Raman spectrometer and method for measuring Raman spectrum

Abstract

To provide a spectroscope device capable of effectively removing / reducing an influence of fluorescent light from spectrum data.SOLUTION: A Raman spectroscope device comprises means 11 for setting a region irradiated with excitation light on a sample, means 16 for setting a taking-in region for light on the sample, a spectroscope 17 for spectral detection on light from the taking-in region, and processing means 30 for spectrum data. The taking-in region setting means 16 sets the taking-in region so that spatial resolution on the sample is high in primary measurement and the spatial resolution on the sample is low in base measurement. The data processing means 30 includes a coefficient calculation part 35 which calculates a base line coefficient on the basis of variation in intensity of a fluorescent component of spectrum data on the base measurement corresponding to differences of measurement conditions, and a difference spectrum calculation part 34 which calculates a difference spectrum between the result obtained by multiplying spectrum data of the base measurement by a base line coefficient and spectrum data of the primary measurement.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for measuring a Raman spectrum, and more particularly to a method for correcting the baseline of measured spectrum data. [Background technology]

[0002] When a substance is excited by light of a specific wavelength, it scatters light of several wavelengths different from the wavelength of the excitation light (Raman scattered light). The difference between the wavenumber of the excitation light and the wavenumber of the Raman peak is called the Raman shift, and is determined by the vibration and rotation of molecules in the substance. Therefore, a Raman spectrum, with the Raman shift on the horizontal axis and the Raman peak intensity on the vertical axis, can be used to identify the substance.

[0003] On the other hand, Raman scattered light is 10 times stronger than Rayleigh scattered light with the same wavelength as the excitation light. -6 The light is about twice as weak as the light emitted from the substance, and the spectrum measurement of the Raman scattered light is significantly affected by the fluorescence from the substance.

[0004] The effects of fluorescence in conventional Raman spectrum measurements can be explained in two ways. First, because the fluorescent components span a relatively wide frequency range, the spectral baseline rises, obscuring weak Raman peaks. Second, the fluorescent components form interference fringes in optical elements such as films inside the analyzer, distorting the baseline into a wavy shape. These baseline rises and distortions are thought to be the cause of a lower success rate in spectral searches for qualitative analysis of samples.

[0005] Therefore, in Raman spectroscopy, it is particularly important to remove or reduce the influence of fluorescence, i.e., to properly correct the baseline.

[0006] A common method for avoiding fluorescence is to change the wavelength of the excitation light to a longer wavelength. For example, with 532 nm excitation light, depending on the substance, a large amount of fluorescence may be generated, obscuring the Raman peak. However, by changing the wavelength of this excitation light to, for example, 1064 nm, it is possible to suppress the generation of fluorescence components, and in some cases, the Raman peak may be visible.

[0007] There have also been various proposals for methods of correcting the baseline of a measured spectrum. For example, Patent Document 1 discloses a method in which shape data such as a semicircle or semiellipse is set for the measured spectrum data, and the baseline is estimated by software, thereby correcting the baseline to be flat. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 4966337 Summary of the Invention [Problem to be solved by the invention]

[0009] However, even with conventional methods, there are cases where the baseline rise caused by fluorescent components cannot be fully eliminated, and there are also cases where the baseline distortion caused by fluorescent interference fringes cannot be fully eliminated.The object of the present invention is to provide a Raman spectrometer and a method for measuring Raman spectra that can effectively eliminate or reduce the influence of fluorescence on the measured spectrum using a novel method that differs from conventional methods. [Means for solving the problem]

[0010] That is, the Raman spectroscopic device according to the present invention: A Raman spectroscopic device comprising: a light source for excitation light of a single wavelength; a stage for placing a sample; an irradiation area setting means for setting an irradiation area of ​​the excitation light on the sample; a take-up area setting means for setting a take-up area of ​​the light on the sample; a spectroscope for spectrally detecting light from the take-up area; and a data processing means for processing spectral data based on the detection value from the spectroscope, At least one of the light source, the irradiation area setting means, and the capture area setting means sets a measurement condition for the measurement in which the ratio of the Raman peak to the fluorescent component in the spectrum data is Compared to the base measurement setting at least one of the capture area, the illumination area, and the excitation light intensity to be large; and The aforementioned As a measurement condition for the base measurement, the ratio of the Raman peak to the fluorescent component in the spectrum data is Compared with the above measurement configured to set at least one of the capture area, the illumination area, and the excitation light intensity to be small; The data processing means a coefficient calculation unit that calculates a rate of change in intensity of a fluorescent component of the spectral data of the base measurement according to differences in measurement conditions, and calculates a baseline coefficient based on the rate of change in intensity; The method is characterized by including a difference spectrum calculation unit that calculates a difference spectrum between the spectrum data of the base measurement to which the baseline coefficient has been added and the spectrum data of the main measurement.

