JP2023032387A - Raman spectroscope device and method for measuring raman spectrum - Google Patents

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

The present invention relates to a method of measuring Raman spectra, and more particularly to a method of correcting the baseline of measured spectral data.

When a substance is excited by light of a specific wavelength, it scatters light of some wavelength 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 Raman shift, which is determined according to the vibration, rotation, etc. of molecules in the substance. Therefore, a Raman spectrum in which the horizontal axis represents the Raman shift and the vertical axis represents the Raman peak intensity can be used to identify the substance.

On the other hand, the Raman scattered light is about 10 ⁻⁶ times weaker than the Rayleigh scattered light of the same wavelength as the excitation light, and the spectrum measurement of the Raman scattered light is greatly affected by the fluorescence from the substance. .

The influence of fluorescence in conventional Raman spectrum measurement is divided into the following two and explained. First, since the fluorescence component covers a relatively broad band, the baseline of the spectrum rises and the weak Raman peak is buried. The second problem is that the fluorescence component forms interference fringes by an optical element such as a film in the analyzer, which distorts the baseline into a wavy shape. These baseline elevations and deformations are considered to be the cause of a lower correct answer rate in spectral searches for qualitative analysis of samples.

Therefore, in Raman spectrum measurement, removal/mitigation of the influence of fluorescence, that is, appropriate correction of the baseline, is particularly important.

As a general method for avoiding fluorescence, there is a method of changing the wavelength of excitation light to the longer wavelength side. For example, in the case of 532 nm excitation light, depending on the substance, a large fluorescent component may be generated and the Raman peak may be buried. 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 it is possible to confirm the Raman peak.

Various proposals have also been made for methods of correcting the baseline of the measured spectrum. is software-estimated, the baseline is corrected to be flat.

Japanese Patent No. 4966337

However, even with the conventional method, there are cases where the elevation of the baseline due to the fluorescence component cannot be sufficiently removed, and the deformation of the baseline due to the interference fringes of the fluorescence cannot be sufficiently removed. An object of the present invention is to provide a Raman spectrometer and a Raman spectrum measurement method that can effectively remove or reduce the influence of fluorescence on a measurement spectrum by a novel method different from conventional methods. That's what it is.

That is, the Raman spectroscopic device according to the present invention is
A light source for excitation light of a single wavelength, a stage on which a sample is placed, an irradiation area setting means for setting an irradiation area of the excitation light on the sample, and a capture area setting for setting a light capturing area on the sample. means, a spectroscope for spectrally detecting light from the capture region, and a data processing means for processing spectral data based on detection values from the spectroscope, wherein
At least one of the light source, the irradiation region setting means, and the capture region setting means sets the capture region so that the ratio of the Raman peak to the fluorescence component in the spectral data is relatively large as the measurement conditions for the main measurement. , the illumination region, and excitation light intensity, and as measurement conditions for base measurement, the acquisition region so that the ratio of Raman peaks to fluorescence components in the spectral data is relatively small; configured to set at least one of the irradiation area and excitation light intensity;
The data processing means are
a coefficient calculation unit that calculates an intensity change rate of a fluorescence component of base measurement spectral data according to a difference in measurement conditions and calculates a baseline coefficient based on the intensity change rate; It is characterized by including a difference spectrum calculation unit for calculating a difference spectrum between the spectrum data to which the coefficient is assigned and the spectrum data of the main measurement.

The capture region setting means may be configured to narrow the area of the capture region as the measurement condition of the main measurement and widen the area of the capture region as the measurement condition of the base measurement.

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

The irradiation area setting means sets an offset amount between the center of the irradiation area and the center of the capture area to be zero or small as the measurement condition of the main measurement, and sets the offset amount between the center of the irradiation area and the center of the irradiation area as the measurement condition of the base measurement. It may be configured to increase the amount of offset from the center of the capturing area.

The light source may be configured to reduce the intensity of the excitation light as the measurement condition for the main measurement and to increase the intensity of the excitation light as the measurement condition for the base measurement.

