Raman spectrum. In addition, the microscopic Raman spectrometer 1 is provided with an optical system X that irradiates the sample S with the excitation laser light L1 or L2 emitted from one of the selected laser oscillators 2 or 3 and guides the weak Raman scattered light R1 or R2 emitted from the sample S to the spectroscope 10.
[0018] The two laser oscillators 2 and 3 each emit excitation laser lights L1 and L2 having different wavelengths. One laser oscillator 2 emits excitation laser light L1 having a wavelength of 532 nm, and the other laser oscillator 3 emits excitation laser light L2 having a wavelength of 785 nm. The optical system X, which irradiates the sample S with excitation laser light L1 or L2 emitted from these two laser oscillators 2 and 3, respectively, and guides the Raman scattered light R1 or R2 emitted from the sample S to the spectrometer 10, is composed of a plurality (7) of reflective mirrors (plane mirrors) M1, M2, M3, M4, M5, M6, and M7, two long-pass filters LPF1 and LPF2, one dichroic mirror DM, two focusing lenses 4 and 5, and one microscope objective lens 6.
[0019] Next, the internal structure of the spectrometer 10 will be described below based on Figure 2.
[0020] Figure 2 is a cross-sectional view showing the internal structure of the spectrometer. The spectrometer 10 shown in the figure is constructed by housing two diffraction gratings 12 and 13 (the same number as the laser oscillators 2 and 3) and one imaging lens 14 within a rectangular box-shaped case 11, and two optical systems I and II that guide the Raman scattered light R1 or R2 incident from one of the two incident apertures 11a and 11b formed on the top surface of the case 11 to one of the diffraction gratings 12 or 13.
[0021] Here, one optical system I is composed of a reflective mirror 15, a collimator lens 16, and a diffraction grating 17, while the other optical system II is composed of a reflective mirror 15 and a collimator lens 18, with one reflective mirror 15 being shared by optical systems I and II. By sharing one reflective mirror 15 between the two optical systems I and II in this way, the configuration of the spectrometer 10 can be simplified, making the spectrometer 10 smaller, more compact, and less expensive. Furthermore, by employing a configuration in which only one imaging lens 14 is provided, and the Raman scattered light R1 or R2, which is guided to the diffraction grating 12 or 13 by the optical system I or II and spectrally separated, is collected by a single common imaging lens 14 and imaged onto the detection surface 20a of the CCD detector 20, the structure of the spectrometer 10 can be simplified, making the spectrometer 10 smaller, more compact, and lowering the cost.
[0022] Next, we will explain the operation of the micro-Raman spectrometer 1 configured as described above.
[0023] In Figure 1, for example, if one of the laser oscillators 2 on the short wavelength side is selected and this laser oscillator 2 is driven (lit), then excitation laser light L1 with a wavelength of 532 nm, as shown by the dashed line in Figure 1, is emitted from this laser oscillator 2. The excitation laser light L1 is then sequentially reflected by the reflection mirrors M1, M2, and M3 and guided to the long-pass filter LPF1, where it is reflected again and guided to the reflection mirror M4, and after being reflected again by the diclock mirror DM and the reflection mirrors M5 and M6, it is reflected towards the sample S. Here, the excitation laser light L1 with a wavelength of 532 nm directed towards the sample S is focused by the objective lens 6, thereby obtaining a high spatial resolution on the order of micrometers.
[0024] As described above, when the excitation laser light L1 with a wavelength of 532 nm emitted from one of the laser oscillators 2 irradiates the sample S, a weak Raman scattered light R1 is emitted from the sample S. This Raman scattered light R1 is sequentially reflected by the reflection mirrors M6 and M5, the dichroic mirror DM, and the reflection mirror M4, as shown by the solid lines in Figure 1, and reaches the long-pass filter LPF1. Then, this Raman scattered light R1 passes through the long-pass filter LPF1, is reflected by the reflection mirror M7, is focused by the focusing lens 4, and is introduced into the inside of the spectrometer 10 through one of the entrance apertures 11a that opens into the case 11 of the spectrometer 10.
[0025] On the other hand, if the other laser oscillator 3 on the longer wavelength side is selected and this laser oscillator 3 is driven (lit), an excitation laser beam L2 with a wavelength of 785 nm, as shown by the dashed line in Figure 1, is emitted from this laser oscillator 3. This excitation laser beam L2 is then sequentially reflected by the reflection mirrors M2 and M3 and guided to the long-pass filter LPF2. Subsequently, the excitation laser light L2 is reflected by the long-pass filter LPF2, passes through the dichroic mirror DM, and reaches the reflection mirror M5. This reflection mirror M5 and reflection mirror M6 then sequentially reflect the light towards the sample S. Here, the excitation laser light L2 with a wavelength of 785 nm directed towards the sample S is focused by the objective lens 6, thereby obtaining a high spatial resolution on the order of micrometers (in this embodiment, a diameter of 5 μm).