[0011] The capture area setting means may be configured to narrow the area of ​​the capture area as a measurement condition for the main measurement, and to widen the area of ​​the capture area as a measurement condition for the base measurement.

[0012] The capture area setting means may have an opening for limiting the light captured by the spectroscope, and may be configured to change the shape or size of the opening.

[0013] The irradiation area setting means may be configured to set the offset amount between the center of the irradiation area and the center of the capture area to zero or small as a measurement condition for the main measurement, and to set the offset amount between the center of the irradiation area and the center of the capture area to large as a measurement condition for the base measurement.

[0014] The light source may be configured so that the measurement condition for the main measurement is a low intensity of excitation light, and the measurement condition for the base measurement is a high intensity of excitation light.

[0015] Furthermore, an exposure time setting means may be provided for setting an exposure time of the sample to excitation light, and the exposure time setting means may be configured to set the exposure time to be long as a measurement condition for the main measurement, and to set the exposure time to be short as a measurement condition for the base measurement.

[0016] Further, the method for measuring a Raman spectrum according to the present invention comprises: A method for measuring a Raman spectrum by capturing light from a sample excited with excitation light of a single wavelength into a spectrometer, comprising: The method includes an irradiation area setting step of setting an irradiation area of ​​the excitation light on the sample, a capture area setting step of setting a light capture area on the sample, an irradiation step of irradiating the excitation light, a step of acquiring spectral data based on detection values ​​from a spectroscope, and a data processing step of processing the spectral data, In at least one of the irradiation area setting step, the capture area setting step, and the irradiation step, The measurement conditions for this measurement are as follows: the ratio of the Raman peak to the fluorescent component in the spectrum data is Compared to the base measurement setting at least one of the capture area, the illumination area, and the excitation light intensity to be large; and The aforementioned As a measurement condition for the base measurement, the ratio of the Raman peak to the fluorescent component in the spectrum data is Compared with the above measurement and setting at least one of the capture area, the irradiation area, and the excitation light intensity so that the area is small; The data processing procedure includes: a coefficient calculation procedure for calculating a rate of change in intensity of the fluorescent component of the spectral data of the base measurement according to differences in measurement conditions, and calculating a baseline coefficient based on the rate of change in intensity; and a difference spectrum calculation step of calculating a difference spectrum between the spectrum data of the base measurement to which the baseline coefficient is applied and the spectrum data of the main measurement, It is characterized by:

[0017] The coefficient calculation step may calculate the baseline coefficient based on spectrum data obtained by a plurality of base measurements under different measurement conditions.

[0018] The data processing step may include, after the difference spectrum calculation step, a baseline flattening step for flattening the baseline of the difference spectrum.

[0019] The measurement method of the present invention may be carried out after measuring the Raman spectrum with the wavelength of the excitation light changed to a longer wavelength side. [Effects of the Invention]

[0020] As described above, by performing Raman spectrum measurements, it is possible to obtain Raman spectrum data in which baseline rise (the influence of fluorescent components over a wide wavenumber range) and baseline distortion (the influence of interference fringe components due to fluorescence) have been corrected, thereby improving the accuracy rate of material analysis using such Raman spectrum data. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a schematic diagram illustrating the configuration of a Raman spectroscopic device according to an embodiment. [Figure 2] FIG. 10 is a diagram showing the difference in capture area between two types of openings in the device. [Figure 3] FIG. 2 is a diagram showing a procedure for measuring Raman spectra using the device. [Figure 4]FIG. 1 is a Raman spectrum actually measured under the first measurement condition. [Figure 5] FIG. 10 is a diagram of a Raman spectrum actually measured under the second measurement conditions. [Figure 6] FIG. 10 is a diagram of a Raman spectrum actually measured under the third measurement condition. [Figure 7] FIG. 10 is an explanatory diagram of verification using spatial offsets. [Figure 8] FIG. 10 is a diagram showing the results of calculating the distribution of the fluorescence area and the Raman area in the verification. [Figure 9] 1A and 1B show spectral data measured using a rectangular slit and a pinhole slit, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0022] Preferred embodiments of the present invention will be described below with reference to the drawings. Fig. 1 is a schematic diagram of a microscopic laser Raman spectrophotometer (hereinafter referred to as a Raman spectrophotometer), which is one embodiment of the Raman spectrophotometer of the present invention. The Raman spectrophotometer is a device for spectrally detecting Raman scattered light from a sample S when irradiated with excitation light (laser light) and acquiring a Raman spectrum. Measurement targets in this embodiment are a wide range, including solid samples (such as PEEK resin), powder samples (such as cellulose powder), and liquid samples (such as colored liquids such as whiskey).

