Calibration device, Raman spectroscopy apparatus, and wavenumber 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

[Technical Field]

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

[0002] Raman spectroscopy is useful for in-situ analysis of products, prototypes, and biological samples because it allows for the label-free and non-destructive identification of the types and states of molecules contained in a sample. In Raman spectroscopy, the spectrum of Raman scattered light, or Raman spectrum, is used to identify the types and states of molecules.

[0003] Non-Patent Document 1 discloses a calibration method for Raman scattered light. In Non-Patent Document 1, standard samples and neon lamps are measured and fitted using a polynomial. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] 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] [Overview of the project]

[0005] The Raman shift corresponds to the intrinsic vibrational energy of a molecule, and the wavenumber [cm] -1 This is shown by ]. Since the natural vibrational modes of a molecule are determined by the type and state of the molecule, the type and state of the molecule can be analyzed using the horizontal axis of the Raman spectrum, i.e., the wavenumber, as a clue. The wavenumber has the following relationship with the excitation wavelength [nm] and scattering wavelength [nm]. Wave number [cm -1 ]=10 7 / (incident wavelength)−10 7 / (scattered wavelength)

[0006] In the most widely used dispersive Raman spectroscopy instruments, a spectrometer is used, and therefore the peak wavelength of the Raman scattered light is detected. More specifically, because the wavelength is dispersed by the spectroscopic element, the position on the dispersion axis of the detection space at 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] This disclosure has been made in view of the above points, and aims to provide a calibration device, a Raman spectrometer, and a wavenumber calibration method that can perform wavenumber calibration of Raman spectroscopic measurements with high accuracy.

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

[0009] The calibration device described above may further include a drive mechanism for driving the holder so that the laser light from the Raman spectrometer is irradiated onto the plurality of materials in sequence.

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

[0011] In the calibration device described above, the drive mechanism may be configured to rotate the holder around a rotation axis offset from the optical axis of the objective lens of the Raman spectrometer.

[0012] In the calibration device described above, the plurality of materials may be placed on a cover glass, and the Raman scattered light generated by the materials may be incident on the objective lens of the Raman spectrometer via the cover glass.

[0013] The calibration device described above further comprises a base plate supporting the drive mechanism, and a fiber for propagating light for intensity calibration of a Raman spectrometer may be connected to the base plate.

[0014] The calibration device described above may further include a lamp light source that generates lamp light having multiple emission lines.

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

[0016] The Raman spectroscopy apparatus according to this embodiment comprises 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 removablely provided in the optical path of the laser light, an objective lens into which the lamp light and Raman scattered light from the one or more materials are incident, a spectrometer that spectrally analyzes and detects the lamp light and the Raman scattered light from the objective lens, and a processing unit that calculates the calibration wavelength axis of the spectrometer 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 calibration wavelength axis, and converts the calibration wavelength axis to a wavenumber axis using the incident wavelength.

[0017] In the Raman spectroscopy apparatus described above, the plurality of materials may be inorganic materials.

[0018] The Raman spectroscopy apparatus described above may further include a holder for holding the plurality of materials, and a drive mechanism for driving the holder so that the laser light is incident on the plurality of materials in sequence.

[0019] In the Raman spectroscopy apparatus described above, the drive mechanism may be configured to rotate the holder around a rotation axis offset from the optical axis of the objective lens.

[0020] In the above Raman spectroscopic measurement device, the holder has an opening through which the lamp light passes, and a filter having a transmittance corresponding to the wavelength may be disposed in the opening.

[0021] In the above Raman spectroscopic measurement device, the plurality of materials may be installed on the cover glass, and the Raman scattered light may be incident on the objective lens through the cover glass.

[0022] The wavenumber calibration method according to the present embodiment includes steps of spectroscopically measuring Raman scattered light from one or a plurality of materials irradiated with laser light by a Raman spectroscopic measurement device; spectroscopically measuring lamp light having a plurality of emission lines by the Raman spectroscopic measurement device; calculating a calibration 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 calibration wavelength axis; and converting the calibration 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 spectroscopic measurement device, and a wavenumber calibration method capable of performing wavenumber calibration of Raman spectroscopic measurement with high accuracy.