[0026] As described above, when the excitation laser light L2 with a wavelength of 785 nm emitted from the other laser oscillator 3 is irradiated onto the sample S, weak Raman scattered light R2 is emitted from the sample S. This Raman scattered light R2 is reflected sequentially by the reflection mirrors M6 and M5, as shown by the solid lines in Figure 1, and then sequentially passes through the dichroic mirror DM and the long-pass filter LPF2 before reaching the reflection mirror M7. Then, this Raman scattered light R2 is reflected by the reflective mirror M7, focused by the focusing lens 5, and introduced into the inside of the spectrometer 10 through the other entrance aperture 11b that opens into the case 11 of the spectrometer 10. Furthermore, of the two laser oscillators 2 and 3, which emit excitation laser beams L1 and L2 with different wavelengths, one is selected to emit excitation laser beams L1 or L2 such that the wavelength of the fluorescence emitted from the sample S does not overlap with the wavelength of the Raman scattered light R1 or R2. The selected laser oscillator 2 or 3 is then selectively driven (lit).
[0027] As described above, when the Raman scattered light R1 or R2 is focused by the entrance aperture 11a or 11b and introduced into the spectrometer 10, the Raman scattered light R1 or R2 is guided to the diffraction grating 12 or 13 by the optical system I or II shown in Figure 2, which is provided in the spectrometer 10, and is spectrally separated by wavelength.
[0028] In other words, as shown in Figure 2, when one Raman scattered light R1 passes through one of the entrance apertures 11a and is introduced into the case 11 of the spectrometer 10, this Raman scattered light R1 is reflected by the reflective mirror 15 that constitutes the optical system I, and then passed through the collimator lens 16, where it is parallelized to become a parallel beam of light. Then, this parallel beam of light is redirected by the diffraction grating 17 and guided to the diffraction grating 12, where it is spectrally separated by wavelength upon passing through the diffraction grating 12.
[0029] On the other hand, when the Raman scattered light R2 from the other side, which is focused by passing through the other entrance aperture 11b, is introduced into the case 11 of the spectrometer 10, this Raman scattered light R2 is reflected by the common reflective mirror 15 that constitutes the optical system II, and then parallelized by passing through the collimator lens 18 to become a parallel beam of light. This parallel beam of light is then guided to the diffraction grating 13, where it is spectrally separated by wavelength upon passing through the diffraction grating 13.
[0030] Incidentally, in this embodiment, while a spatial resolution of 5 μm in diameter is achieved on the sample S, the wavelength resolution of the spectrometer 10 is selected to be approximately 0.3 μm, balancing the minimum required value with the energy to be obtained. In this embodiment, the diameters of the entrance apertures 11a and 11b, which correspond to a wavelength resolution of 0.3 μm, are approximately 30 μm. Therefore, in order to direct the Raman scattered light R1 and R2 from a 5 μm diameter region of the sample S into the entrance apertures 11a and 11b, respectively, the diameters of the Raman scattered light R1 and R2 emitted from the sample S should be enlarged by approximately six times to image them into the entrance apertures 11a and 11b. Therefore, the F-numbers of the Raman scattered light R1 and R2 incident on the spectrometer 10 do not need to be very small (there is no need to incident a bright beam of light).
[0031] As described above, when the Raman scattered light R1 or R2 is spectrally separated by the diffraction grating 12 or 13, the spectrally separated Raman scattered light R1 or R2 is imaged onto the detection surface 20a of the CCD detector 20 by the imaging lens 14. The CCD detector 20 then detects the spectrally separated Raman scattered light R1 or R2 for each wavelength (wavenumber) and converts it into photoelectric signals. The signal obtained by the CCD detector 20 is then converted into a Raman shift value (wavenumber shift value) by the personal computer (PC) 30 and displayed as a Raman spectrum.
[0032] Incidentally, in this embodiment, the F-number of the Raman scattered light R1 and R2 incident on the spectrometer 10 is approximately 6. Considering that this is 6 / 2.3 = 2.6 times the F-number of 2.3 in a typical bright Raman spectrometer, the parallel luminous flux of the Raman scattered light R1 and R2 can be as small as 1 / 2.6 ≈ 0.38, or about 40%. Therefore, Raman scattered light R1 and R2 can be incident in tandem on a single imaging lens 14, allowing the diameter of the imaging lens 14 to be kept small, thereby making the imaging lens 14 smaller and more compact, and ultimately enabling the entire spectrometer 10 to be made smaller and more compact.
[0033] Here, an example of a Raman spectrum is shown in Figure 3.
[0034] Figure 3 shows the Raman spectrum of cyclohexane, where (a) is the spectrum when excited at an excitation wavelength of 532 nm, and (b) is the spectrum when excited at an excitation wavelength of 785 nm. Note that the horizontal axis of the Raman spectra shown in Figures 3(a) and (b) represents the Raman shift (cm²). -1 The vertical axis represents Raman intensity.
[0035] As is clear from Figures 3(a) and (b), in both the case of excitation at an excitation wavelength of 532 nm and the case of excitation at an excitation wavelength of 785 nm, a peak in Raman intensity appears at the same Raman shift (wavenumber). However, the intensity ratio differs depending on the excitation wavelength, with excitation at an excitation wavelength of 785 nm showing a higher intensity peak value than excitation at an excitation wavelength of 532 nm.