Furthermore, the exposure time setting means for setting the exposure time of the excitation light to the sample is provided, and the exposure time setting means sets the exposure time to be long as the measurement condition of the main measurement, and sets the exposure time to be long as the measurement condition of the base measurement. , the exposure time may be set short.

Further, the method for measuring a Raman spectrum according to the present invention is
A method for measuring a Raman spectrum by capturing light from a sample excited with a single wavelength excitation light into a spectroscope,
Irradiation area setting procedure for setting the excitation light irradiation area on the sample, capture area setting procedure for setting the light capture area on the sample, irradiation procedure for irradiating the excitation light, and detection values from the spectrometer and a data processing procedure for processing said spectral data,
In at least one of the irradiation area setting procedure, the capturing area setting procedure and the irradiation procedure,
As measurement conditions for this measurement, at least one of the capture region, the irradiation region, and the excitation light intensity is set so that the ratio of the Raman peak to the fluorescence component in the spectral data is relatively large; setting at least one of the capture region, the irradiation region, and the excitation light intensity as measurement conditions for the base measurement so that the ratio of Raman peaks to fluorescence components in the spectral data is relatively small; Do two ways,
Said data processing procedure comprises:
A coefficient calculation procedure for calculating the intensity change rate of the fluorescence component of the base measurement spectrum data according to the difference in measurement conditions, and calculating a baseline coefficient based on the intensity change rate; including a difference spectrum calculation procedure for calculating a difference spectrum between the spectrum data to which the coefficient is assigned and the spectrum data of the main measurement;
It is characterized by

In the coefficient calculation procedure, the baseline coefficient may be calculated based on spectrum data obtained by a plurality of base measurements under different measurement conditions.

The data processing procedure may include, after the difference spectrum calculation procedure, a baseline flattening procedure for flattening the baseline of the difference spectrum.

The measurement method of the present invention may be performed after changing the wavelength of the excitation light to the longer wavelength side and measuring the Raman spectrum.

If the Raman spectrum measurement is performed as shown above, the Raman spectrum data corrected for baseline elevation (effect of fluorescence component over a wide wavenumber range) and baseline deformation (effect of interference fringe component due to fluorescence) can be obtained. can be obtained, and the correct answer rate of substance analysis using such Raman spectrum data can be improved.

1 is a schematic configuration diagram of a Raman spectrometer according to one embodiment; FIG. It is a figure which shows the difference of the taking-in area|region by two types of opening parts in the said apparatus. It is a figure which shows the procedure of Raman spectrum measurement using the said apparatus. It is a Raman spectrum diagram actually measured under the first measurement condition. FIG. 10 is a diagram of Raman spectra actually measured under the second measurement conditions; It is a figure of the Raman spectrum actually measured on the 3rd measurement conditions. FIG. 10 is an explanatory diagram of verification using a spatial offset; The figure which shows the result of having calculated the distribution of the fluorescence area and the Raman area in the said verification. FIG. 4 shows spectral data measured using a rectangular slit and a pinhole slit, respectively;

Preferred embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic configuration diagram of a microscopic laser Raman spectrophotometer (hereinafter referred to as a Raman spectrophotometer), which is an embodiment of the Raman spectrometer of the present invention. The Raman spectrometer is a device for spectroscopically detecting Raman scattered light from the sample S accompanying irradiation of excitation light (laser light) to obtain a Raman spectrum. A wide range of objects to be measured in this embodiment include solid samples (such as PEEK resin), powder samples (such as cellulose powder), and liquid samples (such as colored liquids such as whiskey).

FIG. 1 shows the configuration that forms the framework of the Raman spectrometer. That is, the Raman spectroscopic apparatus comprises a confocal optical system comprising a movable stage 11 for a sample, an objective lens 12, and a beam splitter (BS) for reflecting excitation light toward the sample and transmitting light from the sample S. 13 (or a dichroic mirror DM), a rejection filter 14 for cutting Rayleigh light, an imaging lens 15, an aperture switching device 16, a spectroscope 17, and a CCD detector .

The Raman spectrometer also includes a laser wavelength switching device 21, a beam spot position changing device 22, and a laser control device 23 as components of an excitation optical system. The laser control device 23 includes an exposure time setting section 24 and a laser intensity setting section 25 .