Brief Description of Drawings

[0024] [Figure 1] It is a diagram showing the overall configuration of a spectroscopic measurement device. [Figure 2] It is a perspective view showing the configuration of a calibration device. [Figure 3] It is a perspective view showing the configuration of a calibration device. [Figure 4] It is a diagram showing the emission line spectrum of a neon lamp. [Figure 5] It is a wavenumber spectrum showing the Raman shift of silicon. [Figure 6] It is a diagram showing a spectrum obtained by calibrating measurement results of different Raman microscopes. [Figure 7] It is a table showing the specifications of three Raman spectroscopic measurement devices. [Figure 8] This figure shows the spectral measurement results on the calibration wavenumber axis. [Figure 9] This is a schematic side view illustrating an example of a calibration device. [Figure 10] This is a photograph showing the device configuration for a modified example. [Figure 11] This is a photograph showing the device configuration for a modified example. [Modes for carrying out the invention]

[0025] The following describes embodiments to which the present invention may be applied. The following description illustrates embodiments of the present invention, and the present invention is not limited to these embodiments. For clarity of explanation, the following description has been omitted and simplified as appropriate. Furthermore, those skilled in the art will be able to easily modify, add, and transform each element of the following embodiments within the scope of the present invention. In each figure, elements denoted by the same reference numerals indicate similar elements, and their descriptions are omitted as appropriate.

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

[0027] The calibration device 100 is equipped with a sample substrate 140, such as a standard sample substrate. The calibration device 100 is detachably installed on the Raman spectrometer 10. The calibration device 100 is attached to the sample stage of the Raman spectrometer 10 only when measuring a standard spectrum for calibration. When measuring an actual sample spectroscopically, 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 an Nd / YVO4 laser that emits CW (Continuous Wave) laser light with a wavelength of 532 nm. Of course, the light source 30 may be other laser light sources. 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 spectrometer 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 as to illuminate a line-shaped sample area where the slit of the spectrometer 60 is imaged. Alternatively, an optical scanner may illuminate a line-shaped area on a standard sample by scanning the laser light L1. Furthermore, a laser line filter or the like may be included to prevent light of a different wavelength from the laser light L1 from entering the standard sample or the spectrometer 60. An edge filter or the like may be included to block the laser wavelength so that the laser light L1 does not enter the spectrometer 60. Since known configurations can be used for the optical system 40, a detailed explanation is omitted.

[0030] The laser light L1 propagated by the optical system 40 enters the objective lens 50. The objective lens 50 focuses the laser light L1 onto the sample substrate 140 of the calibration device 100. The objective lens 50 forms a focal point for the laser light L1 on the sample substrate 140. This generates Raman scattered light L2 on the sample substrate 140. A portion of the Raman scattered light L2 generated on the sample substrate 140 enters the objective lens 50. The Raman scattered light L2 from the objective lens 50 becomes detection light L4 and enters the optical system 40. The optical system 40 propagates the detection light L4 to the spectrometer 60.

[0031] The detected light L4 from the optical system 40 is incident on the spectrometer 60. The spectrometer 60 comprises a spectroscopic unit 61 and a photodetector 62, and performs spectral measurement of the detected light L4. The spectroscopic unit 61 is equipped with a slit and a wavelength-dispersing element, etc. The wavelength-dispersing element is a diffraction grating or a prism. Furthermore, the spectroscopic unit 61 may also be equipped with a concave mirror or a lens. The spectroscopic unit 61 disperses the detected 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 equipped with multiple pixels arranged in an array. The photodetector 62 is 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 to the processing unit 70 according to the amount of light detected by each pixel. The processing unit 70 is a personal computer or the like, and stores the value of the detection signal in memory or the like, associating it with the XY coordinates of the pixels.

[0033] For example, the photodetector 62 is a cooled CCD camera with multiple pixels arranged in the X and Y directions. The pixels of the photodetector 62 are arranged along the 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 of the spectroscopic unit 61 (X direction). The distribution of light intensity in the X direction of the photodetector 62 will show 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 detected light L4. The photodetector 62 outputs detection data for each pixel to the processing unit 70.

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

[0035] Lamp light L3 from lamp light source 160 enters the calibration device 100 via filter 150. Filter 150 has different transmittances depending on the wavelength. 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 spectrometer 60. Thus, the lamp light L3, like the Raman scattered light L2, is spectrally measured in the spectrometer 60.

