Raman microspectrometer

Abstract

To provide a Raman microspectrometer that can achieve size and cost reduction through simplification of the structure of a spectroscope, and can constantly analyze a sample with high accuracy.SOLUTION: A Raman microspectrometer comprises: laser oscillators (excitation light sources) that emit excitation laser light (excitation light) of different wavelengths; a spectroscope 10 that uses a diffraction grating 12 or 13 to disperse Raman scattered light R1 or R2 radiated from a sample as a result of emission of the excitation laser light from the laser oscillator; and a CCD detector 20 that detects and subjects to photoelectric conversion the Raman scattered light R1 or R2 dispersed by the spectroscope 10. The spectroscope 10 comprises incident apertures 11a, 11b and multiple (two) optical systems I, II that guide the Raman scattered light R1 or R2 that makes incident from the incident apertures 11a, 11b to the diffraction grating 12 or 13. The mulitiple light beams dispersed by a diffraction grating 12 or 13 enter in tandem to a single image formation lens 14.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a Raman microspectroscopic device that can obtain a high spatial resolution on the order of μm by focusing excitation light using an objective lens of a microscope. [Background technology]

[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 some of it becomes weak Raman scattered light that has a different wavelength from the irradiated light due to molecular vibration. Since the frequency of this Raman scattered light coincides with the inherent vibrational frequency of the molecules, the Raman scattered light appears at a certain wave number (Raman shift) based on the vibration and rotation of the molecules that make up the sample. A Raman spectrometer is a device that analyzes the molecular-level structure of a sample by detecting the Raman spectrum obtained by dispersing this Raman scattered light. This Raman spectrometer is composed of an excitation light source, a spectrometer, and a detector, and various proposals have been made regarding this device (see, for example, Patent Document 1).

[0003] In addition, a proposal has been made regarding a microscopic Raman spectroscopic device that can obtain high spatial resolution on the order of μm by focusing irradiation light (excitation light) using a microscope objective lens in the optical system of the Raman spectroscopic device (see, for example, Patent Document 2).

[0004] Incidentally, since samples have various fluorescent properties with respect to ultraviolet light and visible light, the samples emit fluorescent light according to the irradiated light. In Raman spectroscopy, if the sample has strong autofluorescence, the wavelengths of the Raman scattered light and the fluorescent light overlap, causing the very weak Raman peaks in the Raman spectrum to be buried in the fluorescent spectrum, making it impossible to analyze the sample with high accuracy.

[0005] Therefore, Patent Document 3 proposes a micro-Raman spectrometer that performs fluorescence observation as a preliminary measurement of a sample, predicts what wavelength of fluorescence the sample will emit in response to what excitation wavelength of laser light, and selects an excitation laser light such that the wavelength of the fluorescence does not overlap with the wavelength of the Raman scattered light, thereby enabling highly accurate analysis of the sample. Specifically, this micro-Raman spectrometer is equipped with a plurality of excitation laser light sources and an automatic laser switch for switching between these excitation laser light sources, and the spectrometer is provided with an aperture switching means for selecting the most appropriate one from a plurality of apertures, a diffraction grating switching means for switching from a plurality of diffraction gratings to an appropriate one according to the excitation laser light, and a detector switching means for switching from a plurality of CCD detectors to an appropriate one according to the excitation laser light. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2006-113021 A [Patent Document 2] JP 2017-207522 A [Patent Document 3] Patent No. 6788298 Summary of the Invention [Problem to be solved by the invention]

[0007] However, in the micro-Raman spectrometer proposed in Patent Document 3, a spectrometer is provided with a plurality of apertures (entrance openings), diffraction gratings, and CCD detectors (the same number as the number of excitation laser light sources), and these are switched by an aperture switching means, a diffraction grating switching means, and a detector switching means for switching according to excitation laser light of different wavelengths, which results in a problem that the structure of the spectrometer becomes complicated, resulting in an increase in size and cost.

[0008] In addition, each switching means for physically switching between the aperture, diffraction grating, and detector is provided with a movable part, and problems such as the positional reproducibility of these movable parts may adversely affect the results of sample analysis.

