Calibration device, raman spectroscopy measurement device, and wave number calibration method

Abstract

A spectroscopy measurement device according to the present embodiment comprises: a lamp light source (160) for generating lamp light having a plurality of emission lines; a plurality of inorganic materials; an objective lens (50) on which the lamp light and Raman scattered light from the plurality of inorganic materials impinge; a spectrometer (60) that spectrally detects the lamp light and the Raman scattered light from the objective lens (50); and a processing unit (70) that calculates a calibration wavelength axis of the spectrometer (60) on the basis of the wavelengths of the plurality of emission lines, calculates the incident wavelength of the laser light on the basis of the Raman bands of the Raman scattered light on the calibration wavelength axis, and converts the calibration wavelength axis to a wavenumber axis using the incident wavelength.

Description

Calibration device, Raman spectrometer, and wavenumber calibration method

[0001] The present invention relates to a calibration device, a Raman spectroscopic measurement apparatus, and a wavenumber calibration method, and more particularly to a technique for calibrating a wavenumber axis in Raman spectroscopic measurement.

[0002] Raman spectroscopy is useful for in-situ analysis of products, prototypes, and biological samples, as it can identify the type and state of molecules contained in a sample without labeling or destructively. The spectrum of Raman scattered light, or the Raman spectrum, is used to identify the type and state of molecules in Raman spectroscopy.

[0003] Non-Patent Document 1 discloses a method for calibrating Raman scattered light, in which a standard sample and a neon lamp are measured and fitted with a polynomial.

[0004] https: / / www.misasa.okayama-u.ac.jp / ~masami / pukiwiki / index.php?%E3%83%A9%E3%83%9E%E3%83%B3%E3%81%AE%E6%B3%A2%E6%95%B0%E6%A0%A1%E6%AD%A3%E3%81%AB%E3%81%A4%E3%81%84%E3%81%A6 Internet search [Searched September 30, 2022]

[0005] The Raman shift corresponds to the inherent vibrational energy of a molecule and is expressed in wave numbers [cm －１ ]. The natural vibration mode of a molecule is determined by the type and state of the molecule, so the type and state of the molecule can be analyzed using the horizontal axis of the Raman spectrum, i.e., the wave number, as a clue. The wave number is related to the excitation wavelength [nm] and scattering wavelength [nm] by the following formula: Wave number [cm －１ ]=10 ７ / (incident wavelength)-10 ７ / (scattered wavelength)

[0006] Dispersive Raman spectrometers, the most widely used type, use a spectrometer to detect the peak wavelength of Raman scattered light. More specifically, because wavelength dispersion is performed by a spectroscopic element, the position on the dispersion axis in the detection space of the photodetector corresponds to the wavelength, i.e., the wavenumber. Therefore, it is desirable to convert the position on the dispersion axis to the wavenumber with higher accuracy.

[0007] The present disclosure has been made in consideration of the above points, and aims to provide a calibration device, a Raman spectroscopic measurement apparatus, and a wavenumber calibration method that are capable of performing wavenumber calibration in Raman spectroscopy with high accuracy.

[0008] The calibration device according to this embodiment is a calibration device for calibrating the wavenumber of a Raman spectrometer, and includes one or more materials and a holder for holding the one or more materials.

[0009] The calibration device may further include a drive mechanism that drives the holder so that the laser light of the Raman spectroscopic measurement apparatus is irradiated onto the plurality of materials in sequence.

[0010] In the above-described calibration device, the plurality of materials may be inorganic materials.

[0011] In the above calibration device, the drive mechanism may rotate the holder around a rotation axis that is offset from the optical axis of the objective lens of the Raman spectroscopic measurement apparatus.

[0012] In the above-described calibration device, the plurality of materials may be placed on a cover glass, and Raman scattered light generated from the materials may be incident on an objective lens of the Raman spectroscopic measurement apparatus via the cover glass.

[0013] The calibration device may further include a base plate that supports the drive mechanism, and a fiber that propagates light for calibrating the intensity of the Raman spectrometer may be connected to the base plate.

[0014] The calibration device may further include a lamp light source that generates lamp light having a plurality of emission lines.

[0015] In the above calibration device, the holder may have an opening through which the lamp light passes, and a filter having a transmittance according to wavelength may be disposed in the opening.

[0016] The Raman spectroscopic measurement device according to this embodiment includes a lamp light source that generates lamp light having a plurality of emission lines, a laser light source that generates laser light, one or more materials that are removably arranged in the optical path of the laser light, an objective lens onto which the lamp light and Raman scattered light from the one or more materials are incident, a spectroscope that disperses and detects the lamp light and the Raman scattered light from the objective lens, and a processing unit that calculates a calibrated wavelength axis of the spectroscope based on the wavelengths of the plurality of emission lines, calculates the incident wavelength of the laser light based on the Raman band of the Raman scattered light on the calibrated wavelength axis, and converts the calibrated wavelength axis into a wave number axis using the incident wavelength.