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

Note that the storage device 40 stores a data processing program for realizing these functional blocks in the spectral data processing device 30, and the spectral data processing device 30 appropriately executes this data processing program to function is exhibited.

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

A laser controller 23 controls selected light sources 26 , 27 . The exposure time setting unit 24 turns on/off the outputs of the light sources 26 and 27 according to the set exposure time. Alternatively, shutters provided on the output windows of the light sources 26 and 27 may be opened and closed. Also, the laser intensity setting unit 25 adjusts the output intensity of the light sources 26 and 27 according to the intensity condition of the excitation light. Alternatively, it is configured to change the intensity of the excitation light by switching on/off a neutral density filter installed on the optical path of the excitation light.

The excitation light forms a minute beam spot on the sample through the objective lens 12, which is the target part of spectrum measurement.

<Details of the confocal optical system>
The movable stage 11 is an XY biaxial stage having a mounting surface parallel to the XY plane, and can position the beam spot on the mounted sample to the focal position of the objective lens 12 .

The objective lens 12 is an example of a collection optical element of the present invention, such as a Cassegrain mirror. The objective lens 12 has a role of forming a beam spot of the excitation light on the sample and a role of condensing the light from the beam spot and its peripheral portion. Rayleigh scattered light contained in the light from the sample S is removed by the rejection filter 14 . The imaging lens 15 forms an image of the condensed light at an opening such as a rectangular slit plate 16A or a pinhole slit plate 16B mounted in a subsequent aperture switching device 16 to form an intermediate image of the beam spot. have a role to play. Although FIG. 1 is an example of a reflection optical system, a Cassegrain mirror or the like may be used to configure a transmission optical system measuring apparatus.

The opening switching device 16 is configured to be able to switch between at least two types of openings. In FIG. 1, as an example, a rectangular slit plate 16A having an elongated opening and a pinhole slit plate 16B having a circular opening are held by a slidable common frame and selected openings are shown. is configured so that it can be installed online. The plate of the opening can have openings of various shapes other than elongated and circular. Also, a plurality of openings having different sizes may be formed for each shape.

As the spectroscope 17, a general dispersive spectroscope using a diffraction grating is used. The diffracted light from the diffraction grating is imaged by the CCD detector 18 in the latter stage, and the spectrum value for each band is detected by the two-dimensionally arranged light-receiving elements of the CCD detector 18 , and the spectral data processing device 30 output to

Here, FIG. 2 shows the difference in the capturing area due to the two types of openings (rectangular slit plate 16A and pinhole slit plate 16B). Here, the beam spot is indicated by the black circle area on the sample. The periphery is indicated by a white circle area larger than the black circle.

Light taken into the spectroscope 17 is restricted by the openings of the rectangular slit plate 16A and the pinhole slit plate 16B. In other words, the light capturing region on the sample is determined by the shape and size of this opening.

In FIG. 2, the slit width of the rectangular slit plate 16A is approximately the same as the image size of the beam spot. are taken into the spectroscope 17 together. On the other hand, in the case of the pinhole slit plate 16B, especially when the aperture is substantially the same as the image of the beam spot, only the light from the beam spot is efficiently taken into the spectroscope 17 due to the confocal effect. , the measurement can be performed with higher spatial resolution than the rectangular slit plate 16A.

This embodiment is characterized by performing at least two spectrum measurements (base measurement and main measurement) under different measurement conditions using the above Raman spectrometer. Examples of measurement conditions are listed in Table 1 below.

Under Condition 1, the base measurement is performed with a low spatial resolution, and the main measurement is performed with a 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 cases in which a rectangular slit or a pinhole slit opening is used to set a light capturing region on the sample.

Condition 1A uses, for example, a rectangular slit with a slit width of 200 μm and a pinhole slit with a diameter of, for example, 100 μm. When using a rectangular slit, the ratio of the beam spot to the capture area on the sample should be small, and when using a pinhole slit, the ratio should be larger than that of the rectangular slit. do it. Alternatively, the diameter of the pinhole slit should be equal to or less than the slit width of the rectangular slit.

