JP7033888B2 - Raman imaging device, Raman imaging system, Raman imaging method, and program for Raman imaging device - Google Patents

Description

The present invention relates to a Raman imaging apparatus that images a sample based on Raman scattered light obtained from the sample.

For example, Raman imaging based on Raman scattered light is performed to analyze and visualize the distribution of the target substance contained in the sample.

Since Raman scattered light has a very weak intensity compared to Rayleigh scattered light, the energy of the excitation light applied to the sample is required to make the Raman scattered light easily detectable by the light detector. Needs to be large.

However, when the energy of the excitation light is increased, the intensity of the fluorescence excited from the sample also increases, and in the spectrum of the measurement light obtained at each measurement point of the sample, the peak of the Raman spectrum is buried in the peak of the fluorescence spectrum. Therefore, it becomes difficult to perform Raman imaging of the target substance in the sample.

In particular, when the sample is a biological sample or the material is placed on paper, the influence of fluorescence becomes remarkable.

Therefore, a fluorescence avoidance method is used in which the peak of fluorescence contained in the measurement light is specified and subtracted from the spectrum of the measurement light.

However, when it is necessary to obtain Raman spectra at each of a plurality of measurement points in a sample as in Raman imaging, it is very troublesome to realize the above fluorescence avoidance method.

This is because the type and amount of the substance that emits fluorescence is different at each measurement point, and the emission intensity of fluorescence is different. Therefore, it is necessary to set an optical system and measurement conditions that can realize fluorescence measurement at all measurement points for each sample.

US Patent Gazette US 8310672

The present invention has been made in view of the above-mentioned problems, and even when the measurement light obtained from each measurement point of the sample contains fluorescence, good Raman imaging without separately specifying the fluorescence. It is an object of the present invention to provide a Raman imaging apparatus capable of obtaining data.

That is, the Raman imaging device according to the present invention is a Raman imaging device that generates Raman imaging data of a sample based on intensity data indicating the intensity of measurement light including Raman scattered light generated at a measurement point in the sample. , A determination unit for determining whether the intensity indicated by the intensity data corresponding to each measurement point in the sample is equal to or higher than the exclusion threshold determined based on the saturation intensity of the light detector in which the measurement light is detected. Image generation that generates Raman imaging data based on the intensity data other than the intensity data determined by the determination unit to indicate the intensity of a value equal to or higher than the exclusion threshold among the intensity data corresponding to each measurement point in the sample. It is characterized by having a part and.

Further, the Raman imaging method according to the present invention is a Raman imaging method that generates Raman imaging data of a sample based on intensity data indicating the intensity of measurement light including Raman scattered light generated at a measurement point in the sample. A determination step for determining whether the intensity indicated by the intensity data corresponding to each measurement point in the sample is equal to or greater than the exclusion threshold determined based on the saturation intensity of the light detector in which the measurement light is detected. Image generation that generates Raman imaging data based on intensity data other than the intensity data determined to indicate the intensity of a value equal to or higher than the exclusion threshold in the determination step among the intensity data corresponding to each measurement point in the sample. It is a Raman imaging method with steps.

In such a case, a fluorescence spectrum having a stronger intensity than the Raman spectrum appears at each measurement point, and Raman imaging is performed for those having a saturation intensity close to or reaching the saturation intensity of the photodetector. It can be prevented from being used for data generation.

Therefore, Raman imaging data can be generated only at the measurement points where the influence of fluorescence is small and the Raman spectrum is dominant, without specifying the fluorescence separately.

In addition, since it is not necessary to obtain individual fluorescence intensity for each measurement point due to the conventional fluorescence avoidance method, it is easy to simplify the optical system for obtaining the measurement light from the sample, and only simple measurement conditions are required. Just set it.

For example, when the target substance for which the presence or absence and the amount thereof are to be specified from the sample generates a characteristic Raman spectrum band in a predetermined wavenumber range, the spectrum derived only from the target substance while eliminating the influence of fluorescence and the like. In order to realize accurate imaging using intensity, the intensity data indicates the spectral intensity, and the range designation unit that specifies the wave number range used in the image generation unit in the spectral intensity. Further, the determination unit is configured to determine whether or not the intensity of each wave number in the spectral intensity is a value equal to or higher than the exclusion threshold within the wave number range specified by the range designation unit. Just do it. The range of the wave number designated by the wave number designation unit may be appropriately designated according to the characteristics of the target substance.

As a specific embodiment for generating Raman image data only at other measurement points by omitting the intensity data of the measurement points where the fluorescence excited from the sample appears, the intensity data indicates the spectral intensity. The determination unit is configured to determine whether or not the intensity of each wave number in the spectral intensity is equal to or higher than the exclusion threshold value, and the image generation unit is set at each measurement point in the sample. Among the corresponding intensity data, it is configured to generate Raman imaging data based on intensity data other than the intensity data for which the intensity of at least one wave number is determined to indicate the intensity equal to or higher than the exclusion threshold by the determination unit. You just have to.

