JP2022122181A - Correction parameter setting method and data correction method - Google Patents

Abstract

To provide a correction parameter setting method and a data correction method that can prevent a difference in result of detection due to individual difference.SOLUTION: A correction parameter setting method sets a parameter p in which a relative error C indicated in the following formula (1) becomes minimum to a correction parameter, based on first light emission spectral data obtained by picking up an image of a first sample with a known light emission intensity by using a first microscope to be a reference, and second light emission spectral data obtained by picking up an image of a second sample whose light emission intensity distribution in a wavelength axis direction is known to be the same as that of the first sample by using a second microscope different from the first microscope. In the following formula, n is the total number of channels, αi is the luminance value of the first light emission spectral data in the i-th channel, and βi is the luminance value of the second light emission spectral data in the i-th channel.SELECTED DRAWING: Figure 1

Description

The present invention relates to a correction parameter setting method and a data correction method for correcting data between microscope devices.

Conventionally, when observing the characteristics of cells or the like contained in a sample, the intensity and spectrum of the light are calculated. An observer checks the characteristics of the sample by looking at the calculated spectrum and the like. For example, Patent Literature 1 uses a microscope to measure the autofluorescence spectrum of cells, calculates the spectrum, and identifies the cell type and the like from the spectrum.

Japanese Patent No. 6422616

By the way, even microscope devices of the same model may have different sensitivities to light due to individual differences between devices. This is due to the difference in sensitivity between individual light-receiving elements, and appears as a difference in luminance value. When observing the properties of cells based on the intensity of the autofluorescence of the cells, even if the intensity of the light emitted by the sample is the same, the brightness values obtained by each microscope device differ. In order to use it for quantitative evaluation, it was necessary to correct the data with appropriate parameters.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a correction parameter setting method and a data correction method capable of suppressing discrepancies in detection results due to individual differences.

ここで、ｎはチャンネル総数、α_iはｉ番目のチャンネルにおける前記第１の発光スペクトルデータの輝度値、β_iはｉ番目のチャンネルにおける前記第２の発光スペクトルデータの輝度値である。 In order to solve the above-described problems and achieve the object, a correction parameter setting method according to the present invention uses a first microscope as a reference to image a first sample whose luminescence intensity is known. Using the first emission spectrum data and a second microscope different from the first microscope, a second sample known to have the same emission intensity distribution in the wavelength axis direction as the first sample is set as a correction parameter based on the second emission spectrum data obtained by imaging .

Here, n is the total number of channels, α _i is the brightness value of the first emission spectrum data in the i-th channel, and β _i is the brightness value of the second emission spectrum data in the i-th channel.

In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are obtained from the first and second samples arranged at one coordinate on a predetermined focal plane. Each generated based on light.

In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are obtained at a plurality of different coordinates on the predetermined focal plane.

The correction parameter setting method according to the present invention is implemented in a plurality of different focal planes in the above invention.

ここで、ｍ₁、ｍ₂は測定点の総数であって、前記第１又は第２の顕微鏡の光軸方向の任意の範囲の総数、ｘ_jに上付きバーを付して表すパラメータは前記第１の顕微鏡の光軸方向のｊ番目の位置における、予め設定された測定範囲の平均輝度値であり、ｙ_kに上付きバーを付して表すパラメータは前記第２の顕微鏡の光軸方向のｋ番目の位置における、予め設定された測定範囲の平均輝度値である。 In the correction parameter setting method according to the present invention, the α _i and β _i are calculated based on the following equations (2) and (3).

Here, m ₁ and m ₂ are the total number of measurement points, the total number of arbitrary ranges in the direction of the optical axis of the first or second microscope, and the parameter indicated by adding a superscript bar to x _j is the above is the average luminance value in a preset measurement range at the j-th position in the optical axis direction of the first microscope, and the parameter represented by y _k with a superscript bar is the parameter in the optical axis direction of the second microscope is the average brightness value in a preset measurement range at the k-th position of .