[0017] In the above Raman spectrometer, the plurality of materials may be inorganic materials.

[0018] The Raman spectroscopic measurement device may further include a holder that holds the plurality of materials, and a drive mechanism that drives the holder so that the laser light is incident on the plurality of materials in sequence.

[0019] In the above Raman spectrometer, the drive mechanism may rotate the holder around a rotation axis that is offset from the optical axis of the objective lens.

[0020] In the above-described Raman spectrometer, the holder may have an opening through which the lamp light passes, and a filter having a transmittance according to wavelength may be disposed in the opening.

[0021] In the above-described Raman spectroscopic measurement device, the plurality of materials may be placed on a cover glass, and the Raman scattered light may be incident on the objective lens via the cover glass.

[0022] The wavenumber calibration method according to this embodiment includes the steps of: performing spectroscopic measurement of Raman scattered light from one or more materials irradiated with laser light using a Raman spectrometer; performing spectroscopic measurement of lamp light having a plurality of emission lines using the Raman spectrometer; calculating a calibrated wavelength axis based on the wavelengths of the plurality of emission lines; calculating the incident wavelength of the laser light based on the Raman band of the Raman scattered light on the calibrated wavelength axis; and converting the calibrated wavelength axis into a wavenumber axis using the incident wavelength.

[0023] According to the present disclosure, it is possible to provide a calibration device, a Raman spectrometer, and a wavenumber calibration method that are capable of performing wavenumber calibration in Raman spectroscopy with high accuracy.

[0024] 1 is a diagram showing the overall configuration of a spectroscopic measurement device; FIG. 2 is a perspective view showing the configuration of a calibration device; FIG. 3 is a perspective view showing the configuration of a calibration device; FIG. 4 is a diagram showing the emission line spectrum of a neon lamp; FIG. 5 is a wavenumber spectrum showing the Raman shift of silicon; FIG. 6 is a diagram showing spectra obtained by calibrating measurement results from different Raman microscopes; FIG. 7 is a table showing the specifications of three Raman spectroscopic measurement devices; FIG. 8 is a diagram showing spectroscopic measurement results on a calibration wavenumber axis; FIG. 9 is a side view schematically showing an example of a calibration device; and FIG. 10 is a photograph showing a device configuration according to a modified example.

[0025] Below, embodiments to which the present invention can be applied are described. The following description is for describing embodiments of the present invention, and the present invention is not limited to the following embodiments. For clarity of explanation, the following description has been omitted and simplified as appropriate. Furthermore, a person skilled in the art would be able to easily modify, add, or convert each element of the following embodiments within the scope of the present invention. Note that elements with the same reference numerals in each drawing indicate similar elements, and descriptions thereof will be omitted as appropriate.

[0026] A calibration device, a Raman spectrometer, and a wavenumber calibration method according to the present embodiment will be described. Fig. 1 is a schematic diagram showing the overall configuration of a Raman spectrometer. The Raman spectrometer 10 includes a light source 30, an optical system 40, an objective lens 50, a spectroscope 60, and a processing unit 70. The Raman spectrometer 10 is equipped with a calibration device 100 for wavenumber calibration.

[0027] The calibration device 100 includes a sample substrate 140 such as a standard sample substrate. The calibration device 100 is detachably installed in the Raman spectrometer 10. The calibration device 100 is attached to a sample stage or the like of the Raman spectrometer 10 only when measuring a standard spectrum for calibration. When performing spectroscopic measurement of an actual sample, the calibration device 100 is removed from the Raman spectrometer 10.

[0028] The light source 30 is a laser light source that generates laser light L1 having a predetermined laser wavelength. For example, the light source 30 is a Nd / YVO4 laser that emits CW (Continuous Wave) laser light with a wavelength of 532 nm. Of course, the light source 30 may be another type of laser light. The light source 30 may be a pulsed laser light source. There may be multiple light sources 30. The laser light L1 from the light source 30 is incident on the optical system 40. The optical system 40 guides the laser light L1 to the objective lens 50 and also guides the detection light L4 from the objective lens 50 to the spectroscope 60.

[0029] The optical system 40 includes optical elements such as filters, mirrors, lenses, beam splitters, and optical scanners. For example, a dichroic mirror that splits the laser light and the detection light according to wavelength can be used as the beam splitter. The optical system 40 may shape the laser light L1 so that it illuminates a linear sample area on which the slit of the spectrometer 60 is imaged. Alternatively, an optical scanner may scan the laser light L1 to illuminate a linear area on the standard sample. The optical system 40 may also include a laser line filter or the like to prevent light of wavelengths different from the laser light L1 from entering the standard sample or the spectrometer 60. The optical system 40 may also include an edge filter or the like to block the laser wavelength so that the laser light L1 does not enter the spectrometer 60. Since a known configuration can be used for the optical system 40, detailed description thereof will be omitted.