[0009] The present invention has been made in view of the above problems, and an object of the present invention is to provide a Raman microspectroscopic instrument which can achieve miniaturization and low cost by simplifying the structure of the spectrometer, and which can consistently analyze samples with high accuracy. [Means for solving the problem]

[0010] The Raman microspectroscopic device according to the present invention comprises: a plurality of excitation light sources each emitting excitation light of a different wavelength; and an objective lens for collecting the excitation light and irradiating the sample with the collected light; a spectrometer that uses a diffraction grating to disperse Raman scattered light emitted from a sample by irradiation of the excitation light from a selected one of the excitation light sources through the one objective lens; a detector that detects the Raman scattered light dispersed by the spectrometer and converts it into an electric signal; A Raman microscope spectroscopic apparatus comprising: The spectrometer includes: A plurality of entrance apertures; an optical system for converting the Raman scattered light incident through the entrance aperture into a parallel beam and guiding the parallel beam to the diffraction grating; an imaging lens that images the plurality of Raman scattered light beams dispersed by the diffraction grating onto the detector; The optical systems are provided in the same number as the entrance apertures, a first imaging lens that receives a plurality of light beams dispersed by the diffraction grating and images the plurality of light beams on the detector; Effect of the Invention

[0011] According to the present invention, the spectrometer corresponds to a plurality of excitation light sources emitting excitation light of different wavelengths, and is provided with entrance apertures in the same number as these excitation light sources, and a plurality of optical systems (the same number as the excitation light sources) that guide the Raman light incident from one of these entrance apertures to a diffraction grating. Therefore, no switching means is required for switching the entrance aperture (aperture) or diffraction grating. Furthermore, since a plurality of light beams can be imaged onto the detector by a single imaging lens, the structure of the spectrometer can be simplified, thereby enabling the spectrometer to be made smaller and less expensive.

[0012] In addition, an excitation light source that emits excitation light of a wavelength that does not overlap with the wavelength of the fluorescence emitted from the sample can be selected, and in the spectrometer, an optical system corresponding to the selected excitation light source can be used to guide the Raman scattered light to a diffraction grating corresponding to the selected excitation light source. This prevents the wavelengths of the Raman scattered light and the fluorescence from overlapping, which can cause the very weak Raman peak of the Raman spectrum to be buried in the fluorescence spectrum, and allows the sample to be analyzed with high precision at all times.

[0013] Furthermore, since the switching means required in the past to physically switch between the aperture, diffraction grating, and detector are no longer necessary, problems such as the position reproducibility of the movable parts of the switching means do not adversely affect the results of sample analysis, which also allows sample analysis to be performed with high precision at all times. [Brief description of the drawings]

[0014] [Figure 1] 1 is a diagram showing the overall configuration of a micro Raman spectroscopic apparatus according to the present invention. [Diagram 2] FIG. 2 is a cross-sectional view showing an internal configuration of a spectroscope of the micro-Raman spectroscopic apparatus according to the present invention. [Diagram 3] FIG. 2 shows Raman spectra of cyclohexane, where (a) is a graph obtained by excitation at an excitation wavelength of 532 nm, and (b) is a graph obtained by excitation at an excitation wavelength of 785 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.

[0016] FIG. 1 is a diagram showing the overall configuration of a micro-Raman spectroscopy apparatus according to the present invention. The illustrated micro-Raman spectroscopy apparatus 1 analyzes the molecular structure, physical properties, etc. of a sample S by using a Raman spectrum obtained by dispersing weak Raman scattered light R1 or R2 emitted from a sample S irradiated with excitation laser light L1 or L2. In particular, the micro-Raman spectroscopy apparatus 1 is capable of local analysis on the order of several μm by focusing the excitation laser light L1 or L2 using an objective lens 6 of a microscope, and is configured as follows.

[0017] That is, the micro-Raman spectroscopic apparatus 1 shown in FIG. 1 basically includes two laser oscillators 2 and 3 as excitation light sources, a spectrometer 10 that disperses Raman scattered light R1 or R2 emitted from a sample S by irradiation with excitation laser light L1 or L2 from a selected one of the laser oscillators 2 or 3, a CCD detector 20 that detects the Raman scattered light dispersed by the spectrometer 10 for each wavelength (wave number) and performs photoelectric conversion, 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. The Raman microspectrometer 1 is also provided with an optical system X that irradiates the sample S with excitation laser light L1 or L2 emitted from a selected one of the laser oscillators 2 or 3, and guides the weak Raman scattered light R1 or R2 emitted from the sample S to a spectrometer 10.