In the correction parameter setting method according to the present invention, in the above invention, the relative error C is calculated based on a value obtained by subtracting the base noise from the α _i and/or β _i .

In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are generated based on fluorescence obtained by irradiating the first and second samples with excitation light, respectively. be.

In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are obtained by irradiating a plurality of excitation lights with different wavelengths, and each fluorescence obtained by the plurality of excitation lights is fluorescence data including spectral profile data composed of emission spectrum data of .

ここで、ｏは使用した励起光の総数である（０＜ｑ≦ｏ）。 In the correction parameter setting method according to the present invention, in the above invention, the parameter p is the sum of the relative errors C obtained by the plurality of excitation _lights , shown in the following equation (4). Calculated.

where o is the total number of excitation lights used (0<q≦o).

ここで、１≦ｉ≦ｎであり、ｎはチャンネル総数、α_iはｉ番目のチャンネルにおける前記第１の発光スペクトルデータの輝度値、β_iはｉ番目のチャンネルにおける前記第２の発光スペクトルデータの輝度値である。 A correction parameter setting method according to the present invention includes first emission spectrum data obtained by imaging a first sample having a known emission intensity using a first microscope as a reference, and the first microscope. Second emission spectrum data obtained by imaging a second sample known to have the same emission intensity distribution in the wavelength axis direction as the first sample using a second microscope different from Based on this, the parameter p _i that minimizes the relative error C _i shown in the following equation (5) is set as the correction parameter.

Here, 1 ≤ i ≤ n, n is the total number of channels, α _i is the luminance value of the first emission spectrum data in the i-th channel, β _i is the second emission spectrum data in the i-th channel is the luminance value of

A data correction method according to the present invention corrects the second emission spectrum data using the correction parameter set by the correction parameter setting method according to the present invention.

ADVANTAGE OF THE INVENTION According to this invention, it is effective in the ability to suppress the deviation|shift of the detection result by individual difference.

FIG. 1 is a diagram schematically showing the schematic configuration of a data correction system according to one embodiment of the present invention. FIG. 2 is a diagram schematically showing the schematic configuration of the reference microscope unit according to one embodiment of the present invention. FIG. 3 is a diagram explaining a scanning method of the microscope unit according to one embodiment of the present invention. FIG. 4 is a diagram illustrating a focused image generated by scanning in a microscope unit according to an embodiment of the invention. FIG. 5 is a diagram schematically showing a schematic configuration of a microscope unit according to one embodiment of the present invention. FIG. 6 is a diagram schematically showing a schematic configuration of a correction parameter setting device according to one embodiment of the present invention. FIG. 7 is a flow chart explaining an example of a correction parameter setting method according to one embodiment of the present invention. FIG. 8 is a diagram showing an example of a sample holding device according to one embodiment of the present invention. FIG. 9 is a diagram (No. 1) showing an example of detection data used in the correction parameter setting method according to the embodiment of the present invention. FIG. 10 is a diagram (Part 1) showing an example of spectra before and after correction processing using correction parameters according to an embodiment of the present invention. FIG. 11 is a diagram (part 2) showing an example of detection data used in the correction parameter setting method according to the embodiment of the present invention. FIG. 12 is a diagram (part 2) showing an example of spectra before and after correction processing using correction parameters according to an embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments for carrying out the present invention (hereinafter referred to as "embodiments") will be described with reference to the accompanying drawings.

(Embodiment)
FIG. 1 is a diagram schematically showing the schematic configuration of a data correction system according to one embodiment of the present invention. The data correction system 1 includes a reference microscope unit 2A including a reference microscope (a confocal laser scanning microscope in this embodiment), a plurality of microscope units (microscope units 2B, 2C, and 2D), and a correction parameter setting device 3. and In the data correction system 1, the correction parameter setting device 3 sets correction parameters based on the data acquired by the reference microscope unit 2A and the data acquired by the microscope unit (one of the microscope units 2B to 2D in FIG. 1). and corrects the data of the microscope unit using this correction parameter.