[0030] The laser light L1 propagated by the optical system 40 is incident on the objective lens 50. The objective lens 50 focuses the laser light L1 on the sample substrate 140 of the calibration device 100. The objective lens 50 forms the focus of the laser light L1 on the sample substrate 140. As a result, Raman scattered light L2 is generated at the sample substrate 140. A portion of the Raman scattered light L2 generated at the sample substrate 140 is incident on the objective lens 50. The Raman scattered light L2 from the objective lens 50 becomes detection light L4 and is incident on the optical system 40. The optical system 40 propagates the detection light L4 to the spectroscope 60.

[0031] The detection light L4 from the optical system 40 is incident on the spectroscope 60. The spectroscope 60 includes a spectroscopic unit 61 and a photodetector 62, and performs spectroscopic measurement of the detection light L4. The spectroscopic unit 61 includes a slit, a wavelength dispersive element, etc. The wavelength dispersive element is a diffraction grating, a prism, etc. Furthermore, the spectroscopic unit 61 may include a concave mirror, a lens, etc. The spectroscopic unit 61 disperses the detection light L4 in the X direction.

[0032] The photodetector 62 is a two-dimensional array photodetector such as a two-dimensional CCD camera. The photodetector 62 is an image sensor having a plurality of pixels arranged in an array. The photodetector 62 is, for example, a two-dimensional photodetector such as a CCD (Charge Coupled Device) camera or a CMOS (Complementary Metal Oxide Semiconductor) image sensor. The photodetector 62 outputs a detection signal corresponding to the amount of light detected by each pixel to the processing unit 70. The processing unit 70 is, for example, a personal computer, and stores the value of the detection signal in a memory or the like in association with the XY coordinates of the pixel.

[0033] For example, the photodetector 62 is a cooled CCD camera in which a plurality of pixels are arranged in the X and Y directions. The pixels of the photodetector 62 are arranged along a direction corresponding to the slit. Therefore, one arrangement direction of the pixels of the photodetector 62 coincides with the slit length direction (Y direction), and the other arrangement direction coincides with the dispersion direction (X direction) of the spectroscopic unit 61. The distribution of light intensity in the X direction of the photodetector 62 indicates the distribution of the Raman spectrum. In other words, the pixel address (pixel position) on the light receiving surface of the photodetector 62 corresponds to the wavelength of the detection light L4. The photodetector 62 outputs detection data for each pixel to the processing unit 70.

[0034] Furthermore, a lamp light source 160 is disposed above the calibration device 100. The lamp light source 160 generates lamp light L3 having a plurality of emission lines. The lamp light source 160 is, for example, a Ne (neon) lamp, an Ar (argon) lamp, or a Kr (krypton) lamp. The wavelengths of each emission line are known. In this example, a neon lamp is used as the lamp light source 160.

[0035] Lamp light L3 from the lamp light source 160 enters the calibration device 100 via a filter 150. The filter 150 has different transmittance depending on the wavelength. The filter 150 adjusts the relative light intensity at each emission line.

[0036] A portion of the lamp light L3 from the lamp light source 160 passes through the calibration device 100 and enters the objective lens 50. The lamp light L3 from the objective lens 50 propagates through the optical system 40 as detection light L4. The optical system 40 guides the detection light L4 to the spectroscope 60. Therefore, the lamp light L3 is spectroscopically measured by the spectroscope 60, similar to the Raman scattered light L2.

[0037] The positions of the lamp light source 160 and the filter 150 are not particularly limited. The lamp light source 160 and the filter 150 can also be provided separately from the calibration device 100. For example, the lamp light source 160 may be disposed in front of the spectroscope 60 at a position shifted from the optical path of the detection light L4. As long as the lamp light L3 from the lamp light source 160 is incident on the spectroscope 60, the position of the lamp light source 160 is not particularly limited.

[0038] Next, a detailed configuration of the calibration device 100 will be described with reference to Fig. 2 and Fig. 3 in addition to Fig. 1. Fig. 2 and Fig. 3 are perspective views schematically showing the appearance of the calibration device 100. Fig. 2 is a view of the calibration device 100 seen from the objective lens 50 side, and Fig. 3 is a view of the calibration device 100 seen from the lamp light source side.

[0039] The calibration device 100 includes a first plate 110, a second plate 120, a cover glass 130, and a sample substrate 140. The sample substrate 140 is formed of a plurality of standard samples 141 to 144. Here, the plurality of standard samples 141 to 144 are configured as separate substrates. As shown in FIG. 3 , the second plate 120 includes a rotation axis 122 and an opening 125. The first plate 110 and the second plate 120 form a rotating disk that rotates the standard samples 141 to 144. The first plate 110 and the second plate 120 rotatably hold the cover glass 130 and the standard samples 141 to 144.

[0040] As shown in FIG. 1, standard samples 141-144 are placed on a cover glass 130. The cover glass 130 is a transparent substrate such as synthetic quartz. Synthetic quartz does not emit strong Raman scattered light, making it suitable for calibration. Alternatively, the cover glass 130 may be made of calcium fluoride, which emits narrow-line Raman scattered light. The standard samples 141-144 are formed of different inorganic materials. For example, standard sample 141 is Si, standard sample 142 is 6H—SiC, and standard sample 143 has a triangular diamond and a trapezoidal Si. Standard sample 144 is a calcium fluoride substrate. The SiC and Si are opaque, while the diamond and calcium fluoride substrate are transparent.