[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 be provided separately from the calibration device 100. For example, the lamp light source 160 may be positioned in front of the spectrometer 60, offset 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 spectrometer 60, the position of the lamp light source 160 is not particularly limited.

[0038] Next, the detailed configuration of the calibration device 100 will be described using Figure 1, as well as Figures 2 and 3. Figures 2 and 3 are schematic perspective views showing the external appearance of the calibration device 100. Figure 2 is a view of the calibration device 100 from the objective lens 50 side, and Figure 3 is a view of the calibration device 100 from the lamp light source side.

[0039] The calibration device 100 comprises a first plate 110, a second plate 120, a cover glass 130, and a sample substrate 140. The sample substrate 140 is formed from a plurality of standard samples 141 to 144. Here, the plurality of standard samples 141 to 144 are each configured as a separate substrate. As shown in Figure 3, the second plate 120 is provided with a rotation axis 122 and an opening 125. The first plate 110 and the second plate 120 constitute a rotating disc for rotating 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 Figure 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 is suitable for calibration because it does not emit strong Raman scattering light. Alternatively, the cover glass 130 may be calcium fluoride or the like, which emits Raman scattering light with a narrow line width. Standard samples 141-144 are made 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. SiC and Si are opaque, while the diamond and calcium fluoride substrates are transparent.

[0041] Standard samples 141-144 are inorganic materials with known Raman shifts. Standard samples 141-144 are arranged in the same plane perpendicular to the optical axis of the objective lens 50. Since standard samples 141-144 are formed as separate substrates, they are each placed on the cover glass 130. Standard samples 141-144 may also be formed as a single sample substrate 140. Standard samples 141-144 are arranged in the optical path of the laser beam so as to be removable. For example, when the calibration device 100 is attached to the sample stage, etc., standard samples 141-144 are inserted into the optical path. When the calibration device 100 is removed from the sample stage, etc., standard samples 141-144 are removed from the optical path.

[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. As shown in Figures 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 beam L1 to 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 Figure 1, a drive mechanism 123 is attached to the rotation axis 122. The drive mechanism 123 has 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 drive of the drive mechanism 123. In other words, the processing unit 70 controls the rotation timing and rotation speed of the drive mechanism 123. As the drive mechanism 123 rotates the calibration device 100, the irradiation position of the laser beam L1 on the calibration device 100 changes. In other words, the laser beam L1 is irradiated sequentially onto the standard samples 141 to 144.

[0045] As shown in Figure 3, the second plate 120 is provided with an opening 125. Lamp light from the lamp light source 160 passes through the opening 125. As shown in Figure 1, a filter 150 (omitted in Figure 3) is installed in the opening 125. Therefore, the lamp light L3 passes through the opening 125 and enters the objective lens 50. A standard sample 144 may or may not be located directly below the filter 150. 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 drive 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 in sequence. In addition, lamp light L3 is also incident on the objective lens 50. The detected light L4 includes 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 detected light L4. The spectral measurement results of the detected light L4 become calibration data that includes the spectrum of the lamp light L3 and the spectra of the Raman scattered light L2 from the standard samples 141-144.

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

[0048] Figure 5 shows the Raman shift spectrum when standard sample 141 is silicon. In Figure 5, the horizontal axis represents the Raman shift [cm]. -1The vertical axis represents light intensity. By using highly crystalline inorganic materials such as Si, a narrow-linewidth peak can be obtained. Of course, the wavenumber of the peak differs for each 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 has a processor and memory. For example, the processing unit 70 has memory that stores detection data associated with pixel addresses. The processor of the processing unit 70 performs the processing described later by executing a program stored in memory.

[0050] The processing unit 70 calculates the calibration wavelength axis of the spectrometer 60 based on the wavelengths of multiple emission lines. The lamp light source 160 has almost no variation in emission lines. Therefore, the pixel position (pixel address) in the X direction of the photodetector 62 can be associated with the wavelength. In other words, the processing unit 70 finds the pixel position that corresponds to the peak wavelength of the measured spectrum of the lamp light L3. Then, the processing unit 70 associates the pixel position that corresponds to the peak wavelength with the wavelength of the emission line. The relationship between the pixel position of the photodetector 62 and the wavelength is approximated by a polynomial or the like. For example, the least squares method can be used for the polynomial approximation. The processing unit 70 finds an approximation formula that replaces the pixel position with the wavelength. In this way, the processing unit 70 can find the calibration 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 calibration 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 wavenumber of the Raman shift is constant regardless of the laser wavelength (incident wavelength).