Conditions 1B and 1C change the size of the rectangular slit and the pinhole slit. For example, for the same measurement position on the sample, the base measurement uses a larger aperture and the main measurement uses a smaller aperture.

When a pinhole slit is used in this measurement, the light capturing area of the pinhole slit may be aligned with the beam spot, or the light capturing area may be completely included in the beam spot area. . In this embodiment, these conditions are set by operating the opening switching device 16 .

Condition 2 changes the spatial offset by changing the spacing between the beam spot and the light capture area. The base measurement is offset and the above interval is increased. In this measurement, the interval is set to zero in the matched state, ie, the interval is reduced even in the offset state. Note that the beam spot may be fixed and the position of the capturing area may be moved. Alternatively, the capturing area may be fixed and the position of the beam spot may be moved by operation of the beam spot repositioning device 22 of FIG.

Condition 3 increases the intensity of the excitation light in the base measurement and decreases the intensity of the excitation light in the main measurement. This condition setting is executed by the operation of the laser intensity setting section 25 in FIG.

As a matter common to each condition, using the exposure time setting unit 24 in FIG. The exposure time should be longer than the actual exposure time.

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

The measurement method according to the present embodiment may be started after optimizing the wavelength of the excitation light, which is a conventional technique for avoiding fluorescence. The wavelength switching of excitation light uses the laser wavelength switching device 21 of FIG. Even if the wavelength of the excitation light is changed to the long wavelength side and the Raman spectrum is measured, the influence of fluorescence may not be sufficiently removed, but by performing the measurement method of the present embodiment, such fluorescence can be eliminated. In some cases, the impact can be eliminated or mitigated.

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

Base measurement is performed (step S16). In the base measurement, first, when excitation light from the light sources 26 and 27 is irradiated to the beam spot position, only the light from the sample that has passed through the opening of the wide rectangular slit plate is taken into the spectroscope 17 . Since the spectroscope 17 detects spectral values for each band, the spectral data processing device 30 reads the detected values. The exposure time for base measurement should be short.

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 this measurement, the exposure time is longer than in the base measurement.

After the spectral data processing device 30 finishes reading the detection values of the base measurement and the main measurement according to the above procedure, the data processing by the spectral data processing device 30 is executed.

The spectrum data SP10 and SP12 of base measurement and main measurement are shown in the upper left of FIG. For base measurements, a 200 μm wide rectangular slit was used with an exposure time of 1 second. In this measurement, a rectangular slit with a width of 100 µm was used and the exposure time was 10 seconds.

In the data processing (steps S22 to S40) of FIG. 3, first, the baseline coefficient A is calculated by the function of the baseline coefficient calculator 35 (step S22). Here, a wavenumber region 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 fluorescence component (for example, the spectral area corresponding to the fluorescence component) occurring in that wavenumber region is calculated. do. Then, the baseline coefficient A is calculated based on this intensity change rate.

Next, using the baseline coefficient A, a difference spectrum between the spectrum data of the main measurement and the spectrum data of the base measurement is calculated (step S24). Here, the spectral data of the base measurement is multiplied by the baseline coefficient A, and the difference from the spectral data of the main measurement is obtained. The above processing is the baseline correction according to the present embodiment.

Through the processing up to this point, the effects of fluorescence (interference fringes, etc.) that are difficult to remove by conventional methods are removed, but some fluorescence components may remain. The fluorescence components remaining at this stage are easily removed by, for example, conventional software-based baseline flattening processing disclosed in Patent Document 1 (step S30).

In the measurement example of FIG. 4, no Raman peak occurs in the wavenumber region of 2500 to 2000 cm ⁻¹ of the two spectral data SP10 and SP12. Fluorescence areas A10 and A12 in these wavenumber regions were calculated, respectively, 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 of FIG. 4 (step S24). This difference spectrum SP14 is obtained by performing a baseline flattening process (step S30) by existing software after calculating the difference spectrum. For comparison, the upper right portion of FIG. 4 also shows comparative data Ref14 obtained by performing only baseline flattening processing on the spectral data of this measurement. It can be seen that the difference spectrum SP14 has less noise and interference fringes.