[0041] The standard samples 141 to 144 are inorganic materials whose Raman shifts are known. The standard samples 141 to 144 are arranged in the same plane perpendicular to the optical axis of the objective lens 50. The standard samples 141 to 144 are formed as separate substrates, and are therefore arranged on the cover glass 130. The standard samples 141 to 144 may also be formed as an integrated sample substrate 140. The standard samples 141 to 144 are arranged so that they can be inserted into or removed from the optical path of the laser light. For example, the standard samples 141 to 144 are inserted into the optical path by attaching the calibration device 100 to a sample stage or the like. The standard samples 141 to 144 are removed from the optical path by removing the calibration device 100 from the sample stage or the like.

[0042] The first plate 110 and the second plate 120 are holders that hold the cover glass 130 and the standard samples 141-144. For example, the cover glass 130 is fixed to the first plate 110 by adhesive or the like. As shown in FIGS. 2 and 3, the first plate 110 and the second plate 120 are circular substrates of approximately the same size. The objective lens 50 focuses the laser light L1 at the position where the sample substrate 140 is located. In other words, the focal plane of the objective lens 50 coincides with the plane where the standard samples 141-144 are located.

[0043] The second plate 120 is provided with a rotation axis 122. The rotation axis 122 extends from the second plate 120 to the opposite side of the objective lens 50. The rotation axis 122 is parallel to the optical axis of the objective lens 50. As shown in FIG. 1 , a drive mechanism 123 is attached to the rotation axis 122. The drive mechanism 123 includes a rotary motor such as a stepping motor. The drive mechanism 123 rotates the calibration device 100 around the rotation axis 122. The rotation axis 122 is located at the center of the circular second plate 120. The rotation axis 122 is offset from the optical axis of the objective lens 50. In other words, the optical axis of the objective lens 50 is offset from the center of the circular second plate 120.

[0044] The processing unit 70 controls the driving of the driving mechanism 123. That is, the processing unit 70 controls the rotation timing and rotation speed of the driving mechanism 123. The driving mechanism 123 rotates the calibration device 100, thereby changing the irradiation position of the laser light L1 on the calibration device 100. That is, the laser light L1 is irradiated onto the standard samples 141 to 144 in order.

[0045] As shown in FIG. 3 , the second plate 120 has an opening 125. Lamp light from the lamp light source 160 passes through the opening 125. As shown in FIG. 1 , a filter 150 (omitted in FIG. 3 ) is installed in the opening 125. Therefore, lamp light L3 passes through the opening 125 and enters the objective lens 50. Note that a standard sample 144 may be located directly below the filter 150, but this is not essential. For example, by providing a transparent standard sample 144, the lamp light passes through the standard sample 144 and enters the objective lens 50.

[0046] The driving mechanism 123 rotates the calibration device 100, causing the standard samples 141-144 to move sequentially into the field of view of the objective lens 50. Raman scattered light L2 from the multiple standard samples 141-144 is incident on the objective lens 50 sequentially. Furthermore, lamp light L3 is also incident on the objective lens 50. The detection light L4 contains the lamp light L3 and the Raman scattered light L2 from the multiple standard samples 141-144. The processing unit 70 performs wavenumber calibration based on the spectral measurement results of the detection light L4. The spectroscopic measurement results of the detection light L4 become calibration data containing the spectrum of the lamp light L3 and the spectrum of the Raman scattered light L2 from the standard samples 141-144.

[0047] FIG. 4 is a graph showing the emission line spectrum of a neon lamp. In FIG. 4, the horizontal axis represents wavelength, and the vertical axis represents the relative intensity of the emission lines. Lamp light has multiple emission lines. Therefore, the spectrometer 60 measures each of these emission lines. The filter 150 is, for example, a bandpass filter, and adjusts the relative intensities of the emission lines. For example, the filter 150 can reduce the difference in relative intensities by blocking some of the emission lines with high relative intensities. By aligning the relative intensities of the emission lines, it is possible to simultaneously measure the neon reference band over a wide wavelength range.

[0048] 5 is a spectrum showing the Raman shift when the standard sample 141 is silicon. In FIG. 5, the horizontal axis represents the Raman shift [cm －１ ], and the vertical axis represents light intensity. By using an inorganic material with high crystallinity such as Si, a peak with a narrow line width can be obtained. Of course, the wave number of the peak differs depending on the inorganic material.

[0049] The calibration process in the processing unit 70 will be described below. The processing unit 70 is an information processing device such as a personal computer, and includes a processor, memory, etc. For example, the processing unit 70 includes a memory that stores detection data in association with pixel addresses. The processor of the processing unit 70 executes a program stored in the memory to perform the process described below.