[0018] The two laser oscillators 2 and 3 respectively emit excitation laser light L1 and L2 of different wavelengths, with the laser oscillator 2 emitting excitation laser light L1 with a wavelength of 532 nm and the other laser oscillator 3 emitting excitation laser light L2 with a wavelength of 785 nm. An optical system X, which irradiates a sample S with excitation laser light L1 or L2 emitted from these two laser oscillators 2 and 3, respectively, and guides Raman scattered light R1 or R2 emitted from the sample S to a spectrometer 10, is composed of multiple (seven) reflecting mirrors (flat 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 in detail below with reference to FIG.

[0020] FIG. 2 is a cross-sectional view showing the internal configuration of the spectrometer. The spectrometer 10 shown in the figure is configured by accommodating, within a rectangular box-shaped case 11, two diffraction gratings 12 and 13 (the same number as the laser oscillators 2 and 3), one imaging lens 14, and two optical systems I and II that guide the Raman scattered light R1 or R2 incident from one of two entrance openings 11a and 11b (the same number as the laser oscillators 2 and 3) 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 reflecting mirror 15, a collimator lens 16, and a diffraction grating 17, and the other optical system II is composed of a reflecting mirror 15 and a collimator lens 18, with one reflecting mirror 15 being shared by both optical systems I and II. By sharing one reflecting mirror 15 for 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. Further, only one imaging lens 14 is provided, and as described below, a configuration is adopted in which 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 dispersed therein is collected by the single common imaging lens 14 to form an image on the detection surface 20a of the CCD detector 20, thereby simplifying the structure of the spectrometer 10 and making the spectrometer 10 smaller, more compact, and less expensive.

[0022] Next, the operation of the Raman microspectrometer 1 configured as above will be described.

[0023] In FIG. 1, for example, when one of the laser oscillators 2 on the short wavelength side is selected and this laser oscillator 2 is driven (lit), the laser oscillator 2 emits an excitation laser light L1 having a wavelength of 532 nm as indicated by a dashed line in FIG. This excitation laser light L1 is then reflected successively by reflecting mirrors M1, M2, and M3, and directed to the long-pass filter LPF1, and is then reflected by this long-pass filter LPF1 and directed to the reflecting mirror M4, and is then reflected by the dichroic mirror DM and the reflecting mirrors M5 and M6, and is then irradiated towards the sample S. Here, the excitation laser light L1 with a wavelength of 532 nm directed toward the sample S is focused by the objective lens 6, thereby making it possible to obtain a high spatial resolution on the order of μm.

[0024] As described above, when the excitation laser light L1 having a wavelength of 532 nm emitted from one of the laser oscillators 2 is irradiated onto the sample S, weak Raman scattered light R1 is emitted from the sample S. As shown by the solid line in FIG. 1, this Raman scattered light R1 is reflected in sequence by the reflecting mirrors M6 and M5, the dichroic mirror DM, and the reflecting mirror M4 to reach the long-pass filter LPF1. Then, this Raman scattered light R1 passes through the long-pass filter LPF1, is reflected by the reflecting mirror M7, and is then focused by the focusing lens 4 and introduced into the inside of the spectrometer 10 through one of the entrance openings 11a opened in the case 11 of the spectrometer 10.

[0025] On the other hand, when the other laser oscillator 3 on the long wavelength side is selected and this laser oscillator 3 is driven (lit), the excitation laser light L2 having a wavelength of 785 nm, as shown by the dashed line in Fig. 1, is emitted from this laser oscillator 3. Then, this excitation laser light L2 is reflected in turn by the reflecting mirrors M2 and M3 and guided to the long pass filter LPF2. Thereafter, the excitation laser light L2 is reflected by the long-pass filter LPF2, passes through the dichroic mirror DM, and reaches the reflecting mirror M5, and is successively reflected by the reflecting mirror M5 and the reflecting mirror M6 to be irradiated 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 making it possible to obtain a high spatial resolution on the order of μm (a diameter of 5 μm in this embodiment).

[0026] As described above, when the excitation laser light L2 having 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. As shown by the solid line in FIG. 1, this Raman scattered light R2 is reflected in turn by the reflecting mirrors M6 and M5, and then passes through the dichroic mirror DM and the long-pass filter LPF2 in turn, before reaching the reflecting mirror M7. Then, this Raman scattered light R2 is reflected by the reflecting mirror M7, and then collected by the collecting lens 5, and is introduced into the inside of the spectrometer 10 from the other entrance opening 11b opened in the case 11 of the spectrometer 10. Of the two laser oscillators 2, 3 which respectively emit excitation laser light L1, L2 having different wavelengths, one that emits excitation laser light 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 is selected, and the selected one of the laser oscillators 2 or 3 is selectively driven (lit up).