[0023] The basic structure of a Raman spectrometer is shown in Figure 1. That is, the Raman spectrometer comprises a confocal optical system consisting of a movable stage 11 for the sample, an objective lens 12, a beam splitter (BS) 13 (or a dichroic mirror DM) that reflects excitation light toward the sample and transmits light from the sample S, a rejection filter 14 for cutting Rayleigh light, an imaging lens 15, an aperture switching device 16, a spectrometer 17, and a CCD detector 18.

[0024] The Raman spectrometer also includes, as excitation optical systems, a laser wavelength switching device 21, a beam spot position changing device 22, and a laser control device 23. The laser control device 23 includes an exposure time setting unit 24 and a laser intensity setting unit 25.

[0025] The Raman spectrometer also includes a spectral data processing unit 30 and a storage device 40. The spectral data processing unit 30 is configured as an arithmetic processing unit such as a computer, and has, as functional blocks, a baseline correction unit 31, a baseline flattening processing unit 32, and a noise removal unit 33. The baseline correction unit 31 includes a difference spectrum calculation unit 34 and a baseline coefficient calculation unit 35, which are characteristic of the present invention.

[0026] The storage device 40 stores a data processing program for causing the spectral data processing device 30 to realize these functional blocks, and the spectral data processing device 30 executes this data processing program as appropriate to perform these functions.

[0027] <Details of the excitation optical system> The laser wavelength switching device 21 is equipped with a plurality of switchable light sources 26, 27 of different wavelengths. The device is configured so that excitation light from the light sources 26, 27 of the selected wavelength is output toward the beam spot position changing device 22. The beam spot position changing device 22 can be configured, for example, with a pair of alignment mirrors. The pair of alignment mirrors can adjust the optical axis of the excitation light from the light sources 26, 27, and can move the position of the beam spot on the sample without changing the positional relationship between the sample S and the objective lens 12.

[0028] The laser control device 23 controls the selected light sources 26 and 27. The exposure time setting unit 24 turns on / off the output of the light sources 26 and 27 according to the set exposure time. Alternatively, it may open and close shutters provided in the output windows of the light sources 26 and 27. The laser intensity setting unit 25 adjusts the output intensity of the light sources 26 and 27 according to the intensity conditions of the excitation light. Alternatively, it is configured to change the intensity of the excitation light by switching on / off a neutral density filter provided in the optical path of the excitation light.

[0029] The excitation light passes through an objective lens 12 on the sample to form a minute beam spot, which is the target area for spectrum measurement.

[0030] <Details of the confocal optical system> The movable stage 11 is an XY two-axis stage having a mounting surface parallel to the XY plane, and can position the beam spot on the mounted sample at the focusing position of the objective lens 12.

[0031] The objective lens 12 is an example of a focusing optical element of the present invention, such as a Cassegrain mirror. The objective lens 12 serves to form a beam spot of excitation light on the sample and to focus light from the beam spot and its surrounding area. Rayleigh scattered light contained in the light from the sample S is removed by a rejection filter 14. The imaging lens 15 focuses the focused light on an aperture, such as a rectangular slit plate 16A or a pinhole slit plate 16B, mounted in the aperture switching device 16 downstream, to form an intermediate image of the beam spot. Note that while FIG. 1 illustrates an example of a reflective optical system, a measurement device using a transmissive optical system, such as a Cassegrain mirror, may also be configured.