[0052] When the incident wavelength (laser wavelength) and scattering wavelength (detection wavelength) are expressed in units of [nm], the Raman shift wavenumber [cm] -1 ] is shown by the following equation (1). Wave number [cm -1 ]=10 7 / (Incident wavelength) - 10 7 / (Scattered wavelength) ··· (1)

[0053] Standard samples 141 to 144 have known wavenumbers of Raman bands [cm -1 . Therefore, the processing unit 70 determines the peak wavelength of the spectrum on the calibration wavelength axis as the scattered wavelength of the Raman scattered light. The processing unit 70 can obtain the laser wavelength from the scattered wavelength on the calibration wavelength axis. That is, since the wavenumber of the Raman band of the inorganic material is known, substituting the scattered wavelength on the calibration wavelength axis into the scattered wavelength in Equation (1) indicates that the incident wavelength in Equation (1) is the laser wavelength. The processing unit 70 obtains the laser wavelength using the calibration wavelength axis.

[0054] The processing unit 70 can calculate the laser wavelength for each of the plurality of standard samples. By doing so, the processing unit 70 can accurately estimate the laser wavelength. For example, the processing unit 70 may obtain the incident wavelength with the scattered wavelengths of the plurality of standard samples according to Equation (1) and use their average value as the laser wavelength.

[0055] Then, the processing unit 70 converts the calibration wavelength axis to a wavenumber axis using the incident wavelength (laser wavelength) of the laser light. Specifically, the wavelength corresponding to each pixel position is obtained using the above approximate formula. The laser wavelength and the wavelength indicated by each pixel are substituted into the incident wavelength and the scattered wavelength in Equation (1), respectively. By doing so, a wavenumber axis indicating the wavenumber of the Raman shift for each pixel position is obtained. The calibrated wavenumber axis is data in which the pixel positions of the photodetector 62 and the wavenumbers are associated.

[0056] The processing unit 70 obtains the wavenumber of the Raman shift of the Raman spectrum of the actual sample using the wavenumber axis (also referred to as the calibrated wavenumber axis) calculated in this way. The processing unit 70 detects the Raman spectrum of the actual sample and obtains the pixel position where the detection intensity peaks. Then, the processing unit 70 converts the pixel position that peaks into a wavenumber. Thereby, the state and type of the molecule can be accurately identified.

[0057] As described above, inorganic materials are used as standard samples 141-144. 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 as standard samples 141-144, 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. In addition, the spectrum of the lamp light has many narrow linewidth 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 possess 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. By using multiple reference bands obtained from several inorganic materials, the excitation wavelength can be calculated accurately. Furthermore, the standard sample may be made from a material other than an inorganic material.

[0059] Furthermore, the wavenumber of the Raman shift can be calculated appropriately even when the laser wavelength fluctuates. Stable spectroscopic measurements can be performed even when the measurement date or measurement equipment differs. In particular, solid-state lasers, whose oscillation wavelength depends on the laser cavity, have a more unstable oscillation wavelength (laser wavelength) compared to gas lasers. In this embodiment, the wavenumber spectrum of the Raman shift can be measured appropriately even when using a laser light source with an unstable oscillation wavelength.

[0060] Furthermore, wavenumber calibration can be performed using the calibration device 100 described above before the actual spectroscopic measurement of the sample. Alternatively, wavenumber calibration can be performed using the calibration device 100 described above after the spectroscopic measurement of the actual sample.

[0061] Spectroscopic measurements of Raman scattered light and lamp light from standard samples 141-144 may be performed with separate camera exposures or with a single camera exposure. For example, during the exposure time of the photodetector 62, the lamp light source 160 is turned on, and the calibration device 100 is rotated while continuously irradiating it with laser light. The photodetector 62 then integrates the Raman scattered light and lamp light, and the Raman scattered light and lamp light can be spectrally measured together. This yields 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 position of each peak.