For measurement points that are strongly affected by fluorescence, it is possible to reduce the computational load and speed up the output of Raman imaging data by not analyzing the presence or absence and amount of the target substance in the sample based on the Raman spectrum. To store measurement data, for each measurement point in the sample, measurement data consisting of position data indicating the position of the measurement point in the sample and intensity data corresponding to the measurement point at the position indicated by the position data is stored. Further, the image generation unit sets a measurement point corresponding to the measurement data including the intensity data determined by the determination unit to indicate the intensity of the value equal to or higher than the exclusion threshold as the analysis non-use point. Corresponding to the setting unit that sets the measurement point corresponding to the measurement data including the intensity data determined not to be the intensity data indicating the intensity of the value equal to or higher than the exclusion threshold in the determination unit as the analysis use point, and each analysis use point. Based on the analysis unit, which inputs only the measurement data, analyzes based on the Raman spectrum indicated by the intensity data in the measurement data, and outputs the presence or absence or amount of the target substance at each analysis point, and the analysis result of the analysis unit. , An output unit for outputting Raman imaging data, and an output unit may be provided.

Even if the target substance in the sample is known and the corresponding Raman spectrum is known, or if the target substance in the sample is unknown and the corresponding Raman spectrum is unknown, the distribution state of the substance in the sample can be accurately determined. In order to obtain it as Raman imaging data, the analysis unit may be configured to be analyzed by multivariate analysis based on the intensity data.

For example, even if the measurement light contains Raman scattered light and fluorescence, if it is known that the Raman spectrum appears only in the wave frequency region lower than the peak of fluorescence, the influence of fluorescence further appears in the Raman image data. To prevent this, the intensity data indicates the spectral intensity, and the image generation unit is configured to generate Raman imaging data based only on the spectral intensity at an intensity of a predetermined wave number or less. Just do it.

A Raman measuring device comprising a light source that irradiates a sample with light and the light detector that detects the intensity of the measured light including the Raman scattered light generated at each measurement point of the sample, and the Raman according to the present invention. If it is a Raman imaging system equipped with an imaging device, the optical system of the Raman measuring device is simplified, and the measurement conditions suitable for each measurement point of the sample do not need to be sequentially set, and the sample is good. Raman imaging can be performed.

Even if the measurement light contains fluorescence, only the Raman spectrum in the wave frequency region lower than the fluorescence is detected by the light detector so that Raman imaging data excluding the influence of fluorescence can be generated. It is sufficient that the Raman measuring device further includes a filter through which only the measurement light generated at each measurement point of the sample, which has a predetermined wave number or less, passes through.

In order to be able to enjoy the same effect as the Raman imaging device according to the present invention in the existing Raman imaging device, for example, by simply updating the program, the measurement light including the Raman scattered light generated at the measurement point in the sample. It is a program for a Raman imaging device that generates Raman imaging data of a sample based on the intensity data indicating the intensity of the sample, and the intensity indicated by the intensity data corresponding to each measurement point in the sample is the detected light of the measurement light. Of the determination unit that determines whether the value is equal to or higher than the exclusion threshold determined based on the saturation intensity of the detector and the intensity data corresponding to each measurement point in the sample, the value equal to or higher than the exclusion threshold in the determination unit. An image generation unit that generates Raman imaging data based on intensity data other than the intensity data determined to indicate the intensity of the above, and a program for a Raman imaging device that exerts the function as a computer may be used.

The program for the Raman imaging device is not limited to being electronically distributed, and may be recorded on a program recording medium such as a CD, DVD, HDD, or flash memory.

As described above, according to the Raman imaging apparatus according to the present invention, among the intensity data of the measurement light detected at each measurement point, the measurement point where the influence of fluorescence is strong and the Raman spectrum is difficult to distinguish is excluded. Therefore, Raman imaging data can be generated only at the measurement points where only the Raman spectrum to be targeted appears. Therefore, good imaging and visualization of the sample can be realized based on the Raman spectrum.

The schematic diagram which shows the Raman imaging apparatus and Raman imaging system which concerns on 1st Embodiment of this invention. The schematic functional block diagram which shows the structure of the Raman imaging apparatus of 1st Embodiment. The flowchart which shows the generation procedure of Raman imaging data by the Raman imaging apparatus of 1st Embodiment. The schematic diagram which shows the specific example of the determination by the exclusion threshold by the Raman imaging apparatus of 1st Embodiment. An example of the intensity data of the measured light detected from the sample by the Raman imaging apparatus of the first embodiment. Details of the sample to be Raman imaged by the Raman imaging apparatus of the first embodiment. Raman imaging obtained from a sample by the Raman imaging apparatus of the first embodiment. The schematic diagram which shows the Raman imaging apparatus and Raman imaging system which concerns on 2nd Embodiment of this invention.