[0032] The aperture switching device 16 is configured to be able to switch between at least two types of apertures. As an example, FIG. 1 shows a configuration in which a rectangular slit plate 16A with an elongated aperture and a pinhole slit plate 16B with a circular aperture are held in a common sliding frame so that the selected aperture can be installed online. The aperture plate can be formed with apertures of various shapes other than elongated or circular. Furthermore, multiple apertures of different sizes may be formed for each shape.

[0033] A typical dispersive spectrometer using a diffraction grating is used as the spectrometer 17. Diffracted light from the diffraction grating is imaged on a downstream CCD detector 18, and spectral values ​​for each band are detected by a group of two-dimensionally arranged light-receiving elements in the CCD detector 18 and output to a spectral data processor 30.

[0034] Here, the difference in capture area between the two types of apertures (rectangular slit plate 16A and pinhole slit plate 16B) is shown using Figure 2. Here, the beam spot is shown as a black circle on the sample, and its periphery is shown as a white circle that is larger than the black circle.

[0035] The light that is taken into the spectroscope 17 is limited by the openings of the rectangular slit plate 16A and the pinhole slit plate 16B. In other words, the light taking-in area on the sample is determined by the shape and size of this opening.

[0036] 2, the slit width of rectangular slit plate 16A is approximately the same as the size of the image of the beam spot, but in the case of rectangular slit plate 16A, not only the light from the beam spot but also much of the light from its periphery is taken into spectroscope 17. On the other hand, in the case of pinhole slit plate 16B, especially when its opening is approximately the same as the image of the beam spot, only the light from the beam spot is efficiently taken into spectroscope 17 due to the confocal effect, allowing measurement with higher spatial resolution than rectangular slit plate 16A.

[0037] This embodiment is characterized in that at least two types of spectrum measurements (base measurement and main measurement) are performed under different measurement conditions using the above-described Raman spectroscopic device. Table 1 below lists examples of measurement conditions.

[0038] [Table 1]

[0039] Under condition 1, the base measurement is performed with low spatial resolution, and the main measurement is performed with high spatial resolution. For example, a wide capture area and a narrow capture area are set for the same measurement position on the sample. Conditions 1A to 1C in Table 1 are used when a rectangular slit or pinhole slit opening is used to set the light capture area on the sample.

[0040] Condition 1A uses, for example, a rectangular slit with a slit width of 200 μm and a pinhole slit with a diameter of 100 μm. When using a rectangular slit, the beam spot should occupy a smaller proportion of the capture area on the sample. Conversely, when using a pinhole slit, the proportion should be larger than that of the rectangular slit. Alternatively, the diameter of the pinhole slit should be equal to or smaller than the slit width of the rectangular slit.

[0041] In conditions 1B and 1C, the size of the rectangular slit or pinhole slit is changed. For example, for the same measurement position on the sample, a slit with a larger opening is used in the base measurement and a slit with a smaller opening is used in the main measurement.

[0042] When a pinhole slit is used in this measurement, the light capture area of ​​the pinhole slit may be aligned with the beam spot, or the light capture area may be completely within the beam spot. In this embodiment, these conditions are set by the operation of the aperture switching device 16.

[0043] In condition 2, the spatial offset is changed by changing the distance between the beam spot and the light capture area. In the base measurement, an offset state is established and the above distance is increased. In the main measurement, a coincidence state is established, meaning the above distance is set to zero, or the distance is reduced even in the offset state. Note that the beam spot may be fixed and the position of the capture area may be moved. Alternatively, the capture area may be fixed and the position of the beam spot may be moved by operating the beam spot position change device 22 in Figure 1.

[0044] Condition 3 is to increase the intensity of the excitation light in the base measurement and decrease the intensity of the excitation light in the main measurement. This condition setting is performed by the operation of the laser intensity setting unit 25 in FIG.

[0045] A common feature of each condition is that the exposure time of the excitation light in this measurement should be set longer than that in the base measurement using the exposure time setting unit 24 in FIG. 1 so that a Raman peak of an appropriate height is obtained.

[0046] <Method for measuring Raman spectra> Figure 3 shows the procedure for measuring Raman spectra by changing the spatial resolution (for example, condition 1B in Table 1). Figures 4 to 6 show the results of measuring Raman spectra of a powdered cellulose sample under three specific measurement conditions.