[0062] 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 large cover glass 130. Therefore, it can be used with Raman spectrometers that assume observation through the cover glass 130. For example, it can be used with Raman spectrometers that use objective lenses with short working distances or immersion objective lenses. Of course, there only needs to be two or more inorganic material standard samples. Also, by placing multiple standard samples with a thickness less than or equal to the depth of focus of the objective lens, the drive mechanism 123 can be omitted. In other words, since the laser light is focused onto 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] Figure 6 shows the spectra obtained by wavenumber calibration of different Raman microscopes using the calibration device 100 described above. The horizontal axis represents the calibration wavenumber axis, and the vertical axis represents the detection intensity. Here, three Raman microscopes are identified as Raman microscopes A to C. Figure 6 shows the emission lines of a neon lamp and the Raman shift of an inorganic material as calibration spectral data. Here, the inorganic materials calcium fluoride (CaF2), silicon (Si), silicon carbide (SiC), and diamond are used as standard samples. Alternatively, inorganic materials such as magnesium fluoride (MgF2), sapphire (α-Al2O3), and calcite (CaCo3) can also be used as standard samples. By selecting standard samples and lamps so that the Raman bands and emission line spectra 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; it may also be a gas. For example, chemically stable materials such as nitrogen gas or oxygen gas can be used as the standard sample. When using a gas, a transparent container filled with the gas should 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 differ in their laser light source, spectrometer 60, camera, and objective lens. The data acquisition time is also different. The wavenumber calibration method described above allows for appropriate calibration of the wavenumber axes of Raman microscopes A to C with different specifications. Therefore, measurement errors between microscopes can be suppressed. Even when using different Raman microscopes, sample classification and identification can be performed appropriately. Consequently, the state and type of molecules can be accurately identified.

[0066] Figure 8 shows the spectroscopic measurement results on a calibrated wavenumber axis. The horizontal axis represents the calibration wavenumber axis, and the vertical axis represents the detection intensity. Here, polystyrene or ethanol is used as the actual sample. Here, the wavenumbers of the spectroscopic measurement results from Raman microscopes A to C described above are calibrated. The wavenumber spectra of the Raman shift for the calibration method according to this embodiment (indicated as "new device") and the calibration method of the comparative example (indicated as "conventional") are also shown. Raman spectra obtained by performing wavenumber calibration multiple times on each of Raman microscopes A to C are shown.

[0067] Figure 8 shows the phenyl ring at a wavenumber of 1004 [cm²]. -1 The wavenumber spectrum around the Raman band of [ ] is shown in magnified view. In the calibration method of the comparative example, a shift in the wavenumber of the Raman band occurs in 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 of this embodiment, the shift in the wavenumber of the Raman band is small. Therefore, calibration can be performed with higher accuracy.

[0068] In this way, standard spectra with multiple wavenumber reference bands and wavelength reference bands can be obtained stably and easily. Therefore, wavenumber calibration of Raman spectrometers can be performed appropriately. This can promote the application of Raman microscopes to the fields of life sciences and medicine.

[0069] Furthermore, the processing unit 70 may control the measurement timing. For example, the processing unit 70 may control the drive 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 of the photodetector 62 of the spectrometer 60. The processing unit 70 controls these timings by 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 spectra that clearly identify each reference band can be obtained. Thus, stable calibration becomes possible. When the measurement is completed, the processing unit 70 stops the operation of the drive mechanism 123 and the like.

[0070] Furthermore, in order to balance the strength, multiple filters 150 may be provided in the opening 125, and there may be areas in the opening 125 where there are no filters. Multiple filters 150 with different characteristics may be prepared and partially placed in the opening 125. Alternatively, there may be parts of the opening 125 where no filters 150 are provided.

[0071] Figure 9 is a schematic side view showing 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. In other words, an objective lens (not shown) is installed on the underside of the calibration device 100.