[0050] The processing unit 70 calculates the calibrated wavelength axis of the spectrometer 60 based on the wavelengths of the multiple emission lines. The lamp light source 160 has almost no fluctuation in emission lines. Therefore, it is possible to associate the pixel positions (pixel addresses) in the X direction of the photodetector 62 with wavelengths. In other words, the processing unit 70 determines the pixel positions at which the peak wavelength of the measured spectrum of the lamp light L3 is obtained. Then, the processing unit 70 corresponds the pixel positions at which the peak wavelength is obtained with the wavelengths of the emission lines. The relationship between the pixel positions of the photodetector 62 and the wavelengths is approximated using a polynomial or the like. For example, the least squares method can be used for the polynomial approximation. The processing unit 70 calculates an approximation equation that replaces the pixel positions with wavelengths. In this way, the processing unit 70 can determine the calibrated wavelength axis.

[0051] Next, the processing unit 70 calculates the incident wavelength of the laser light based on the Raman band of the Raman scattered light on the calibrated wavelength axis. In the light source 30, the oscillation wavelength (laser wavelength) of the laser light L1 may fluctuate. When the laser wavelength (incident wavelength) fluctuates, the scattering wavelength (detection wavelength) of the Raman scattered light changes. On the other hand, the wave number of the Raman shift is constant regardless of the laser wavelength (incident wavelength).

[0052] When the units of the incident wavelength (laser wavelength) and the scattering wavelength (detection wavelength) are expressed in [nm], the wave number of the Raman shift [cm －１ ] is expressed by the following formula (1): Wave number [cm －１ ]=10 ７ / (incident wavelength)-10 ７ / (scattered wavelength)...(1)

[0053] Standard samples 141 to 144 have Raman band wave numbers [cm －１ ] is known. Therefore, the processing unit 70 determines the peak wavelength of the spectrum on the calibrated wavelength axis as the scattering wavelength of the Raman scattered light. The processing unit 70 can determine the laser wavelength from the scattering wavelength on the calibrated wavelength axis. In other words, since the wavenumber of the Raman band of the inorganic material is known, when the scattering wavelength on the calibrated wavelength axis is substituted for the scattering wavelength in equation (1), the incident wavelength in equation (1) indicates the laser wavelength. The processing unit 70 determines the laser wavelength using the calibrated wavelength axis.

[0054] The processing unit 70 can calculate the laser wavelength for each of the plurality of standard samples. In this way, the processing unit 70 can accurately estimate the laser wavelength. For example, the processing unit 70 may use equation (1) to determine the incident wavelength using the scattered wavelengths of the plurality of standard samples, and use the average value of these as the laser wavelength.

[0055] The processing unit 70 then converts the calibrated wavelength axis into a wavenumber axis using the incident wavelength of the laser light (laser wavelength). Specifically, the wavelength corresponding to each pixel position is found using the above approximation formula. The laser wavelength and the wavelength indicated by each pixel are substituted into the incident wavelength and scattered wavelength in formula (1), respectively. In this way, a wavenumber axis indicating the wavenumber of the Raman shift for each pixel position is found. The calibrated wavenumber axis becomes data in which the pixel position of the photodetector 62 is associated with the wavenumber.

[0056] The processing unit 70 uses the wavenumber axis calculated in this manner (also referred to as the calibrated wavenumber axis) to determine the wavenumber of the Raman shift in the Raman spectrum of the actual sample. The processing unit 70 detects the Raman spectrum of the actual sample and determines the pixel position where the detected intensity peaks. The processing unit 70 then converts the pixel position where the peak occurs into a wavenumber. This allows for accurate identification of the state and type of molecules.

[0057] As described above, inorganic materials are used for the standard samples 141 to 144. Inorganic materials are more chemically stable and non-toxic than organic materials. Because the standard samples are easy to handle, calibration can be performed simply. Furthermore, by using highly crystalline inorganic materials for the standard samples 141 to 144, the linewidth of the Raman band can be narrowed. Therefore, the peak position of the reference band can be detected with high accuracy, enabling highly accurate calibration. Furthermore, the spectrum of lamp light has many narrow emission lines and is stable. Therefore, the peak position of the reference band can be detected with high accuracy, enabling stable and highly accurate calibration.

[0058] Inorganic materials with high crystallinity and narrow Raman spectral linewidths have only a few strong reference bands. Using a small number of reference bands obtained from only one inorganic material makes it difficult to accurately calculate the excitation wavelength. Using multiple reference bands obtained from multiple inorganic materials makes it possible to accurately calculate the excitation wavelength. Furthermore, the standard sample may be made of a material other than an inorganic material.

[0059] Furthermore, even if the laser wavelength fluctuates, the wavenumber of the Raman shift can be calculated appropriately. Stable spectroscopic measurement is possible even if the measurement date or measurement device is different. In particular, in a solid-state laser whose oscillation wavelength depends on the laser cavity, the oscillation wavelength (laser wavelength) is more unstable than in a gas laser. In this embodiment, even if a laser light source with an unstable oscillation wavelength is used, the wavenumber spectrum of the Raman shift can be measured appropriately.