The Raman imaging apparatus 100 and the Raman imaging system 200 according to the first embodiment of the present invention will be described with reference to each figure.

As shown in FIG. 1, the Raman imaging system 200 is detected by the Raman measuring device 101 and the Raman measuring device 101, which irradiate the sample S with light and detect the measurement light including the Raman scattered light from the sample S. It is provided with a Raman imaging apparatus 100 that generates Raman imaging data of the sample S based on the intensity of the measured light.

The Raman imaging apparatus 100 is used for imaging ink containing single-walled carbon nanotubes (hereinafter, also referred to as single-walled CNTs) coated on copy paper as sample S, for example. More specifically, characters are also written on the copy paper even with ink, and the ink containing a single-layer CNT using gelatin as a dispersant is applied on the copy paper further above the portion to which the ink is applied. Has been done.

As shown in FIG. 1, the Raman measuring device 101 transmits only a predetermined wavelength component among the light source 11 that irradiates the sample S with laser light as excitation light and the excitation light emitted from the light source 11. A one-band bus filter B11, a half mirror 12 provided so as to reflect the excitation light to the sample S and transmit the measurement light generated from the sample S, and a half mirror 12 provided between the half mirror 12 and the sample S. The objective lens 13, the Rayleigh cut filter 14 that cuts the Rayleigh scattering component of the measurement light transmitted through the half mirror 12, the condenser lens 15 that collects the measurement light after passing through the Rayleigh cut filter 14, and the focal point of the lens. A slit 16 provided in the above, a grating 17 that disperses the measurement light that has passed through the slit 16, and an optical detector 18 that is a multi-channel detector such as a two-dimensional CCD that detects the measurement light dispersed by the grating 17. It is provided with a stage 19 on which the sample S is placed and the measurement point at which the excitation light is irradiated on the sample S is changed. By changing the position of the stage 19 (the position of the XY plane), the excitation light in the sample S is scanned, and the intensity of the measurement light generated at each measurement point of the sample S is detected by the photodetector 18.

It should be noted that a part of the configuration of the Raman measuring device 101 is also configured by using the function of the computer C that realizes the function of the Raman imaging device 100 described later. That is, in the computer C described later, the function as the measurement position command unit 1A for controlling the position of the stage 19 of the Raman measuring device 101 is also exhibited.

Here, the wavelength of the excitation light emitted from the light source 11 is the wavelength at which Raman scattered light having a wave number corresponding to the radial breathing mode (hereinafter also referred to as RBM) in which the single-layer CNT expands and contracts in the radial direction is generated. It is set to. Specifically, the sample S is irradiated with a single-wavelength laser beam having a wavelength of 355 nm or more and 830 nm or less, or a laser beam having a combination of a plurality of wavelengths within the wavelength range described above. The wavelength is set according to the detected diameter of the single-walled CNT. That is, the wavelength of the laser beam can be appropriately set based on the fact that the wave number of the Raman band appearing as RBM is proportional to the reciprocal of the diameter of the single layer CNT.

When the sample S is irradiated with laser light having a wavelength such as these, fluorescence is excited by the excitation light from the copy paper forming the sample S and the ink juice applied to the copy paper, and the photodetector 18 excites the fluorescence. The spectrum will be detected together with the Raman band of the RBM of the single layer CNT.

The function of the Raman imaging device 100 is realized by a so-called computer C provided with various input / output means such as a CPU, a memory, an A / D converter, a D / A converter, and a display and a keyboard. That is, when the program for the Raman imaging device 100 stored in the memory is executed and various devices collaborate, the Raman imaging device 100 has at least the measurement data storage unit 2 and the range designation unit 3 as shown in FIG. , The function as the determination unit 4 and the image generation unit 5.

If necessary, the Raman imaging apparatus 100 includes a copy sheet or a copy paper constituting the sample S within the wave number range designated by the range designation unit 3 among the spectral intensities of each measurement point stored in the measurement data storage unit 2. The determination unit 4 determines that the influence of the excited fluorescence in the ink juice is strong. Based on the determination result of the determination unit 4, the image generation unit 5 does not use the measurement points that are strongly affected by fluorescence, and the single layer in the sample S is based only on the spectral intensities obtained at the other measurement points. It is configured to generate Raman imaging data for CNTs.

Each part will be described in detail.