[0072] A calibration device 100 is installed on the underside of the base plate 80. A drive mechanism 123 is provided on the upper side of 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 rotating 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] Figure 9 shows a calibration device 100 mounted on an inverted microscope, but the calibration device 100 can also be mounted on microscopes other than inverted microscopes. The calibration device 100 can also be mounted on upright microscopes and stereomicroscopes. For example, when mounting the calibration device 100 on an upright microscope, the configuration of the calibration device 100 and the drive mechanism 123 only needs to be inverted vertically. In other words, the drive mechanism 123 only needs to be installed below the calibration device 100. Also, the drive mechanism 123 is not limited to a rotary motor, but may be a linear motor or the like. In other words, the drive mechanism 123 just needs to move multiple standard samples into the field of view of the objective lens 50.

[0075] Variation Furthermore, a fiber lamp for Raman spectroscopy intensity calibration can also be attached to the calibration device 100. Figures 10 and 11 are photographs showing a modified version of the calibration device 100. 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 the optical fiber 201. The input end of the optical fiber 201 is connected to the standard light source 200, and the output end 202 is connected to the connection port 83. The standard light source 200 is a lamp light source for Raman spectrometer intensity calibration. The standard light source 200 generates light for Raman spectrometer intensity calibration. The optical fiber 201 then propagates this light for Raman spectrometer intensity calibration. Therefore, the light for Raman spectrometer intensity calibration propagates through the optical fiber 201 and is emitted from the output 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 Raman spectroscopy intensity calibration, the exit end 202 of the optical fiber 201 is moved into the field of view of the objective lens 50. In other words, by moving the base plate 80 relative to the objective lens 50, wavenumber calibration and Raman spectroscopy intensity calibration can be performed, respectively. Thus, simple calibration is possible.

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

[0079] Furthermore, since the Raman shift of a material is temperature-dependent, the processing unit 70 may use the material's temperature for calibration. In other words, the temperature of a standard sample can be measured and used for calibration. For example, a temperature sensor such as a thermocouple or resistance thermometer can be placed near the standard sample. The processing unit 70 can then use the Raman shift of the peak of the reference spectrum at the measurement temperature to achieve more accurate calibration.

[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 and supplied to a computer using various types of non-transitory computer-readable medium. Non-transitory computer-readable mediums include various types of tangible storage mediums. Examples of non-transitory computer-readable mediums 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 memory (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 using various types of transient computer-readable mediums. Examples of transient computer-readable mediums include electrical signals, optical signals, and electromagnetic waves. Transitory computer-readable mediums can be supplied to a computer via wired communication channels such as electric wires and optical fibers, or via wireless communication channels.

[0081] Although the present invention has been specifically described above based on embodiments, it goes without saying that the present invention is not limited to the above embodiments and can be modified in various ways without departing from its essence.

[0082] This application claims priority based on Japanese Patent Application No. 2022-166762, filed on 18 October 2022, and incorporates all of its disclosures herein. [Explanation of symbols]

[0083] 10. Raman Spectrometer 30 light source 40 Optical system 50 objective lens 60 spectrometer 61 Spectroscopic section 62 Photodetector 70 Processing Unit 80 Base Plate 81 Legs 110 Plate 1 120 Second Plate 122 Rotation axis 123 Drive mechanism 125 Opening 130 Cover glass 140 Sample substrates 141-144 Standard Samples 150 filters 160 Lamp light source L1 laser light L2 Raman scattering light L3 Lamp Light L4 detection light

Claims

1. A calibration device for calibrating the wavenumber of a Raman spectrometer having a microscope objective lens, A plurality of standard samples, whose Raman shift is known, are placed at the focal plane of the microscope objective lens in the optical path of the laser beam of the Raman spectrometer, A single cover glass on which the aforementioned multiple standard samples are placed, The laser light from the microscope objective lens is incident on the standard sample through the cover glass, and the cover glass and the holder for holding the plurality of standard samples are provided. The holder is driven by a drive mechanism that drives the holder so that the laser light from the microscope objective lens is irradiated sequentially onto the plurality of standard samples. A calibration device in which Raman scattered light generated by the standard sample is incident on the microscope objective lens via the cover glass.

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

3. The calibration device according to claim 2, wherein the cover glass is fitted with the standard samples of diamond, CaF2, Si, and SiC.

4. The drive mechanism rotates the holder around a rotation axis offset from the optical axis of the microscope objective lens, The aforementioned holder is, A first plate is positioned on the microscope objective lens side of the cover glass and to which the cover glass is fixed, The calibration device according to claim 1, further comprising a second plate to which the aforementioned rotating shaft is attached.