[0060] Note that wavenumber calibration may be performed before spectroscopic measurement of an actual sample using the calibration device 100. Alternatively, wavenumber calibration may be performed using the calibration device 100 after spectroscopic measurement of an actual sample.

[0061] Spectroscopic measurements of the Raman scattered light and lamp light of the standard samples 141-144 may be performed using separate camera exposures, or may be performed using a single camera exposure. For example, during the exposure time of the photodetector 62, the lamp light source 160 is turned on, and laser light is continuously irradiated while the calibration device 100 is rotated. The photodetector 62 then integrates the Raman scattered light and lamp light, allowing spectroscopic measurement of the Raman scattered light and lamp light together. This provides a standard spectrum of the detection light L4, which includes the Raman scattered light and lamp light. The processing unit 70 can then perform wavenumber calibration by determining the positions of each peak.

[0062] The standard samples 141-144 may be formed from a single inorganic material substrate, the sample substrate 140. The sample substrate 140 is then placed on a single large cover glass 130. This allows for compatibility with Raman spectroscopy measurement devices that require observation through the cover glass 130. For example, it is also compatible with Raman spectroscopy measurement devices that use objective lenses with short working distances or immersion objective lenses. Of course, two or more inorganic material standard samples are sufficient. Furthermore, by placing multiple standard samples at a thickness equal to or less than the focal depth of the objective lens, the drive mechanism 123 can be omitted. In other words, because the laser light is focused on multiple inorganic materials, the photodetector 62 can simultaneously detect Raman scattered light from multiple inorganic materials. The spectrometer 60 can detect Raman scattered light from multiple standard samples while the standard samples remain fixed.

[0063] FIG. 6 shows spectra showing the results of wavenumber calibration of different Raman microscopes using the calibration device 100. The horizontal axis shows the calibration wavenumber axis, and the vertical axis shows the detected intensity. Here, the three Raman microscopes are identified as Raman microscopes A to C. FIG. 6 shows the emission lines of a neon lamp and the Raman shifts of inorganic materials as spectral data for calibration. Here, calcium fluoride (CaF), an inorganic material, is used as a standard sample. ２ ), silicon (Si), silicon carbide (SiC), and diamond are used as standard samples. Alternatively, magnesium fluoride (MgF), which is an inorganic material, is used as a standard sample. ２ ), sapphire (α-Al ２ O ３ ), calcite (CaCo ３ By selecting a standard sample and lamp so that the Raman band and emission line spectrum do not overlap, the calibration device can be optimized to match the excitation wavelength.

[0064] Furthermore, the material used as the standard sample is not limited to a solid, but may also be a gas. For example, chemically stable nitrogen gas or oxygen gas can be used as the standard sample material. When using a gas, a transparent container filled with the gas can be placed in the calibration device.

[0065] Figure 7 is a table showing the specifications of Raman microscopes A to C. As shown in Figure 8, Raman microscopes A to C have different laser light sources, spectrometers 60, cameras, and objective lenses. The data acquisition times are also different. The wavenumber calibration method described above allows the wavenumber axes of Raman microscopes A to C, which have different specifications, to be properly calibrated. This reduces measurement errors between microscopes. Even when different Raman microscopes are used, sample classification, discrimination, and the like can be properly performed. Therefore, the state and type of molecules can be accurately identified.

[0066] FIG. 8 is a diagram showing the results of spectroscopic measurement on a calibrated wavenumber axis. The horizontal axis represents the calibrated wavenumber axis, and the vertical axis represents the detected intensity. Here, polystyrene or ethanol is used as the actual sample. Here, the wavenumbers of the spectroscopic measurement results using the above-mentioned Raman microscopes A to C are calibrated. Also shown are the wavenumber spectra of the Raman shift obtained using the calibration method according to this embodiment (denoted as "new device") and the calibration method according to the comparative example (denoted as "conventional"). The Raman spectra shown are obtained by performing wavenumber calibration multiple times using each of the Raman microscopes A to C.

[0067] In FIG. 8, the wave number of the phenyl ring is 1004 [cm －１ ] is shown enlarged, showing the wavenumber spectrum around the Raman band. In the calibration method according to the comparative example, a difference in the wavenumber of the Raman bands occurs among the three Raman microscopes A to C. More specifically, the wavenumber of the peak in Raman microscope C is different from the wavenumber of the peaks in Raman microscopes A and B. In contrast, in the calibration wavenumber axis according to the present embodiment, the difference in the wavenumber of the Raman bands is smaller. Therefore, calibration can be performed with higher accuracy.

[0068] In this way, it is possible to stably and easily obtain standard spectra with multiple wavenumber reference bands and wavelength reference bands. This allows for appropriate wavenumber calibration of Raman spectroscopy instruments, thereby promoting the application of Raman microscopes in the fields of life science and medicine.