The measurement data storage unit 2 stores the measurement data of each measurement point of the sample S detected by the Raman measuring device 101. The measurement data shows the position data indicating the position coordinates of the measurement point instructed to the stage 19 in the measurement position command unit 1A and the spectral intensity detected by the optical detector 18 at the measurement point corresponding to the position data. It consists of intensity data. That is, every time the measurement point is changed in the Raman measuring device 101, the measurement data corresponding to the measurement point is accumulated in the measurement data storage unit 2.

The range designation unit 3 designates the wave number range used for Raman imaging in the image generation unit 5 among the measured spectral intensities. The first embodiment aims to image the distribution of single-walled CNTs, which is a target substance, in a sample for each diameter. Therefore, the range designation unit 3 designates the wavenumber range so that only the spectral intensity in the Raman band corresponding to the RBM of the single-walled CNT is used in the image generation unit 5. Since the Raman spectrum corresponding to RBM is generated in a wave number region lower than the fluorescence generated by the copy paper or ink, the influence of such fluorescence can be reduced. Further, since RBM is a Raman band peculiar to single-walled CNTs, it is possible to accurately image the distribution of single-walled CNTs without considering the spectral intensity of the wave number region other than RBM. In other words, when you want to image the distribution of a target substance other than single-walled CNTs, if there is a peculiar Raman band corresponding to the target substance, the range specification unit 3 specifies the range in the wave number region. May be good.

The determination unit 4 determines whether or not the value of the intensity of each wave number in the designated spectral region designated by the range designation unit 3 is equal to or higher than the exclusion threshold value in the spectral intensity indicated by the intensity data constituting the measurement data corresponding to each measurement point. judge. By configuring the determination unit 4 in this way, for example, even if the intensity is smaller than the exclusion threshold value at each wave number of the RBM Raman band and the intensity is higher than the exclusion threshold value at a wave number higher than the RMB Raman band, it will be described later. There is no problem that the image generation unit 5 is no longer used. The exclusion threshold is set based on the saturation intensity of the photodetector 18 of the Raman measuring device 101. In the first embodiment, an intensity 10% to 20% smaller than the saturation intensity is set as the exclusion threshold. The exclusion threshold can be appropriately set based on the saturation intensity, and is not limited to this example.

Of the intensity data included in the measurement data corresponding to each measurement point in the sample S, the image generation unit 5 determines the measurement data of the measurement point when the intensity of one wave number in the determination unit 4 is equal to or higher than the exclusion threshold. Is not used, and Raman imaging data of sample S is generated based on other measurement data.

Specifically, the image generation unit 5 is based on the setting unit 51 that sets the measurement data of the measurement points used to generate the Raman imaging data based on the determination result of the determination unit 4, and the setting result of the setting unit 51. Analysis of the analysis unit 52 and the analysis unit 52 that analyze the measurement data of some measurement points and output the value regarding the presence or absence of the single-layer CNT that is the target substance OB at the measurement point or the amount of the single-layer CNT. It includes an output unit 53 that outputs Raman imaging data based on the result.

The setting unit 51 sets a measurement point corresponding to the measurement data including the intensity data determined by the determination unit 4 to indicate the intensity of the value equal to or higher than the exclusion threshold value as the analysis non-use point. Further, the measurement point corresponding to the measurement data including the intensity data determined by the determination unit 4 to be not the intensity data indicating the intensity of the value equal to or higher than the exclusion threshold is set as the analysis use point. For example, the setting unit 51 assigns an identifier indicating whether the analysis is not used or the analysis used point to the measurement data corresponding to each measurement point.

Only the measurement data corresponding to the analysis use point is input to the analysis unit 52. The intensity data of the measurement data input to the analysis unit 52 is subjected to multivariate analysis by the analysis unit 52, and the presence or absence of a single-layer CNT and the number of single-layer CNTs at the measurement point set as the analysis use point are determined. It is output. For multivariate analysis, various methods such as PCA, MCR, and CLS are used. In addition, it does not matter whether there is a teacher or no teacher.

Further, the analysis unit 52 performs multivariate analysis by limiting the spectral intensity indicated by the intensity data to the spectral intensity of a predetermined wave number or less, and outputs the number of single-layer CNTs present at the analysis use point in the sample S. I try to do it. More specifically, the analysis unit 52 is configured to perform multivariate analysis using only the spectral intensity in the Raman band of the RBM within the wave number range specified by the range designation unit 3 in the intensity data. There is. In other words, the intensities of all wavenumbers included in the intensity data are not used in the multivariate analysis, and the analysis is performed only with the intensity data in the limited wavenumber region. Since the purpose of the first embodiment is to detect the single-layer CNT which is the target substance OB in the sample S, the wavenumber region corresponding to the Raman band of RBM is used for multivariate analysis. That is, the predetermined wave number is set to 400 cm ^-1 .