[0036] As is clear from the above explanation, according to the micro-Raman spectrometer 1 of this embodiment, the spectrometer 10 is equipped with two entrance apertures 11a and 11b, the same number as the two laser oscillators 2 and 3 that emit excitation laser light of different wavelengths, and two optical systems I and II (the same number as the laser oscillators 2 and 3) that guide the Raman scattered light R1 or R2 incident from one of these entrance apertures 11a and 11b to the diffraction grating 12 or 13. As a result, a switching means for switching between the entrance apertures 11a and 11b and the diffraction gratings 12 and 13 is not required, and consequently the structure of the spectrometer 10 is simplified, resulting in miniaturization and cost reduction of the spectrometer 10.
[0037] Furthermore, by selecting laser oscillators 2 and 3 that emit excitation laser light L1 and L2 at wavelengths that do not overlap with the fluorescence wavelength emitted from the sample S, the spectrometer 10 can guide the Raman scattered light R1 or R2 to the corresponding diffraction grating 12 or 13 using optical system I or II corresponding to the selected laser oscillator 2 or 3. Therefore, the wavelengths of the Raman scattered light R1 and R2 and the fluorescence overlap, preventing the very weak Raman peaks in the Raman spectrum from being buried in the fluorescence spectrum. This prevents such problems and ensures that the analysis of sample S is always performed with high accuracy.
[0038] Furthermore, since the switching means required to physically switch the entrance apertures 11a and 11b, diffraction gratings 12 and 13, and CCD detector 20, which were previously necessary, are no longer required, the analysis results of the sample S will not be adversely affected by issues such as the positional reproducibility of the movable parts of the switching means, and thus the analysis of the sample S can always be performed with high accuracy.
[0039] In the embodiments described above, the present invention was applied to a micro-Raman spectrometer 1 equipped with two laser oscillators 2 and 3 that emit excitation laser light L1 and L2 of different wavelengths, respectively. However, the present invention is similarly applicable to a micro-Raman spectrometer equipped with three or more laser oscillators that emit excitation laser light of different wavelengths.
[0040] Furthermore, in the embodiments described above, we explained that excitation laser light L1 has a wavelength of 532 nm and laser light L2 has a wavelength of 785 nm as examples of excitation laser light with different wavelengths. However, any other wavelength (for example, 488 nm, 633 nm, etc.) can be selected as the wavelength of the laser light.
[0041] Furthermore, 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 as the excitation light source.
[0042] Furthermore, the present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the technical idea described in the claims, specification, and drawings. [Explanation of Symbols]
[0043] 1. Micro-Raman Spectrometer 2,3 Laser oscillator (excitation light source) 4.5 Focusing lens 6. Objective lens 10 spectrometer 11. Spectrometer case 11a,11b entrance aperture 12, 13, 17 Diffraction gratings 14. Imaging lens 15 Reflective mirror 16,18 Collimator lens 20 CCD detectors (detectors) 20a Detection surface of the CCD detector 30. Personal Computers (PCs) I, II Optical Systems DM Dichroic Mirror L1, L2 excitation laser light (excitation light) M1-M7 Reflective Mirrors R1, R2 Raman scattered light S sample X optical system
Claims
1. Multiple excitation light sources, each emitting excitation light of different wavelengths, and a single objective lens that focuses the excitation light and irradiates the sample with it. A spectrometer that spectrally analyzes Raman scattered light emitted from a sample by irradiation with excitation light from one selected excitation light source through one objective lens using a diffraction grating, A detector that detects the Raman scattered light spectrally separated by the spectrometer and performs photoelectric conversion, A micro-Raman spectrometer equipped with, The aforementioned spectrometer is, Multiple entrance apertures, An optical system that directs the Raman scattered light incident from the aforementioned incident aperture into a parallel beam and guides it to the diffraction grating, The system comprises an imaging lens that frames the beams of multiple Raman scattered light spectrally separated by the diffraction grating onto the detector, The optical system is provided in the same number as the number of the entrance apertures, A micro-Raman spectrometer characterized in that one imaging lens receives multiple light beams spectrally separated by the diffraction grating and forms an image on the detector.
2. The micro-Raman spectrometer according to claim 1, characterized in that the light beam focused by one imaging lens is imaged onto the detection surface of a common detector.
3. Each of the multiple optical systems includes a reflective mirror that reflects the Raman scattered light incident from the incident aperture, The micro-Raman spectrometer according to claim 1, further comprising a collimator lens that parallelizes the light reflected by the reflective mirror and guides it to the diffraction grating.
4. The micro-Raman spectrometer according to claim 3, characterized in that one of the reflective mirrors is shared among multiple optical systems.
5. The micro-Raman spectrometer according to any one of claims 1 to 4, characterized in that the excitation light source comprises a laser oscillator that emits excitation laser light with a wavelength of 532 nm and a laser oscillator that emits excitation laser light with a wavelength of 785 nm.