[0069] The processing unit 70 may also control the timing of measurement. For example, the processing unit 70 may control the driving mechanism 123, the light source 30, and the spectrometer 60 so that each timing is synchronized. The processing unit 70 controls the rotation timing and speed of the calibration device. The processing unit 70 controls the opening and closing timing of the shutter of the light source 30. The processing unit 70 controls the timing of the start of measurement by the photodetector 62 of the spectrometer 60. The processing unit 70 controls these timings using software or the like. In this way, the intensity balance between the lamp light and the Raman scattered light is achieved in the photodetector 62, and a spectrum in which each reference band can be clearly identified can be obtained. This enables stable calibration. Then, when the measurement is completed, the processing unit 70 stops the operation of the driving mechanism 123 and the like.

[0070] Furthermore, in order to achieve a balanced strength, multiple filters 150 may be provided in the opening 125, or a portion of the opening 125 may have an area without a filter. Multiple filters 150 with different characteristics may be prepared and the filters 150 may be partially disposed in the opening 125. Alternatively, a portion of the opening 125 may have a portion where no filter 150 is provided.

[0071] 9 is a side view schematically illustrating the configuration of an embodiment of the calibration device 100. Here, an embodiment is shown in which the calibration device 100 is mounted on an inverted microscope. That is, an objective lens (not shown) is installed below the calibration device 100.

[0072] The calibration device 100 is installed below the base plate 80. A drive mechanism 123 is provided above the base plate 80. The drive mechanism 123 is a rotary motor, and is fixed to the base plate 80. The base plate 80 supports the drive mechanism 123 and the calibration device 100. Furthermore, a lamp light source 160 is attached to the base plate 80. The rotation shaft 122 passes through the base plate 80.

[0073] Furthermore, a lamp light source 160 is attached to the underside of the base plate 80. Legs 81 are provided at the ends of the base plate 80. The legs 81 are fixed to the stage of a microscope or the like. The legs 81 may have a height adjustment function so that the standard sample is positioned within the sample plane of the Raman spectrometer.

[0074] Although FIG. 9 shows the calibration device 100 mounted on an inverted microscope, the calibration device 100 can also be mounted on microscopes other than inverted microscopes. The calibration device 100 can also be mounted on an upright microscope or a stereo microscope. For example, when mounting the calibration device 100 on an upright microscope, the configuration of the calibration device 100, the drive mechanism 123, etc., only needs to be inverted upside down. In other words, the drive mechanism 123 only needs to be installed below the calibration device 100. Furthermore, the drive mechanism 123 is not limited to a rotary motor, and may also be a linear motor, etc. In other words, the drive mechanism 123 only needs to move multiple standard samples into the field of view of the objective lens 50.

[0075] Modifications Furthermore, a fiber lamp for calibrating the intensity of a Raman spectrometer can be attached to the calibration device 100. Figures 10 and 11 are photographs showing a calibration device 100 according to a modification. Figure 10 is a photograph showing the upper side of the calibration device 100, and Figure 11 is a photograph showing the lower side.

[0076] As shown in Figure 10, the base plate 80 is provided with a connection port 83 for connecting an optical fiber 201. The incident end of the optical fiber 201 is connected to a standard light source 200, and the exit end 202 is connected to the connection port 83. The standard light source 200 serves as a lamp light source for calibrating the intensity of the Raman spectrometer. The standard light source 200 generates light for calibrating the intensity of the Raman spectrometer. The optical fiber 201 then propagates the light for calibrating the intensity of the Raman spectrometer. Therefore, the light for calibrating the intensity of the Raman spectrometer propagates through the optical fiber 201 and is emitted from the exit end 202.

[0077] When performing wavenumber calibration, the calibration device 100 is placed in the field of view of the objective lens 50. When performing intensity calibration of the Raman spectrometer, the exit end 202 of the optical fiber 201 is moved into the field of view of the objective lens 50. That is, by moving the base plate 80 relative to the objective lens 50, wavenumber calibration and intensity calibration of the Raman spectrometer can both be performed. This allows for simple calibration.

[0078] The number of calibration materials may be one or more. For example, only a single standard sample may be provided in the calibration device 100. Then, the processing unit 70 may perform calibration based on the Raman spectrum obtained from the single standard sample.

[0079] Furthermore, because the Raman shift of a material depends on temperature, the processing unit 70 may perform calibration using the temperature of the material. That is, the temperature of the standard sample can be measured and used for calibration. For example, a temperature sensor such as a thermocouple or a resistance thermometer is placed near the standard sample. The processing unit 70 can achieve more accurate calibration by using the Raman shift at the measured temperature of the peak of the reference spectrum.