[0027] When the Raman scattered light R1 or R2 is collected by the entrance aperture 11a or 11b and introduced into the spectrometer 10 in the above manner, 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 FIG. 2 provided in the spectrometer 10 and separated into individual wavelengths.

[0028] That is, as shown in FIG. 2, when one of the Raman scattered lights R1 that has passed through one of the entrance openings 11a is introduced into the case 11 of the spectrometer 10, this Raman scattered light R1 is reflected by the reflecting mirror 15 that constitutes the optical system I, and then passes through the collimator lens 16 to be collimated into a parallel beam. Then, the direction of this parallel light beam is changed by the diffraction grating 17 and is guided to the diffraction grating 12, where it passes through and is separated into light beams of different wavelengths.

[0029] On the other hand, when the other Raman scattered light R2 that has been collected 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 reflecting mirror 15 that constitutes the optical system II, and then is collimated into a parallel beam by passing through the collimator lens 18. This parallel light beam is then guided to the diffraction grating 13, and is separated into light beams of different wavelengths by passing through the diffraction grating 13.

[0030] In the present embodiment, a spatial resolution of 5 μm diameter is achieved on the sample S, while the wavelength resolution of the spectrometer 10 is selected to be about 0.3 μm, taking into account the balance between the minimum required value and the desired energy. In this embodiment, the diameter of the entrance apertures 11a and 11b corresponding to this wavelength resolution of 0.3 μm is approximately 30 μm. Therefore, in order to cause the Raman scattered light R1 and R2 from a region of the sample S with a diameter of 5 μm to be incident on the entrance apertures 11a and 11b, respectively, the diameter of the Raman scattered light R1 and R2 emitted from the sample S can be enlarged by approximately six times and imaged at the entrance apertures 11a and 11b. For this reason, the F value of the Raman scattered lights R1 and R2 incident on the spectrometer 10 does not need to be so small (it is not necessary to cause a bright light beam to be incident).

[0031] As described above, when the Raman scattered light R1 or R2 is dispersed by the diffraction grating 12 or 13, the dispersed Raman scattered light R1 or R2 is imaged on the detection surface 20a of the CCD detector 20 by the imaging lens . The CCD detector 20 then detects the dispersed Raman scattered light R1 or R2 for each wavelength (wavenumber) and performs photoelectric conversion. The signal obtained by this CCD detector 20 is converted by a personal computer (PC) 30 into a Raman shift value (wavenumber shift value) and displayed as a Raman spectrum.

[0032] In the present embodiment, the F-number of the Raman scattered light R1, 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 light flux of the Raman scattered light R1, R2 can be as small as 1 / 2.6 ≒ 0.38, which is just under 40%. As a result, the Raman scattered light R1 and R2 can be incident in tandem on a single imaging lens 14, and the diameter of the imaging lens 14 can be kept small, making the imaging lens 14 smaller and more compact, and ultimately making the entire spectrometer 10 smaller and more compact.

[0033] An example of a Raman spectrum is shown in FIG.

[0034] FIG. 3 shows Raman spectra of cyclohexane, where (a) is a graph obtained when excited with an excitation wavelength of 532 nm, and (b) is a graph obtained when excited with an excitation wavelength of 785 nm. The horizontal axis of the Raman spectra shown in Fig. 3(a) and (b) is the Raman shift (cm -1 ), and the vertical axis is the Raman intensity.

[0035] As is clear from Figures 3(a) and (b), a Raman intensity peak appears at the same Raman shift (wavenumber) when excited with an excitation wavelength of 532 nm or 785 nm. However, the intensity ratio differs depending on the excitation wavelength, and excitation with an excitation wavelength of 785 nm shows a higher intensity peak value than excitation with an excitation wavelength of 532 nm.

[0036] As is clear from the above explanation, according to the micro-Raman spectroscopic apparatus 1 of this embodiment, the spectrometer 10 is provided with two entrance openings 11a, 11b, the same number as the two laser oscillators 2, 3, which emit excitation laser light of different wavelengths, and a plurality of two optical systems I, II (the same number as the laser oscillators 2, 3) that guide the Raman scattered light R1 or R2 incident from one of these entrance openings 11a, 11b to the diffraction grating 12 or 13. Therefore, no switching means is required for switching the entrance openings 11a, 11b or the diffraction gratings 12, 13. As a result, the structure of the spectrometer 10 is simplified, and the spectrometer 10 can be made smaller and less expensive.