The output unit 53 outputs the position data of the analysis use point used in the analysis unit 52 and the Raman imaging data in which the color is set according to the number of single-layer CNTs of the analysis use point output by the analysis unit 52. do. The output unit 53 treats the position corresponding to the analysis non-use point as toothless data and performs transparent processing, or sets the color in the same manner as when the single-walled CNT does not exist.

The operation of the Raman imaging system 200 configured in this way will be described with reference to the flowchart shown in FIG.

First, the spectral intensity of each measurement point of the sample S is measured by the Raman measuring device 101 (step S1). The position of the measured measurement point and the spectral intensity obtained at the measurement point are stored as measurement data in the measurement data storage unit 2 of the Raman imaging apparatus 100 (step S2). Specifically, the stage 19 is sequentially moved so that the measurement point of the sample S is irradiated with the excitation light, the spectral intensity at that time is measured, and the measurement data is taken into the measurement data storage unit 2.

Next, the range designation unit 3 designates the wave number range used in the determination unit 4 and the image generation unit 5 in each intensity data (step S3). For example, it is specified in the wave number range input by the user by the input means of the computer.

Regarding the spectral intensity indicated by the measurement data obtained at the measurement point, the determination unit 4 determines whether or not the intensity of each wave number within the wave number range designated by the range designation unit 3 is equal to or greater than the exclusion threshold value (step S4).

When the determination unit 4 determines that the intensity of one wave number is equal to or higher than the exclusion threshold value, the setting unit 51 sets the measurement point corresponding to the measurement data as the analysis non-use point, and Raman imaging. (Step S5).

On the other hand, when the determination unit 4 determines that the intensities of all wave numbers are less than the exclusion threshold, the setting unit 51 sets the measurement points corresponding to the measurement data as the analysis use points and performs Raman imaging. To be used (step S6).

Here, the operation by the range designation unit 3, the determination unit 4, and the setting unit 51 will be further described with a simple example. FIG. 4A shows that there are measurement points 1 to 9 in a part of the sample region, and FIG. 4B shows the spectral intensities obtained at the measurement points 1 to 9, respectively. The wavenumber region shown in the two thin lines of FIG. 4B is the region designated by the wavenumber designation unit, and the dotted line indicates the exclusion threshold value. The intensity spectrum of No. 4 exceeds the exclusion threshold in the region outside the Raman band of RBM, but since there is no intensity exceeding the exclusion threshold in the wave number range specified by the range designation unit 3, at the measurement point No. 4. The obtained measurement data will be used by the image generation unit 5. On the contrary, the measurement points No. 1, 3, and 5 are not used in the image generation unit 5 because they have an intensity equal to or higher than the exclusion threshold within the wave number range specified by the range specification unit 3.

The analysis unit 52 performs multivariate analysis only on the measurement points set as the analysis use points and not excluded, and outputs a value indicating the presence or absence of single-walled CNTs or the abundance thereof at the measurement points ( Step S6). Here, the analysis unit 52 multivariately analyzes only the data in the region having a predetermined wave number or less among the output data corresponding to each analysis use point. FIG. 5 shows an example of spectral intensity data designated as an analysis point and subjected to multivariate analysis. As shown in the spectrum of FIG. 5, only the region containing the Raman band corresponding to the RBM of the single-walled CNT in the wavenumber region of 400 cm ^-1 or less is the target of the multivariate analysis. There is almost no influence of fluorescence in the wavenumber region of 400 cm ^-1 or less, and the spectrum corresponding to RBM appears almost. The portion where the spectral intensity is large at a wave number larger than 400 cm ^-1 is the portion where the intensity is increased within the exclusion threshold due to the influence of the fluorescence excited in the copy paper or ink that constitutes the sample S. .. The intensity in the wavenumber region larger than 400 cm ^-1 is ignored and not used in multivariate analysis.

Based on the analysis result of the analysis unit 52, the output unit 53 outputs Raman imaging data showing an image in which colors are set according to the amount of single-walled CNTs for analysis points (step S7). Here, since there is no multivariate analysis result for the analysis non-used points, for example, the color when the single-walled CNT does not exist is set. In this way, Raman imaging data showing an image in which the entire measurement region of the sample S is color-coded according to the abundance of single-walled CNTs is generated and displayed on the display.

Next, the experimental results of imaging the distribution of the single-walled CNTs in the sample S by the Raman imaging apparatus 100 and the Raman imaging system 200 of the first embodiment are shown. FIG. 6A is an optical micrograph showing the sample S, and FIG. 6B shows the actual measurement results of what kind of Raman spectrum appears from each of the copy paper, the single-walled CNT, and the ink. In the graph, the left end portion is a portion corresponding to the Raman band of RBM and is a wave number region designated by the range designation unit 3.