In the data processing of FIG. 3, an existing software-based noise removal process (step S40) may be finally executed as needed. For example, spectrum data may be subjected to inverse Fourier transform, and the obtained power spectrum may be subjected to a low-pass filter to remove, as noise, fluctuations in the spectrum of high-frequency components appearing on the baseline of the spectrum data.

In the measurement example of FIG. 4, noise removal processing (step S40) was performed on the difference spectrum SP14 of this embodiment after the baseline flattening processing. The lower part of FIG. 4 shows the difference spectrum SP16 with an improved SN ratio due to noise removal. Moreover, both existing noise removal processing and interference fringe removal processing were applied to the comparison data Ref14. The results are shown together as comparative data Ref16. While the comparison data Ref16 has interference fringes (wavy deformation of the baseline) that are not removed by the existing data processing, the difference spectrum SP16 of this embodiment has such interference fringes greatly reduced. It can be seen that

FIG. 5 shows spectrum data SP22 of this measurement using a rectangular slit with a width of 50 μm. The base measurement data are the same as in FIG. Comparing the difference spectra SP24 and SP26 calculated from these spectrum data with the comparison data Ref24 and Ref26, the difference spectrum SP24 according to the present embodiment has less noise and interference fringes than the comparison data Ref24. It can be seen that the difference spectrum SP26 after noise removal has more interference fringes than the comparison data Ref26.

Furthermore, FIG. 6 shows difference spectra SP34 and SP36 together with comparative data Ref34 and Ref36, based on spectral data SP32 of this measurement using a pinhole slit with a diameter of 100 μm. As in FIG. 5, the difference spectrum according to this embodiment is superior to the comparison data Ref34 and Ref36 in terms of noise and interference fringes.

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

<Verification by spatial offset>
A first feature of the present invention is that, in the Raman spectrum measuring method of the present invention, two spectrum measurements (main measurement and base measurement) are performed on the same sample under different measurement conditions. In the main measurement, set the measurement conditions so that "the ratio of the Raman peak to the fluorescence component in the spectrum data is relatively large", and in the base measurement, set the measurement conditions so that "the above ratio is relatively small". .

As a specific example, the measurement condition 1 is to change the area of the light capturing region (measurement field of view) on the sample, and the measurement condition 2 is to change the irradiation region (beam spot) of the excitation light on the sample and the capturing region. Changing the positional relationship, measurement condition 3, is to change the intensity of the excitation light.

The second feature is to offset the fluorescence component contained in the spectral data of the main measurement by taking the difference between the spectral data obtained under two different measurement conditions. Here, since it is spectral data of the same sample, it may be difficult to cancel out only the fluorescence component even if the measurement conditions are changed (maybe the desired Raman peak is also canceled out)? There may be a question.

In response to this, the inventors performed "verification by spatial offset" as shown in FIG. Here, PEEK resin was used as a sample. An objective lens 12 with a magnification of 100 was used so that the excitation light with a wavelength of 532 nm formed a beam spot with a diameter of about 1 μm on the sample. In addition, the spatial resolution on the sample was set to correspond to 1 μm (capture area). When the offset amount between the center of the beam spot on the sample and the center of the light capturing area (the same circular area with a diameter of 1 μm was used) was gradually increased from 0 μm to 5 μm and each spectrum was measured, the peak of the Raman peak There was a difference between how the height changed and how the height of the fluorescent component changed. As shown in FIG. 7, the offset moves the optical axis of the excitation light (black area) by 0 to 5 μm with respect to the optical axis of the measurement light without changing the positional relationship between the sample S and the objective lens 12. It depends on how you make it.

Even with an offset of 3 μm or 5 μm, a fluorescence component covering a wide wavenumber range (a wide range of elevation of the baseline) can be confirmed, but Raman peaks can hardly be confirmed even with an offset of 3 μm. A Raman peak can be confirmed if the offset amount is up to 1 μm.