[0047] The measurement method according to this embodiment may be started immediately after optimizing the wavelength of the excitation light, which is a conventional method for avoiding fluorescence. The wavelength of the excitation light is switched using the laser wavelength switching device 21 shown in FIG. 1. Even if the wavelength of the excitation light is changed to a longer wavelength and a Raman spectrum is measured, it may not be possible to sufficiently eliminate the influence of fluorescence. However, by performing the measurement method according to this embodiment, it may be possible to eliminate or reduce the influence of such fluorescence.

[0048] First, a measurement position on a sample placed on the movable stage 11 is specified (step S10). Next, the movable stage 11 is operated to align the beam spot position with the measurement position, thereby setting the beam spot (step S12). Next, the aperture switching device 16, which is equipped with a wide rectangular slit plate and a narrow rectangular slit plate, is operated to set the wide rectangular slit on the optical path (step S14).

[0049] A base measurement is then performed (step S16). In the base measurement, first, excitation light from light sources 26 and 27 is irradiated onto the beam spot position, and only the light from the sample that passes through the opening of the wide rectangular slit plate is captured by spectrometer 17. Spectrometer 17 detects the spectral values ​​for each band, and the detected values ​​are read into spectrum data processor 30. Note that the exposure time in the base measurement is short.

[0050] After the base measurement is completed, the aperture switching device 16 is operated to set a narrow rectangular slit plate on the optical path (step S18), and the main measurement is performed in the same manner as the base measurement (step S20). In the main measurement, the exposure time is made longer than in the base measurement.

[0051] After the spectrum data processing device 30 has read the detected values ​​of the base measurement and the main measurement through the above procedure, the spectrum data processing device 30 executes data processing.

[0052] The spectral data SP10 and SP12 from the base measurement and the actual measurement are shown in the upper left of Figure 4. In the base measurement, a rectangular slit with a width of 200 μm was used, with an exposure time of 1 second. In the actual measurement, a rectangular slit with a width of 100 μm was used, with an exposure time of 10 seconds.

[0053] In the data processing (steps S22 to S40) of Fig. 3, first, the baseline coefficient A is calculated (step S22) by the function of the baseline coefficient calculation unit 35. Here, a wavenumber range in which no Raman scattered light occurs in either of the two types of measurement spectra is selected, and the intensity change rate of the fluorescent component occurring in that wavenumber range (for example, the spectral area corresponding to the fluorescent component) is calculated. Then, the baseline coefficient A is calculated based on this intensity change rate.

[0054] Next, the baseline coefficient A is used to calculate the difference spectrum between the spectrum data of the main measurement and the spectrum data of the base measurement (step S24). Here, the spectrum data of the base measurement is multiplied by the baseline coefficient A to obtain the difference from the spectrum data of the main measurement. The above processing is the baseline correction according to this embodiment.

[0055] The processing up to this point removes the effects of fluorescence (such as interference fringes), which were difficult to remove using conventional methods, but some fluorescent components may remain. The remaining fluorescent components at this stage can be easily removed, for example, by conventional software-based baseline flattening processing as described in Patent Document 1 (step S30).

[0056] In the measurement example in Figure 4, the two spectral data SP10 and SP12 are in the 2500–2000 cm -1 No Raman peaks appear in any of the wavenumber ranges. The fluorescence areas A10 and A12 in this wavenumber range were calculated, and a baseline coefficient A was calculated so that the difference between the two fluorescence areas would be zero (step S22). The difference spectrum SP14 using this coefficient A is shown in the upper right corner of Figure 4 (step S24). This difference spectrum SP14 was calculated after baseline flattening processing using existing software (step S30). For comparison, the upper right corner of Figure 4 also shows comparison data Ref14, which was obtained by performing only baseline flattening on the spectral data from this measurement. It can be seen that the difference spectrum SP14 has less noise and interference fringes.

[0057] 3, a final step of the data processing shown in Fig. 3 may be to perform existing software-based noise removal processing (step S40) as needed. For example, by performing an inverse Fourier transform on the spectral data and applying a low-pass filter to the resulting power spectrum, it is possible to remove, as noise, spectral fluctuations of high-frequency components occurring on the baseline of the spectral data.

[0058] In the measurement example of Figure 4, a noise removal process (step S40) was performed on the difference spectrum SP14 of this embodiment after baseline flattening. The lower part of Figure 4 shows the difference spectrum SP16, with an improved S / N ratio due to noise removal. Furthermore, both existing noise removal and interference fringe removal processes were performed on the comparison data Ref14. The results are also shown as comparison data Ref16. It can be seen that while the comparison data Ref16 contains interference fringes (wavy deformation of the baseline) that are not removed by existing data processing, the difference spectrum SP16 of this embodiment shows that such interference fringes have been significantly reduced.