FIG. 2 is a diagram schematically showing the schematic configuration of the reference microscope unit according to one embodiment of the present invention. A reference microscope unit 2A shown in the figure generates data that serves as a reference for parameter setting by the correction parameter setting device 3. Based on the image data acquired by the confocal laser scanning microscope 100, the reference microscope unit 2A identifies the type of target reflected in the image, for example, the type of microorganism, and displays the identification result and the acquired image. is displayed. Here, microorganisms include bacteria, fungi, viruses, microalgae, protozoa, and the like. In the present embodiment, the sample to be imaged is tissue section, tissue, animal cell, plant cell, yeast cell, fungal cell, microalgal cell, bacteria, archaea, virus, or phage. , and the spores, spores, or membrane vesicles they produce.

As shown in FIG. 2, the reference microscope unit 2A integrates a confocal laser scanning microscope 100 that irradiates a laser beam to acquire the autofluorescence of the specimen or the transmitted/reflected light from the specimen, and the microscope unit 2A. A control device 200 for control, an image processing device 300 for generating various data such as intensity data and image data based on light acquired by the confocal laser scanning microscope 100, and image data for display generated by the image processing device 300. and a display device 400 for displaying an image based on the image. In this embodiment, the confocal laser scanning microscope 100 included in the reference microscope unit 2A corresponds to the first microscope.

A confocal laser scanning microscope 100 includes a stage 101, an objective lens 102, a laser light source 103, a lens 104, collimating lenses 105 and 112, a beam splitter 106, imaging lenses 107 and 114, and a confocal pinhole 108. , a detector 109 , scanning mirrors 110 and 113 , and a transmission light source 111 . Hereinafter, the two orthogonal axes on the plane parallel to the specimen mounting surface of the stage 101 are defined as the X axis and the Y axis, and the axis orthogonal to this plane is defined as the Z axis. Note that the Z axis is assumed to be parallel to the optical axis of the objective lens 102 .

A stage 101 carries a specimen. The stage 101 is configured to be movable in the Z-axis direction using a driving source such as a motor under the control of the control device 200 . The specimen is a solution or culture medium containing microorganisms, and is placed on the stage 101 while being held by a holding member such as a petri dish or a slide glass.

The objective lens 102 converges the laser light reflected by the beam splitter 106 toward the stage 101 and converts the light from the specimen on the stage 101 into parallel light to enter the beam splitter 106 .

A laser light source 103 emits laser light having a predetermined wavelength. Specifically, the laser light source 103 emits laser light having a wavelength corresponding to the excitation wavelength for exciting the specimen. The laser light source 103 may have a plurality of light sources each capable of emitting laser light of the wavelength to be used, or may emit white laser light and select the wavelength of the emitted light by a filter. may

The lens 104 emits the laser light emitted by the laser light source 103 as radial laser light.

The collimating lens 105 converts the radial laser light that has passed through the lens 104 into parallel light and emits it to the beam splitter 106 .

Beam splitter 106 passes a portion of the incident light and reflects the remaining light. Specifically, the beam splitter 106 bends part of the light emitted from the laser light source 103 to the objective lens 102 side, and allows part of the light incident from the objective lens 102 to pass through to enter the imaging lens 107 . Let The beam splitter 106 is configured using, for example, a half mirror, and allows half of the incident laser light to pass therethrough and reflects the remaining half of the laser light.

An imaging lens 107 forms an image of the light that has passed through the beam splitter 106 .

The confocal pinhole 108 allows at least part of the light imaged by the imaging lens 107 to pass therethrough. The confocal pinhole 108 is formed with a pinhole 108a which is a hole through which light can pass. Also, the confocal pinhole 108 is provided at a position conjugate with the objective lens 102 . Therefore, in the confocal pinhole 108, the light from the focal plane of the objective lens 102 passes through the pinhole 108a, and the light from the out-of-focus position is blocked. For example, when the spot diameter of the laser light imaged by the imaging lens 107 is 0.2 μm, light from a range of about 0.03 μm ² passes through the pinhole 108a at the imaging position. The diameter of the pinhole 108a and the size of the focal space can be changed.