As shown in FIG. 7, according to the Raman imaging apparatus 100 and the Raman imaging system 200 of the first embodiment, only the portion of the ink containing the single-walled CNT applied on the ink on the copy paper is Raman imaging. You can see that it is done. It can also be seen that fluorescence is not generated from the region dissatisfied with the single-walled CNT.

According to the Raman imaging apparatus 100 and the Raman imaging system 200 of the first embodiment configured in this way, they are black and have the same color like ink containing ink and single-walled CNTs, so that they cannot be visually identified. Even if it is, the shape of the portion to which the single-walled CNT is applied can be clearly obtained from the Raman imaging data.

The reason why the image of the region where the clear single-walled CNT is applied is obtained is that the peak influences the measurement light even if the Raman spectrum and the fluorescence of the copy paper or ink are excited by the excitation light. This is because the measurement points that appear strongly are not analyzed and are not used in the Raman image.

In addition, the Raman band corresponding to the RBM of the single-walled CNT is included, and the multivariate analysis is performed with the intensity data in the low wavenumber region where fluorescence is unlikely to occur. It contributes to clearly obtaining an image of only a part.

Furthermore, since the reciprocal of the diameter of single-walled CNTs is proportional to the peak wavenumber in the Raman band of RBM, multivariate analysis also provides Raman imaging data showing the distribution of single-walled CNTs on paper or ink by diameter. It is also possible to obtain.

Since single-walled CNTs have a unique property of diameter distribution depending on the manufacturing method, if the distribution of single-walled CNTs by diameter can be imaged, it is possible to determine whether the ink containing single-walled CNTs was manufactured in the same manufacturing lot. Can also be identified. Furthermore, if the diameter control synthesis technology for single-walled CNTs advances and the diameter distribution can be freely controlled, the portion itself coated with the ink containing single-walled CNTs can be used as a unique identification tag. That is, if a database recording the diameter distribution of the ink containing the single-walled CNTs is created and the diameter distribution of the single-walled CNTs printed by the Raman Imaging System 200 or the like of the first embodiment is acquired, the transfer identification is performed. Can be realized.

In addition, since the ink containing single-walled CNTs can be printed in various shapes, the ink containing single-walled CNTs can be further applied on a barcode, QR code (registered trademark), etc. and used together as an identification tag. ..

Further, if the excitation light has a wavelength in the visible region, it is possible to cause a component resonance Raman phenomenon of at least a part of the black single-walled CNT and obtain a Raman band corresponding to the RBM of the single-walled CNT. Therefore, it is not necessary to use a light source 11 having a specific wavelength for the light source 11 of the Raman measuring device 101 as in imaging by fluorescence, for example.

Furthermore, Raman imaging data that clearly shows the target substance in the sample can be obtained without specifying the intensity at which the fluorescence of the copy paper or ink that constitutes the sample S is generated at each measurement point. Can be done. Therefore, unlike the conventional Raman imaging device 100, it is necessary to prepare a special optical system for specifying the fluorescence peak, or to set measurement conditions so that the fluorescence peak can be specified at each measurement point. There is no.

Next, the Raman imaging apparatus 100 and the Raman imaging system 200 according to the second embodiment will be described with reference to FIG. The members corresponding to the members described in the first embodiment are designated by the same reference numerals.

In the Raman imaging system 200 of the second embodiment, the configuration of the Raman measuring device 101 is different from that of the first embodiment.

That is, as shown in FIG. 8, the Raman measuring device 101 of the second embodiment has a different configuration after the Rayleigh cut filter 14. Specifically, the Raman measuring device 101 includes a bandpass filter that allows only the light in the wavelength region corresponding to the RBM of the single-layer CNT to pass through the measurement light that has passed through the Rayleigh cut filter 14, and the measurement light that has passed through the bandpass filter. It is provided with a condensing lens 15 for condensing light, and a CCD as an optical detector 18 for detecting the intensity of the measured light condensed by the condensing lens 15. Specifically, the bandpass filter is configured so that only light in the wavenumber region of 400 cm ^-1 or less is transmitted.

In the Raman measuring device 101 of the second embodiment configured in this way, since the spectroscope does not exist, the intensity detected by the photodetector 18 is the intensity of the entire RBM regardless of the diameter of the single layer CNT. It becomes one strength in the form of a shape. In other words, the intensity data shows a single intensity rather than a spectral intensity.

Further, in the low wavenumber region corresponding to RBM, fluorescence from the paper or ink that constitutes the sample S is unlikely to occur in the first place, so that the influence of fluorescence on the obtained intensity data can be further reduced. In addition, the analysis unit 52 does not limit the wave number region to be analyzed as in the first embodiment, but naturally performs analysis only by the intensity of the wave number region corresponding to the RBM of the single-walled CNT, and at each measurement point. Raman imaging data can be obtained that clearly indicate the presence or absence of single-walled CNTs.