[0059] Figure 5 shows the spectral data SP22 from this measurement using a 50-µm-wide rectangular slit. The data from the base measurement is the same as that shown in Figure 4. Comparing the difference spectra SP24 and SP26 calculated from these spectral data with the comparison data Ref24 and Ref26, it is clear that the difference spectrum SP24 according to this embodiment has less noise and interference fringes than the comparison data Ref24, and that the interference fringes in the difference spectrum SP26 after noise removal are significantly reduced compared to the comparison data Ref26.

[0060] Furthermore, Fig. 6 shows difference spectra SP34 and SP36 based on the spectrum data SP32 obtained in this measurement using a pinhole slit with a diameter of 100 µm, along with comparison data Ref34 and Ref36. As in Fig. 5, it can be seen that the difference spectrum obtained in this embodiment is superior to the comparison data Ref34 and Ref36 in terms of noise and interference fringes.

[0061] In the procedure of FIG. 3, the case where the base measurement (step S16) is performed only once has been described. However, the base measurement may be performed multiple times by setting different measurement conditions, and the baseline coefficient A may be calculated based on multiple pieces of spectral data (step S22).

[0062] <Verification by spatial offset> The first feature of the present invention is that the Raman spectrum measurement method of the present invention performs two types of spectrum measurements (main measurement and base measurement) under different measurement conditions on the same sample. In the main measurement, the measurement conditions are set so that "the ratio of the Raman peak to the fluorescent component in the spectral data is relatively large," and in the base measurement, the measurement conditions are set so that "the ratio is relatively small."

[0063] As specific examples, measurement condition 1 is changing the area of ​​the light capture area (measurement field of view) on the sample, measurement condition 2 is changing the positional relationship between the excitation light irradiation area (beam spot) on the sample and the capture area, and measurement condition 3 is changing the intensity of the excitation light.

[0064] The second feature is that the difference between the spectral data obtained under two different measurement conditions is taken to cancel out the fluorescent components contained in the spectral data of this measurement. However, since this is spectral data from the same sample, some may wonder whether it would be difficult to cancel out just the fluorescent components even if the measurement conditions were changed (wouldn't the desired Raman peak also be canceled out?).

[0065] To address this issue, the inventors conducted a "spatial offset verification" as shown in Figure 7. Here, PEEK resin was used as the sample. An objective lens 12 with a magnification of 100 was used so that the excitation light with a wavelength of 532 nm would form a beam spot with a diameter of approximately 1 μm on the sample. The spatial resolution on the sample was also set to be equivalent to 1 μm (capture area). The offset between the center of the beam spot on the sample and the center of the light capture area (both circular areas with a diameter of 1 μm) was gradually increased from 0 μm to 5 μm, and the spectra were measured. Differences were observed between the change in the peak height of the Raman peak and the change in the height of the fluorescent component. The offset was achieved by shifting the optical axis of the excitation light (black area) by 0 to 5 μm relative to the optical axis of the measurement light without changing the relative positions of the sample S and the objective lens 12, as shown in Figure 7.

[0066] Even with an offset of 3 μm or 5 μm, fluorescence components over a wide wavenumber range (a wide rise in the baseline) can be observed, but Raman peaks are barely discernible even with an offset of 3 μm.Raman peaks can be observed with an offset of up to 1 μm.

[0067] Therefore, as shown in Figure 8, 1600 cm -1 The change in the peak area for each offset amount and the band where no Raman peak occurs (2500–2000 cm) were measured. -1 When we investigated the change in the spectral area of ​​the fluorescent component of the beam spot, we confirmed that the fluorescent component (fluorescence area) gradually decreases from its peak at the beam spot, while the Raman peak (Raman area) rapidly decreases from its peak at the beam spot. Note that the graph showing the area for each offset amount in Figure 8 has been normalized by setting the fluorescent area and Raman area at 0 μm to 100.

[0068] The inventors then estimated that while the intensity distribution of fluorescence is a relatively gentle mountain-like distribution with its peak at the beam spot, the intensity distribution of Raman scattered light is a Gaussian distribution with its peak at the beam spot, because the Raman scattered light decreases rapidly with distance from the beam spot and becomes almost zero after a short distance.