5. The drive mechanism further comprises a base plate supporting the drive mechanism, The calibration device according to claim 1, wherein a fiber for propagating light for intensity calibration of a Raman spectrometer is connected to the base plate.

6. The calibration device according to any one of claims 1 to 5, further comprising a lamp light source that generates lamp light having multiple emission lines.

7. The holder has an opening through which the lamp light passes, The calibration device according to claim 6, wherein a filter having different transmittances depending on the wavelength is arranged in the aperture.

8. A lamp light source that generates lamp light having multiple emission lines, The aforementioned lamp light and Raman scattered light are incident on a microscope objective lens, A laser light source that generates laser light, A calibration device that can be placed in the field of view of the microscope objective lens, A spectrometer that spectrally analyzes and detects the lamp light and Raman scattered light from the microscope objective lens, The system includes a processing unit that calculates the calibration wavelength axis of the spectrometer 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 calibration wavelength axis, and converts the calibration wavelength axis to a wavenumber axis using the incident wavelength, The calibration device, Multiple standard samples with known Raman shifts, A single cover glass on which the aforementioned multiple standard samples are placed, The holder for holding the cover glass and the plurality of standard samples is provided so that the laser light from the microscope objective lens is incident on the standard sample through the cover glass, The holder is driven so that the laser light from the microscope objective lens is incident on the plurality of standard samples in sequence, The calibration device can be installed in the optical path of the laser beam so that the standard sample is placed at the focal plane of the microscope objective lens in the optical path of the laser beam. A Raman spectroscopic measuring device in which the Raman scattered light generated by the standard sample is incident on the microscope objective lens via the cover glass.

9. The Raman spectrometer according to claim 8, wherein the plurality of standard samples are inorganic materials.

10. The Raman spectrometer according to claim 9, wherein the standard samples of diamond, CaF2, Si, and SiC are placed on the cover glass.

11. The drive mechanism rotates the holder around a rotation axis offset from the optical axis of the microscope objective lens, The aforementioned holder is, A first plate is positioned on the microscope objective lens side of the cover glass and to which the cover glass is fixed, The aforementioned rotating shaft is attached to a second plate, A Raman spectrometer according to any one of claims 8 to 10.

12. The holder has an opening through which the lamp light passes, The Raman spectrometer according to claim 11, wherein a filter having different transmittances depending on the wavelength is arranged in the aperture.

13. Further comprising a base plate supporting the drive mechanism, The Raman spectrometer according to claim 11, wherein a fiber for propagating light for intensity calibration of the Raman spectrometer is connected to the base plate.

14. A wavenumber calibration method for a Raman spectrometer that performs spectral measurements of Raman scattered light, The steps include: placing a calibration device containing multiple standard samples within the field of view of the microscope objective lens; The steps include: using the Raman spectroscopy apparatus to spectrally measure the Raman scattered light from the plurality of standard samples irradiated with laser light; The steps include: performing spectral measurements of lamp light having multiple emission lines using the aforementioned Raman spectrometer; A step of calculating a calibration wavelength axis based on the wavelengths of the plurality of emission lines, A step of calculating the incident wavelength of the laser light based on the Raman band of the Raman scattered light on the calibration wavelength axis, The system includes the step of converting the calibration wavelength axis to the wavenumber axis using the incident wavelength, The calibration device is A single cover glass on which the aforementioned multiple standard samples are placed, The holder for holding the cover glass and the plurality of standard samples is provided so that the laser light from the microscope objective lens is incident on the standard sample through the cover glass, The holder is driven so that the laser light from the microscope objective lens is irradiated sequentially onto the plurality of standard samples, The Raman shifts of the aforementioned multiple standard samples are known, and they are placed at the focal plane of the microscope objective lens in the optical path of the laser light, A wavenumber calibration method in which Raman scattered light generated by the standard sample is incident on the microscope objective lens via the cover glass.

15. The drive mechanism rotates the holder around a rotation axis offset from the optical axis of the microscope objective lens, The aforementioned holder is, A first plate is positioned on the microscope objective lens side of the cover glass and to which the cover glass is fixed, The wavenumber calibration method according to claim 14, further comprising a second plate to which the aforementioned rotating shaft is attached.

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