The detector 109 includes a reflective diffraction grating that separates incident light into set wavelength bands, and a plurality of photomultiplier tubes (PhotoMultipliers) that photoelectrically convert the obtained light and current-amplify the converted electrical signal. Tube: PMT (hereinafter also referred to as channel). The detector 109 separates the light into 32 light beams having different wavelength bands by, for example, a reflection diffraction grating, and the separated light beams enter 32 photomultiplier tubes. Each photomultiplier tube photoelectrically converts incident light and outputs an electric signal. In this embodiment, an example having 32 photomultiplier tubes will be described, but the number of photomultiplier tubes is not limited to this.

The scanning mirror 110 controls the irradiation position of the laser light on the focal plane P _F of the specimen under the control of the control device 200 . The scanning mirror 110 is composed of, for example, an X-position control mirror and a Y-position control mirror, and guides laser light to a predetermined position on the XY plane. The scanning mirror 110 moves the irradiation position of the laser light along a preset scanning path by changing the angle of each position control mirror under the control of the control device 200 .

The transmission light source 111 emits transmitted light that passes through the stage 101 under the control of the control device 200 . The transmission light source 111 is configured using, for example, a halogen lamp, a laser light source, or the like.

The collimating lens 112 converts the radial light emitted by the transmission light source 111 into parallel light and emits the parallel light to the scanning mirror 113 .

The scanning mirror 113 controls the irradiation position of the light for transmission on the focal plane P _F of the specimen under the control of the control device 200 . The scanning mirror 113, like the scanning mirror 110, is configured using, for example, an X position control mirror and a Y position control mirror.

The imaging lens 114 forms an image of the light that has passed through the scanning mirror 113 .

Next, the configuration of the control device 200 will be described. The control device 200 includes a control section 201 and an input section 202 . Note that the control device 200 includes a recording unit (not shown) that records various information necessary for the operation of the control device 200 .

The control unit 201 comprehensively controls the microscope unit 2 by reading information stored in the recording unit and executing various kinds of arithmetic processing. The control unit 201 has a laser control unit 203 , a scanning control unit 204 and a transmitted light control unit 205 .

The laser control unit 203 controls emission of laser light from the laser light source 103 based on the control program and instruction information received by the input unit 202 . Specifically, the laser control unit 203 controls the emission timing of the laser light and the wavelength of the emitted laser light. The laser control unit 203 performs control to intermittently emit laser light by, for example, pulse control.

The scanning control unit 204 controls the position of the stage 101 in the Z direction and the irradiation position of the laser beam by the scanning mirror 110 based on the control program and the instruction information received by the input unit 202 .

The transmitted light control unit 205 drives and controls the transmission light source 111 based on the control program and the instruction information received by the input unit 202 .

Here, a scanning method by the microscope unit 2A will be described with reference to FIG. FIG. 3 is a diagram explaining a scanning method of the microscope unit according to one embodiment of the present invention. In the confocal laser scanning microscope 100, after scanning the XY plane on the focal plane at a certain Z position and receiving light from the specimen, the Z position is changed, and the XY plane at the changed Z position is scanned. do. For example, scanning is performed for each Z position set in the Z scanning range R _Z shown in FIG. 2, and light (reflected light or autofluorescence) is obtained from a plurality of positions on the focal plane at each Z position. In the confocal laser scanning microscope 100, the configurations of the beam splitter 106 and the detector 109 can be appropriately changed according to the image to be generated.

For example, as shown in FIG. 3, after scanning the focal plane P _F 1 with the laser beam, the stage 101 is moved in the Z-axis direction, and after the movement, the focal plane P _F 2 where the focal point of the laser beam is located is located. Laser light scanning is performed in This is sequentially scanned with focal planes P _F 3, P _F 4, P _F 5, P _F 6, P _F 7, . . . in a preset Z scanning range R _Z .