[0069] Since Raman scattered light is inelastically scattered light and is proportional to the intensity of the excitation light, the distribution of Raman light can be said to be a Gaussian distribution, just like excitation light (laser light). In contrast, fluorescence is light emitted in association with the absorption of light by a substance and tends to saturate above a certain level of excitation light. Therefore, the distribution of fluorescence is thought to be not a Gaussian distribution, but rather a mountain-shaped distribution. In other words, when the same excitation light is applied to a sample, the volume of the sample from which Raman scattered light is detected is smaller than the volume of the sample from which fluorescence is detected.

[0070] The inventors discovered that, due to these differences in the distribution of fluorescence and Raman light on the sample, by performing two different spectral measurements on the same sample under different conditions and taking the difference between the two spectra, it is possible to obtain a good Raman spectrum in which the effects of fluorescence are canceled out and the desired Raman peak remains.

[0071] Several measurement conditions can be set. The common feature is that some measurement conditions are set so that the ratio of the Raman peak to the fluorescent component in the spectrum data is relatively large, and other measurement conditions are set so that the ratio is relatively small.

[0072] As shown in the light distribution in Figure 8, the area near the beam spot has a relatively high ratio of Raman light to fluorescence, which can be called the "Raman light-rich region."Furthermore, the area away from the beam spot has a ratio of Raman light to fluorescence that is almost zero, which can be called the "fluorescence-rich region."

[0073] For example, under measurement condition 1, in this measurement, a relatively narrow region that mainly includes the "Raman light-rich region" is set as the light capture region. Then, in the base measurement, a relatively wide region that mainly includes the "fluorescence-rich region" is set as the light capture region. In other words, by changing the area of ​​the light capture region, the spatial resolution can be varied.

[0074] 7, for example, under measurement condition 2, the "Raman light-rich region" is set as the light capture region in this measurement, and the "fluorescence-rich region" is set as the light capture region in the base measurement. In other words, the positional relationship between the beam spot and the capture region is changed.

[0075] Also, for example, measurement condition 3 involves changing the intensity of the excitation light. In this measurement, the light capture region is set to a "Raman light-rich region" to obtain spectral data in which the ratio of Raman peaks to fluorescent components is relatively large. Then, in the base measurement, the spectrum of the same capture region is measured, but the intensity of the excitation light is increased to saturate the fluorescence, obtaining spectral data in which the ratio of Raman peaks to fluorescent components is relatively small.

[0076] In addition, in the spectral data measured under multiple offset conditions as shown in Figure 7, the fluorescent components have similar shapes (similar shapes along the vertical axis of the spectral data). Furthermore, in the spectral data measured under different spatial resolution conditions (rectangular slit, pinhole slit), as shown in Figure 9, the fluorescent components also have similar shapes. Therefore, in calculating the difference spectrum, if an appropriate baseline coefficient is applied to the spectral data of the base measurement and the difference with the spectral data of the main measurement is taken, the fluorescent components can be completely canceled out. In this invention, the rate of change in intensity of the fluorescent components accompanying changes in measurement conditions is calculated from the fluorescent area of ​​each component, and a baseline coefficient is calculated based on this rate of change in intensity.

[0077] For these reasons, the spectrum measurement method of this embodiment makes it possible to acquire Raman spectrum data in which the rise and deformation of the baseline due to the influence of fluorescence has been corrected. [Explanation of symbols]

[0078] 11 Movable stage (irradiation area setting means), 12 Objective lens, 13 Beam splitter, 14 Rejection filter, 15 Imaging lens, 16 Aperture switching device (capture area setting means), 16A Rectangular slit plate, 16B Pinhole slit plate, 17 Spectrometer, 18 CCD detector, 21 Laser wavelength switching device, 22 Beam spot position changing device (irradiation area setting means), 23 Laser control device, 24 Exposure time setting unit, 25 Laser intensity setting unit, 26, 27 Light source, 30 Spectral data processing device, 31 Baseline correction unit, 32 Baseline flattening processing unit, 33 Noise removal unit, 34 Difference spectrum calculation unit, 35 Baseline coefficient calculation unit, 40 Storage device.