Therefore, as shown in FIG. 8, for the Raman peak near 1600 cm ^-1 , the change in the peak area for each offset amount and the spectral area of the fluorescent component in the band (2500 to 2000 cm ^-1 ) where no Raman peak occurs As a result, it was confirmed that the fluorescence component (fluorescence area) gradually decreased from the beam spot to the apex, while the Raman peak (Raman area) decreased sharply from the beam spot to the apex. The graph showing the area for each offset amount in FIG. 8 is normalized by setting the fluorescence area and the Raman area at 0 μm to 100, respectively.

The inventors have found that the intensity distribution of fluorescence has a relatively gentle mountain shape with the beam spot as the apex, whereas the Raman scattered light intensity distribution has a Gaussian distribution with the beam spot as the apex. estimated. This is because the Raman scattered light sharply decreases with distance from the beam spot position, and becomes almost zero after a short distance.

The Raman scattered light is inelastic scattered light and is proportional to the intensity of the excitation light. Therefore, it can be said that the Raman light has a Gaussian distribution similar to the excitation light (laser light). Fluorescence, on the other hand, is light emitted as light is absorbed by a substance, and tends to be saturated with excitation light above a certain level. Therefore, it is considered that the distribution of fluorescence does not have a Gaussian distribution type but has a mountain shape. In other words, when the same excitation light is applied to the 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.

Since there is such a difference in the distribution of fluorescence and Raman light on the sample, the inventors performed two spectrum measurements under different conditions for the same sample, and took the difference between the two spectra. As a result, the inventors have found that it is possible to obtain a good Raman spectrum in which the influence of fluorescence is canceled and desired Raman peaks remain.

Several measurement conditions can be set. What is common is to set measurement conditions such that the ratio of the Raman peak to the fluorescent component in the spectrum data becomes relatively large and measurement conditions that make the ratio relatively small.

Here, as shown in the distribution of light in FIG. 8, the vicinity of the beam spot has a relatively large ratio of Raman light to fluorescence, and can be called a "Raman light-rich region". At a distance from the beam spot, the ratio of Raman light to fluorescence is almost zero, and can be called a "fluorescence-rich region".

For example, as measurement condition 1, in this measurement, a relatively narrow region mainly including a "Raman light rich region" is set as a light capturing region. Then, in the base measurement, a relatively wide area mainly including the "fluorescence-rich area" is set as the light capturing area. That is, the spatial resolution is made different by changing the area of the light capturing region.

Further, for example, as measurement condition 2, as shown in FIG. Set to capture area. That is, the positional relationship between the beam spot and the capturing area is changed.

Also, for example, as the measurement condition 3, the intensity of the excitation light is changed. In this measurement, the light capturing region is set to the "Raman light rich region" to obtain spectral data in which the ratio of the Raman peak to the fluorescence component is relatively large. Then, in the base measurement, the spectrum of the same uptake region is measured, but the intensity of the excitation light is increased to saturate the fluorescence, thereby obtaining spectral data with a relatively small ratio of Raman peaks to fluorescence components.

In the spectrum data measured under a plurality of conditions of offset amount as shown in FIG. 7, the respective fluorescence components are similar to each other (similar to the vertical axis direction of the spectrum data). In addition, as shown in FIG. 9, even in spectral data measured under different spatial resolution conditions (rectangular slit, pinhole slit), the respective fluorescence components have similar shapes to each other. Therefore, in the calculation of the difference spectrum, if a suitable baseline coefficient is given to the spectrum data of the base measurement and the difference from the spectrum data of the main measurement is taken, the fluorescence component can be canceled cleanly. In the present invention, the rate of change in intensity of the fluorescent component due to changes in the measurement conditions is calculated from each fluorescence area and the like, and the baseline coefficient is calculated based on this rate of change in intensity.

For this reason, according to the spectrum measurement method of the present embodiment, it is possible to obtain Raman spectrum data in which the rise and deformation of the baseline due to the influence of fluorescence have been corrected.