[0080] Some or all of the processing of the processing unit 70 described above may be executed by a computer program. The program described above can be stored in various types of non-transitory computer-readable media and supplied to a computer. Non-transitory computer-readable media include various types of tangible storage media. Examples of non-transitory computer-readable media include magnetic recording media (e.g., flexible disks, magnetic tapes, hard disk drives), magneto-optical recording media (e.g., magneto-optical disks), CD-ROMs (Read Only Memory), CD-Rs, CD-R / Ws, and semiconductor memories (e.g., mask ROMs, PROMs (Programmable ROMs), EPROMs (Erasable PROMs), flash ROMs, and RAMs (Random Access Memory)). The program may also be supplied to a computer by various types of transient computer-readable media. Examples of transient computer-readable media include electric signals, optical signals, and electromagnetic waves. The transient computer-readable medium can supply the program to a computer via a wired communication path such as an electric wire or optical fiber, or via a wireless communication path.

[0081] The invention made by the inventor has been specifically described above based on an embodiment, but it goes without saying that the present invention is not limited to the above embodiment and can be modified in various ways without departing from the gist of the invention.

[0082] This application claims priority based on Japanese Patent Application No. 2022-166762, filed on October 18, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0083] REFERENCE SIGNS LIST 10 Raman spectrometer 30 Light source 40 Optical system 50 Objective lens 60 Spectrometer 61 Spectroscopic section 62 Photodetector 70 Processing section 80 Base plate 81 Leg 110 First plate 120 Second plate 122 Rotation shaft 123 Driving mechanism 125 Opening 130 Cover glass 140 Sample substrate 141 to 144 Standard sample 150 Filter 160 Lamp light source L1 Laser light L2 Raman scattered light L3 Lamp light L4 Detection light

Claims

1. A calibration device for calibrating the wavenumber of a Raman spectroscopy measurement device, comprising: one or more standard samples, which are removably installed in the optical path of the laser light of the Raman spectroscopy measurement device and have a known Raman shift; and a holder for holding the one or more standard samples.

2. The calibration device according to claim 1, further comprising a drive mechanism for driving the holder so that the laser light of the Raman spectroscopy measurement device irradiates the plurality of standard samples in sequence.

3. The calibration device according to claim 1 or 2, wherein the plurality of standard samples are inorganic materials.

4. The calibration device according to claim 2, wherein the drive mechanism rotates the holder around a rotation axis offset from the optical axis of the objective lens of the Raman spectroscopy measurement device.

5. The plurality of standard samples are installed on a cover glass, and the Raman scattered light generated by the standard sample enters the objective lens of the Raman spectroscopy measurement device through the cover glass. The calibration device according to claim 4.

6. The calibration device according to claim 2, further comprising a base plate for supporting the drive mechanism, and a fiber for propagating light for calibrating the intensity of the Raman spectroscopy measurement device is connected to the base plate.

7. The calibration device according to claim 1 or 2, further comprising a lamp light source for generating lamp light having a plurality of emission lines.

8. The holder has an opening through which the lamp light passes, and a filter having different transmittances according to wavelength is disposed in the opening. The calibration device according to claim 7.

9. A lamp light source for generating lamp light having a plurality of emission lines; a laser light source for generating laser light; one or more standard samples having a known Raman shift, which are removably provided in the optical path of the laser light; an objective lens into which the lamp light and the Raman scattered light from the one or more standard samples enter; a spectroscope for spectroscopically detecting the lamp light and the Raman scattered light from the objective lens; and a processing unit for calculating a calibration wavelength axis of the spectroscope based on the wavelengths of the plurality of emission lines, calculating an incident wavelength of the laser light based on the Raman band of the Raman scattered light on the calibration wavelength axis, and converting the calibration wavelength axis to a wavenumber axis using the incident wavelength. A Raman spectroscopy measurement device.

10. The Raman spectroscopy measurement device according to claim 9, wherein the plurality of standard samples are inorganic materials.

11. A holder for holding the plurality of standard samples; The Raman spectroscopic measurement apparatus according to claim 9 or 10, further comprising: a drive mechanism that drives the holder so that the laser light is incident on the plurality of standard samples in order.

12. The Raman spectroscopic measurement apparatus according to claim 11, wherein the drive mechanism rotates the holder around a rotation axis deviated from the optical axis of the objective lens.

13. The holder has an opening through which the lamp light passes, The Raman spectroscopic measurement apparatus according to claim 11, wherein a filter having different transmittances according to wavelengths is disposed in the opening.

14. A plurality of inorganic materials, which are the plurality of standard samples, are placed on a cover glass, The Raman spectroscopic measurement apparatus according to claim 10, wherein the Raman scattered light is incident on the objective lens through the cover glass.

15. A wavenumber calibration method for a Raman spectroscopic measurement apparatus that spectroscopically measures Raman scattered light, comprising: spectroscopically measuring Raman scattered light from one or more standard samples irradiated with laser light by the Raman spectroscopic measurement apparatus; spectroscopically measuring lamp light having a plurality of emission lines by the Raman spectroscopic measurement apparatus; calculating a calibration wavelength axis based on wavelengths of the plurality of emission lines; calculating an incident wavelength of the laser light based on a Raman band of the Raman scattered light on the calibration wavelength axis; converting the calibration wavelength axis into a wavenumber axis using the incident wavelength, and The wavenumber calibration method, wherein Raman shifts of the one or more standard samples are known.