Regarding the scanning method in the XY plane, for example, as shown in FIG. 3, a laser beam is irradiated from one corner of a rectangular focal plane (focal plane P _F 7 in FIG. 3) to form a spot SP receive light from By zigzag scanning this spot SP, it is possible to acquire light corresponding to the number of data for generating one two-dimensional image (focused image) on the focal plane P _F 7 . If the diameter of the spot SP is made approximately equal to the size of one pixel (equivalent to one dot displayed on a monitor), the colors of two-dimensional and three-dimensional images can be expressed in pixel units. Furthermore, it is possible to color the visual information corresponding to the identification information on a pixel-by-pixel basis. The phrase "substantially equal to the size of a pixel" means, for example, when the spot SP is a circle, the size is substantially the same as the size inscribed in the pixel. Note that the scanning path described above is an example, and the scanning path is not limited to this path as long as the focal plane can be scanned. The diameter of the spot SP can be appropriately adjusted by changing the diameter of the confocal pinhole, with the lower limit of approximately 0.2 μm, which is the resolution limit of the optical microscope.

The input unit 202 receives input of various information. The input unit 202 is configured using a user interface such as a keyboard, mouse, and touch panel.

Next, the configuration of the image processing apparatus 300 will be described. The image processing apparatus 300 has a detection signal reception section 301 , a data generation section 302 and a recording section 303 .

The detection signal receiving section 301 receives the electrical signal of each channel from the detector 109 . The detection signal receiving unit 301 associates the received electrical signal of each channel with the position information (laser beam irradiation position) on the scanning surface, and outputs the information to the data generating unit 302 . The detection signal receiving unit 301 may be provided separately for reflected light detection and autofluorescence detection.

The data generator 302 generates data in which the light intensity based on the electrical signal received from the detection signal receiver 301 is associated with the position information on the scanning plane. The data generator 302 includes an autofluorescence data generator 302a, a reflected light data generator 302b, a corresponding data generator 302c, and a reference fluorescent substance data generator 302d.

The autofluorescence data generation unit 302a acquires an electric signal associated with the autofluorescence received by the detection signal receiving unit 301, and obtains an electric signal of each channel, and converts it into one on a predetermined focal plane (the XY plane in FIG. 2). Intensity data and/or fluorescence spectra (spectral data) are generated for each coordinate. The autofluorescence data generation unit 302a generates one fluorescence spectrum for one position on the scanning plane when one excitation light beam is irradiated, and a plurality of different wavelengths of excitation light beams are irradiated at different timings. If so, it produces multiple fluorescence spectra in response to the excitation light. The term “fluorescence spectrum” as used herein means “intensity distribution with respect to wavelength” of autofluorescence generated when a laser beam of a predetermined wavelength is applied as excitation light. Further, the “intensity” referred to here refers to a signal value obtained by photoelectrically converting the autofluorescence obtained, for example. The fluorescence spectrum consists of a waveform that has undergone smoothing processing, for example, by interpolating between plots. In this specification, data composed of a plurality of fluorescence spectra may be referred to as spectral profile data. As used herein, “autofluorescence data” includes any or all of autofluorescence intensity data, spectral data, and spectral profile data. The autofluorescence data generation unit 302a generates autofluorescence data in which fluorescence spectra generated according to excitation wavelengths are associated with each position on the scanning plane (a plurality of coordinates on a predetermined focal plane).

The reflected light data generation unit 302b acquires the detection signal related to the reflected light reflected by the specimen, which is received by the detection signal reception unit 301, and calculates the intensity of the reflected light based on the acquired detection signal and the position on the scanning plane. The reflected light data associated with the information is generated. The reflected light data generation unit 302b sums up the light intensity based on the electrical signal of each channel, for example, and obtains the reflected light intensity at the position on the scanning plane.