11 movable stage (irradiation area setting means), 12 objective lens, 13 beam splitter, 14 rejection filter, 15 imaging lens, 16 aperture switching device (capturing area setting means), 16A rectangular slit plate, 16B pinhole slit plate , 17 spectroscope, 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 spectrum 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 (10)

A light source for excitation light of a single wavelength, a stage on which a sample is placed, an irradiation area setting means for setting an irradiation area of the excitation light on the sample, and a capture area setting for setting a light capturing area on the sample. means, a spectroscope for spectrally detecting light from the capture region, and a data processing means for processing spectral data based on detection values from the spectroscope, wherein
At least one of the light source, the irradiation region setting means, and the capture region setting means sets the capture region so that the ratio of the Raman peak to the fluorescence component in the spectral data is relatively large as the measurement conditions for the main measurement. , the illumination region, and excitation light intensity, and as measurement conditions for base measurement, the acquisition region so that the ratio of Raman peaks to fluorescence components in the spectral data is relatively small; configured to set at least one of the irradiation area and excitation light intensity;
The data processing means are
a coefficient calculation unit that calculates an intensity change rate of a fluorescence component of base measurement spectral data according to a difference in measurement conditions and calculates a baseline coefficient based on the intensity change rate; A difference spectrum calculation unit that calculates a difference spectrum between the spectrum data to which the coefficient is assigned and the spectrum data of the main measurement,
A Raman spectrometer characterized by: 2. The apparatus according to claim 1, wherein said capture region setting means narrows the area of said capture region as the measurement condition of said main measurement and widens the area of said capture region as the measurement condition of said base measurement. A Raman spectrometer, comprising: 3. The apparatus according to claim 2, wherein said capture area setting means has an aperture for limiting the light captured by said spectroscope, and is configured to change the shape or size of said aperture. and Raman spectrometer. 4. The apparatus according to any one of claims 1 to 3, wherein said irradiation area setting means sets an offset amount between the center of said irradiation area and the center of said acquisition area to zero or small as a measurement condition for said main measurement, A Raman spectrometer, wherein as a measurement condition of the base measurement, an offset amount between the center of the irradiation region and the center of the capture region is increased. 5. The apparatus according to claim 1, wherein the light source reduces the intensity of the excitation light as the measurement condition for the main measurement and increases the intensity of the excitation light as the measurement condition for the base measurement. A Raman spectrometer characterized by comprising: 6. The apparatus according to claim 1, further comprising exposure time setting means for setting an exposure time of the excitation light to the sample, wherein said exposure time setting means sets said A Raman spectrometer, wherein a long exposure time is set, and a short exposure time is set as a measurement condition for the base measurement. A method for measuring a Raman spectrum by capturing light from a sample excited with a single wavelength excitation light into a spectroscope,
Irradiation area setting procedure for setting the excitation light irradiation area on the sample, capture area setting procedure for setting the light capture area on the sample, irradiation procedure for irradiating the excitation light, and detection values from the spectrometer and a data processing procedure for processing said spectral data,
In at least one of the irradiation area setting procedure, the capturing area setting procedure and the irradiation procedure,
As measurement conditions for this measurement, at least one of the capture region, the irradiation region, and the excitation light intensity is set so that the ratio of the Raman peak to the fluorescence component in the spectral data is relatively large; setting at least one of the capture region, the irradiation region, and the excitation light intensity as measurement conditions for the base measurement so that the ratio of Raman peaks to fluorescence components in the spectral data is relatively small; Do two ways,
Said data processing procedure comprises:
A coefficient calculation procedure for calculating the intensity change rate of the fluorescence component of the base measurement spectrum data according to the difference in measurement conditions, and calculating a baseline coefficient based on the intensity change rate; including a difference spectrum calculation procedure for calculating a difference spectrum between the spectrum data to which the coefficient is assigned and the spectrum data of the main measurement;
A method for measuring a Raman spectrum, characterized by: 8. The Raman spectrum measuring method according to claim 7, wherein said coefficient calculation step calculates said baseline coefficient based on spectrum data obtained by a plurality of base measurements under different measurement conditions. 9. A method according to claim 7 or 8, wherein said data processing step comprises:
A method for measuring a Raman spectrum, comprising a baseline flattening procedure for flattening a baseline of the difference spectrum after the procedure for calculating the difference spectrum. 10. A method for measuring a Raman spectrum, wherein the method according to any one of claims 7 to 9 is performed after changing the wavelength of the excitation light to the longer wavelength side and measuring the Raman spectrum.

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