Claims

1. A Raman spectroscopic device comprising: a light source for excitation light of a single wavelength; a stage for placing a sample; an irradiation area setting means for setting an irradiation area of ​​the excitation light on the sample; a take-up area setting means for setting a take-up area of ​​the light on the sample; a spectroscope for spectrally detecting light from the take-up area; and a data processing means for processing spectral data based on the detection value from the spectroscope, At least one of the light source, the irradiation area setting means, and the capture area setting means sets, as a measurement condition for the main measurement, at least one of the capture area, the irradiation area, and the excitation light intensity so that the ratio of the Raman peak to the fluorescent component in the spectral data is larger than that in the base measurement; and as a measurement condition for the base measurement, at least one of the capture area, the irradiation area, and the excitation light intensity is set so that a ratio of a Raman peak to a fluorescent component in the spectral data is smaller than that in the main measurement; The data processing means a coefficient calculation unit that calculates a rate of change in intensity of a fluorescent component of the spectral data of the base measurement according to differences in measurement conditions, and calculates a baseline coefficient based on the rate of change in intensity; a difference spectrum calculation unit that calculates a difference spectrum between the spectrum data of the base measurement to which the baseline coefficient has been added and the spectrum data of the main measurement, A Raman spectroscopic device characterized by:

2. 2. The Raman spectroscopic apparatus according to claim 1, wherein the capture area setting means is configured to narrow the area of ​​the capture area as a measurement condition for the main measurement and to widen the area of ​​the capture area as a measurement condition for the base measurement.

3. 3. The Raman spectroscopic apparatus according to claim 2, wherein the capture area setting means has an opening for limiting the light captured by the spectrometer, and is configured to change the shape or size of the opening.

4. 4. The Raman spectroscopic apparatus according to claim 1, wherein the irradiation area setting means is configured to set an offset amount between the center of the irradiation area and the center of the capture area to zero or small as a measurement condition for the main measurement, and to set an offset amount between the center of the irradiation area and the center of the capture area to large as a measurement condition for the base measurement.

5. 5. The Raman spectroscopic apparatus according to claim 1, wherein the light source is configured to reduce the intensity of the excitation light as a measurement condition for the main measurement and to increase the intensity of the excitation light as a measurement condition for the base measurement.

6. 6. The Raman spectroscopic apparatus according to claim 1, further comprising an exposure time setting means for setting an exposure time of the sample to the excitation light, wherein the exposure time setting means is configured to set the exposure time to be long as a measurement condition for the main measurement and to set the exposure time to be short as a measurement condition for the base measurement.

7. A method for measuring a Raman spectrum by capturing light from a sample excited with excitation light of a single wavelength into a spectrometer, comprising: The method includes an irradiation area setting step of setting an irradiation area of ​​the excitation light on the sample, a capture area setting step of setting a light capture area on the sample, an irradiation step of irradiating the excitation light, a step of acquiring spectral data based on detection values ​​from a spectroscope, and a data processing step of processing the spectral data, In at least one of the irradiation area setting step, the capture area setting step, and the irradiation step, As a measurement condition for this measurement, at least one of the capture area, the irradiation area, and the excitation light intensity is set so that the ratio of the Raman peak to the fluorescent component in the spectrum data is larger than that in the base measurement; and As the measurement conditions for the base measurement, at least one of the capture area, the irradiation area, and the excitation light intensity is set so that the ratio of the Raman peak to the fluorescent component in the spectral data is smaller than that in the main measurement; The data processing procedure includes: a coefficient calculation procedure for calculating a rate of change in intensity of the fluorescent component of the spectral data of the base measurement according to differences in measurement conditions, and calculating a baseline coefficient based on the rate of change in intensity; and a difference spectrum calculation step of calculating a difference spectrum between the spectrum data of the base measurement to which the baseline coefficient is applied and the spectrum data of the main measurement, A method for measuring a Raman spectrum.

8. 8. The method for measuring a Raman spectrum according to claim 7, wherein the coefficient calculation step calculates the baseline coefficient based on spectral data obtained by a plurality of base measurements under different measurement conditions.

9. 9. The method according to claim 7, wherein the data processing step comprises: A method for measuring a Raman spectrum, comprising, after the difference spectrum calculation step, a baseline flattening step of flattening the baseline of the difference spectrum.

10. A method for measuring a Raman spectrum according to any one of claims 7 to 9, characterized in that the method is carried out after measuring the Raman spectrum with the wavelength of the excitation light changed to a longer wavelength side.

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