[0037] In addition, a laser oscillator 2, 3 that emits excitation laser light L1, L2 having a wavelength that does not overlap with the wavelength of the fluorescence emitted from the sample S is selected, and in the spectrometer 10, the Raman scattered light R1 or R2 can be guided to the corresponding diffraction grating 12 or 13 using an optical system I or II corresponding to the selected laser oscillator 2 or 3. This prevents the wavelengths of the Raman scattered lights R1, R2 and the fluorescence from overlapping, which would otherwise cause the very weak Raman peaks in the Raman spectrum to be buried in the fluorescence spectrum, making it possible to always analyze the sample S with high precision.

[0038] Furthermore, since the switching means required in the past for physically switching between the entrance apertures 11a, 11b, the diffraction gratings 12, 13, and the CCD detector 20 are no longer necessary, the analysis results of the sample S are not adversely affected by issues such as the position reproducibility of the movable parts of the switching means, which also makes it possible to always analyze the sample S with high precision.

[0039] In the above embodiment, the present invention has been described as being applied to a micro-Raman spectroscopic device 1 having two laser oscillators 2 and 3 that respectively emit excitation laser beams L1 and L2 having different wavelengths. However, the present invention can be similarly applied to a micro-Raman spectroscopic device having three or more laser oscillators that emit excitation laser beams having different wavelengths.

[0040] In addition, in the above embodiment, the excitation laser light L1 having a wavelength of 532 nm and the laser light L2 having a wavelength of 785 nm have been described as examples of excitation laser light having different wavelengths, but any other wavelength (e.g., 488 nm, 633 nm, etc.) can be selected as the wavelength of the laser light.

[0041] Furthermore, as the 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.

[0042] In addition, the present invention is not limited to the application of the embodiments described above, and various modifications are possible within the scope of the claims and the technical ideas described in the specification and drawings. [Explanation of symbols]

[0043] 1. Raman Microscope 2,3 Laser oscillator (excitation light source) 4,5 Condenser lens 6 Objective Lenses 10 spectrometer 11 Spectroscope case 11a,11b entrance aperture 12,13,17 Diffraction grating 14 Imaging Lens 15 Reflective mirror 16,18 Collimator lens 20 CCD detector (detector) 20a CCD detector surface 30 Personal Computer (PC) I, II Optical system DM Dichroic Mirror L1, L2 Excitation laser light (excitation light) M1~M7 Reflective mirrors R1, R2 Raman scattering light S sample X optical system

Claims

1. a plurality of excitation light sources each emitting excitation light of a different wavelength; and an objective lens for collecting the excitation light and irradiating the sample with the collected light; a spectrometer that uses a diffraction grating to disperse Raman scattered light emitted from a sample by irradiation of the excitation light from a selected one of the excitation light sources through the one objective lens; a detector that detects the Raman scattered light dispersed by the spectrometer and converts it into an electric signal; A Raman microscope spectroscopic apparatus comprising: The spectrometer includes: A plurality of entrance apertures; an optical system for converting the Raman scattered light incident through the entrance aperture into a parallel beam and guiding the parallel beam to the diffraction grating; an imaging lens that images the plurality of Raman scattered light beams dispersed by the diffraction grating onto the detector; The optical systems are provided in the same number as the entrance apertures, a first imaging lens that receives a plurality of light beams dispersed by the diffraction grating and images the plurality of light beams on the detector;

2. 3. The Raman microspectroscopic apparatus according to claim 2, wherein the light beam collected by the imaging lens is imaged on a detection surface of the common detector.

3. Each of the optical systems includes a reflecting mirror that reflects the Raman scattered light incident through the entrance aperture; 3. A Raman microspectroscopic spectroscopic apparatus according to claim 1, further comprising a collimator lens for collimating the light reflected by said reflecting mirror and directing the light to each of said diffraction gratings.

4. 4. The Raman microspectroscopic apparatus according to claim 3, wherein the reflecting mirror is shared by a plurality of the optical systems.

5. 5. The micro-Raman spectroscopic apparatus according to claim 2, further comprising, as the excitation light source, a laser oscillator for emitting excitation laser light having a wavelength of 532 nm and a laser oscillator for emitting excitation laser light having a wavelength of 785 nm.

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