The correspondence data generation unit 302c generates correspondence data composed of autofluorescence data and reflected light data at one coordinate on a predetermined focal plane. If the autofluorescence data and the reflected light data are generated at a plurality of coordinates, the corresponding data generation unit 302c generates corresponding data in which the autofluorescence data and the reflected light data are associated with each other for each coordinate. Also, if autofluorescence data is generated by a plurality of excitation lights at the same coordinates, each autofluorescence data is associated with the coordinates.

Here, the significance of associating reflected light data and autofluorescence data at one coordinate on a predetermined focal plane will be described. The intensity of reflected light reflects the presence of an analyte, such as a cell, at one coordinate on a given focal plane. If the specimen (cell) does not exist at the coordinates, the intensity of the reflected light is low, and if the specimen (cell) exists, the reflected light has a high intensity. By acquiring reflected light at high magnification, it is possible to obtain reflected light from the outline of the cell, reflected light from the inside of the cell, and even reflected light from the organelle inside the cell such as the nucleus. In this way, by obtaining information on the presence or absence of a sample (cell) at a certain coordinate or to which part of the sample (cell) a certain coordinate corresponds to, and by using this and the autofluorescence data at the coordinate, It is now possible to perform analyzes (for example, identification and evaluation) at the level of individual cells and intracellular organelles, which has been impossible until now.

The correspondence data generation unit 302c generates image data corresponding to one frame of the display image based on the associated various data. For example, when generating focused image data based on reflected light, the corresponding data generation unit 302c generates a focused image to which luminance information is assigned for each pixel position based on the reflected light data generated by the reflected light data generation unit 302b. One or more data are generated depending on the number of scan planes scanned. In addition, when generating fluorescence image data from autofluorescence generated by irradiated excitation light, the corresponding data generation unit 302c generates fluorescence image data to which luminance information is added for each pixel position based on the fluorescence spectrum, and the scanning surface obtained by scanning the fluorescence image data. Generate one or more depending on the number of The corresponding data generating unit 302c may superimpose color information in accordance with the microbial species identified by the fluorescence spectrum, for example, in addition to the luminance information. The corresponding data generation unit 302c performs image processing using known techniques such as gain processing, contrast processing, and γ correction processing on the generated one-frame two-dimensional image data, and also performs image processing according to the display specifications of the display device 400. Image data for display is generated by performing appropriate processing.

Furthermore, the corresponding data generation unit 302c can generate three-dimensional image data based on the two-dimensional image data. The corresponding data generation unit 302c generates three-dimensional image data by adding luminance information in each frame to a three-dimensional space.

Here, the laser beam irradiation position is associated with the spatial information of the image data generated by the corresponding data generation unit 302c. The spatial position is position information consisting of the position of the pixel on the X axis (X position) and the position of the pixel on the Y axis (Y position) in two dimensions, and in three dimensions, the X position, Y position, and position information consisting of the position of the pixel on the Z axis (Z position). For example, the scanning plane corresponds to a plane orthogonal to the Z axis, and the position on the scanning plane is represented by the X position and Y position on the scanning plane.

Also, the corresponding data generation unit 302c performs sample analysis processing based on the spectral profile data. The analysis process identifies biological kingdoms, phyla, classes, orders, families, genuses, species, breeds, pathogenic types or serotypes, or identifies microbiological strains or substrains, depending on the content of the processing. or assess the state of the sample with respect to the metabolic or physiological state of an unknown or known sample.

FIG. 4 is a diagram illustrating a focused image generated by scanning in a microscope unit according to an embodiment of the invention. The correspondence data generation unit 302c performs image processing based on the intensity of the reflected light in the correspondence data, thereby generating N focused images D based on the light reflected on each focal plane, as shown in FIG. ₁ , D ₂ , . . . , D _N (N is a natural number of 3 or more). The correspondence data generation unit 302c converts the intensity of the reflected light obtained at each position into luminance information, and generates focused image data arranged according to the irradiation position of the laser light. That is, the corresponding data generation unit 302c generates two-dimensional image data including an image generated by the reflected light and position information (for example, Z position) regarding the irradiation position of the laser light.