Even in the second embodiment, when the single intensity is equal to or higher than the exclusion threshold value, the measurement point is set to the analysis non-use point by the operation of the determination unit 4 and the setting unit 51. Therefore, for example, the ink of the single-walled CNT. If a substance that further generates fluorescence is attached on the top, the analysis is not performed and the presence or absence of the single-walled CNT is not determined. Therefore, in the Raman imaging system 200 of the second embodiment as in the first embodiment, it is possible to prevent inaccurate Raman imaging from being performed due to the influence of fluorescence.

Other embodiments will be described.

In the first embodiment, when at least one wave number of the spectral intensity is equal to or higher than the exclusion threshold value, the measurement point from which the intensity data is obtained is set as the analysis non-use point, but for example, two or more. When the wave number of the wave number exceeds the exclusion threshold value, it may be set as an analysis non-use point.

In the first embodiment, when it is intended to image the single-walled CNTs in the sample with the Raman band corresponding to the RBM, the determination unit is omitted and the multivariate is applied only in the wave number region of the Raman band corresponding to the RBM. The analysis may be performed. Even in such a case, fluorescence is unlikely to occur in the wave number region of the Raman band corresponding to RBM, and even if it does occur, it is absorbed by the single-walled CNT itself and does not appear in the measurement light. A clear image of single-walled CNTs can be obtained.

On the contrary, if the measurement point where the intensity exceeding the exclusion threshold is measured is configured to be set as the analysis non-use point, the sample is subjected to multivariate analysis using the intensity of the measured full wave number region. Raman imaging data may be obtained based on the presence or absence and amount of the single layer CNT inside.

The sample may be a biological sample such as a cell or a biological tissue or something else, and Raman imaging data may be obtained according to the presence or absence and amount of the target substance contained in such a sample.

The determination unit was configured to determine whether the measurement data measured by the Raman measuring device was stored in the measurement data storage unit and then whether it was equal to or greater than the exclusion threshold, but the determination was made when only the intensity data was obtained. If it is equal to or more than the exclusion threshold, it may not be stored in the measurement data storage unit as measurement data. On the contrary, even if the measurement data has a wave number point equal to or higher than the exclusion threshold value, if the wave number is other than the designated wave number region designated by the range designation unit, the data is stored in the data storage unit. Alternatively, the wavenumber points may be reduced to the designated wavenumber region, and it may be determined whether or not the spectrum intensity in the designated wavenumber region includes wavenumber points equal to or greater than the exclusion threshold.

In order to scan the excitation light with respect to the sample, the light source or the optical system may be configured so as to change the irradiation position of the excitation light itself instead of using the stage. The excitation light may be irradiated in a predetermined region of the sample so that the intensity of the measured light in the entire predetermined region can be detected in one shot.

In addition, a part of each embodiment may be combined or modified as long as it does not contradict the gist of the present invention.

200 ... Raman imaging system 100 ... Raman imaging device 101 ... Raman measuring device 11 ... Light source 18 ... Photodetector 3 ... Judgment unit 4 ... Image generation unit 41 ...・ Setting unit 42 ・ ・ ・ Analysis unit 43 ・ ・ ・ Output unit

Claims (9)

A Raman imaging device that generates Raman imaging data of a sample based on intensity data indicating the intensity of measurement light including Raman scattered light generated at a measurement point in the sample.
A determination unit for determining whether the intensity indicated by the intensity data corresponding to each measurement point in the sample is equal to or higher than the exclusion threshold value determined based on the saturation intensity of the photodetector in which the measurement light is detected.
Image generation that generates Raman imaging data based on the intensity data other than the intensity data determined by the determination unit to indicate the intensity of a value equal to or higher than the exclusion threshold among the intensity data corresponding to each measurement point in the sample. Department and
For each measurement point in the sample, a measurement data storage unit that stores measurement data consisting of position data indicating the position of the measurement point in the sample and intensity data corresponding to the measurement point at the position indicated by the position data. Prepare,
The image generation unit
A measurement point corresponding to the measurement data including the intensity data determined by the determination unit to indicate the intensity of the value equal to or higher than the exclusion threshold is set as the analysis non-use point, and the intensity of the value equal to or higher than the exclusion threshold is set by the determination unit. A setting unit that sets the measurement point corresponding to the measurement data including the intensity data determined not to be the intensity data indicating the analysis use point, and the setting unit.
An analysis unit that inputs only the measurement data corresponding to each analysis use point, analyzes based on the Raman spectrum indicated by the intensity data in the measurement data, and outputs the presence or absence or amount of the target substance at each analysis use point.
A Raman imaging apparatus including an output unit that outputs Raman imaging data based on the analysis result of the analysis unit. The intensity data shows the spectral intensity,
Further, a range specifying section for specifying the wave number range used in the image generation section among the spectral intensities is provided.
The first aspect of the present invention, wherein the determination unit determines whether or not the intensity of each wave number in the spectral intensity is equal to or higher than the exclusion threshold value within the wave number range specified by the range designation unit. Raman imaging device. The intensity data shows the spectral intensity,
The determination unit is configured to determine whether or not the intensity of each wave number in the spectral intensity is equal to or higher than the exclusion threshold value.
Among the intensity data corresponding to each measurement point in the sample, the image generation unit determines that the intensity of at least one wave number indicates the intensity equal to or higher than the exclusion threshold in the intensity data other than the intensity data. The Raman imaging apparatus according to claim 1 or 2, which is configured to generate Raman imaging data based on the above. The Raman imaging apparatus according to any one of claims 1 to 3, wherein the analysis unit is configured to analyze by multivariate analysis based on intensity data. The intensity data shows the spectral intensity,
The Raman imaging apparatus according to any one of claims 1 to 4, wherein the image generation unit is configured to generate Raman imaging data based only on spectral intensities of a predetermined wave number or less . A Raman measuring device including a light source that irradiates a sample with light and the light detector that detects the intensity of the measured light including the Raman scattered light generated at each measurement point of the sample.
A Raman imaging system comprising the Raman imaging apparatus according to any one of claims 1 to 5. The Raman measuring device
The Raman imaging system according to claim 6, further comprising a filter through which only the measurement light generated at each measurement point of the sample passes through a predetermined wave number or less. A Raman imaging method that generates Raman imaging data of a sample based on intensity data indicating the intensity of measurement light including Raman scattered light generated at a measurement point in the sample.
A determination step for determining whether the intensity indicated by the intensity data corresponding to each measurement point in the sample is equal to or higher than the exclusion threshold value determined based on the saturation intensity of the photodetector in which the measurement light is detected.
Image generation that generates Raman imaging data based on the intensity data other than the intensity data determined to indicate the intensity of the value equal to or higher than the exclusion threshold in the determination step among the intensity data corresponding to each measurement point in the sample. Steps and
For each measurement point in the sample, a measurement data storage step for storing measurement data consisting of position data indicating the position of the measurement point in the sample and intensity data corresponding to the measurement point at the position indicated by the position data. Prepare,
The image generation step
A measurement point corresponding to the measurement data including the intensity data determined to indicate the intensity of the value equal to or higher than the exclusion threshold in the determination step is set as the analysis non-use point, and the intensity of the value equal to or higher than the exclusion threshold is set in the determination step. The setting step to set the measurement point corresponding to the measurement data including the intensity data determined not to be the intensity data indicating the analysis use point, and
An analysis step in which only the measurement data corresponding to each analysis use point is input, analysis is performed based on the Raman spectrum indicated by the intensity data in the measurement data, and the presence or absence or amount of the target substance at each analysis use point is output.
A Raman imaging method comprising an output step for outputting Raman imaging data based on the analysis result of the analysis step. A program for a Raman imaging device that generates Raman imaging data of a sample based on intensity data indicating the intensity of measurement light including Raman scattered light generated at a measurement point in the sample.
A determination unit for determining whether the intensity indicated by the intensity data corresponding to each measurement point in the sample is equal to or higher than the exclusion threshold value determined based on the saturation intensity of the photodetector in which the measurement light is detected.
Image generation that generates Raman imaging data based on the intensity data other than the intensity data determined by the determination unit to indicate the intensity of a value equal to or higher than the exclusion threshold among the intensity data corresponding to each measurement point in the sample. Department and
For each measurement point in the sample, as a measurement data storage unit that stores measurement data consisting of position data indicating the position of the measurement point in the sample and intensity data corresponding to the measurement point at the position indicated by the position data. To bring out the functions of
The image generation unit
A measurement point corresponding to the measurement data including the intensity data determined by the determination unit to indicate the intensity of the value equal to or higher than the exclusion threshold is set as the analysis non-use point, and the intensity of the value equal to or higher than the exclusion threshold is set by the determination unit. A setting unit that sets the measurement point corresponding to the measurement data including the intensity data determined not to be the intensity data indicating the analysis use point, and the setting unit.
An analysis unit that inputs only the measurement data corresponding to each analysis use point, analyzes based on the Raman spectrum indicated by the intensity data in the measurement data, and outputs the presence or absence or amount of the target substance at each analysis use point.
A program for a Raman imaging apparatus including an output unit that outputs Raman imaging data based on the analysis result of the analysis unit.

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