Further, the correspondence data generation unit 302c generates each focused image in the orthogonal coordinate system of the three-dimensional space based on a plurality of focused image data (focused images D ₁ , D ₂ , . . . , D _N ). 3D image data expressing the specimen image according to the brightness in the 3D space is generated by associating the brightness information of the .

The reference fluorescent material data generation unit 302d generates light intensity data of the reference fluorescent material used to set the correction parameters.

A recording unit 303 records various programs including programs for executing operations of the image processing apparatus 300 . The recording unit 303 is configured using a ROM (Read Only Memory) in which various programs and the like are pre-installed, a RAM (Random Access Memory) for recording calculation parameters and the like, and the like.

The display device 400 is configured using liquid crystal or organic EL (Electro Luminescence), and displays an image or the like generated by the image processing device 300 . The display device 400 may display various information generated by the control device 200 .

Next, the configuration of microscope units 2B to 2D will be described with reference to FIG. FIG. 5 is a diagram schematically showing a schematic configuration of a microscope unit according to one embodiment of the present invention. Although FIG. 5 shows the configuration of the microscope unit 2B as a representative, the microscopes 2C and 2D also have the same configuration. As shown in FIG. 5, the microscope unit 2B includes a confocal laser scanning microscope 100, a control device 200 for overall control of the microscope unit 2B, and intensity data and images based on light acquired by the confocal laser scanning microscope 100. It includes an image processing device 300A that generates various data such as data, and a display device 400 that displays an image based on display image data generated by the image processing device 300A. The microscope unit 2B has the same configuration as the reference microscope unit 2A except that it has an image processing device 300A instead of the image processing device 300 of the reference microscope unit 2A. Note that, in the present embodiment, the confocal laser scanning microscope 100 included in the reference microscope units 2B to 2D corresponds to the second microscope.

Next, the configuration of the image processing apparatus 300 will be described. The image processing apparatus 300 has a detection signal reception section 301, a data generation section 302A, and a recording section 303A.

The data generator 302A includes an autofluorescence data generator 302a, a reflected light data generator 302b, a corresponding data generator 302c, a reference fluorescent substance data generator 302d, and a data corrector 302e.

The data correction unit 302e corrects the autofluorescence intensity data generated by the autofluorescence data generation unit 302a according to the correction parameter set by the correction parameter setting device 3. FIG.

The recording unit 303A records various programs including programs for executing operations of the image processing apparatus 300A. The recording unit 303A is configured using a ROM (Read Only Memory) in which various programs and the like are pre-installed, a RAM (Random Access Memory) for recording calculation parameters and the like, and the like.

The recording unit 303A has a correction information recording unit 303a that records the correction parameters set by the correction parameter setting device 3. FIG.

Next, the configuration of the correction parameter setting device 3 will be described with reference to FIG. FIG. 6 is a diagram schematically showing a schematic configuration of a correction parameter setting device according to one embodiment of the present invention. The correction parameter setting device 3 has a fluorescence data acquisition section 31 , a calculation section 32 , a setting section 33 , a control section 34 and a recording section 35 .

The fluorescence data acquisition unit 31 acquires the reference fluorescent substance data generated by the reference fluorescent substance data generation unit 302d of the reference microscope unit 2A and the reference fluorescent substance data generated by the reference fluorescent substance data generation units 302d of the microscopes 2B to 2D. do. The fluorescence data acquisition unit 31 acquires data from each microscope unit via a communication cable or communication network.

ここで、ｎはチャンネル総数（ここではｎ＝３２）、α_iはｉ番目のチャンネルにおける基準顕微鏡ユニット２Ａから取得した蛍光スペクトルデータ（第１の発光スペクトルデータ）の輝度値、β_iはｉ番目のチャンネルにおける顕微鏡ユニット２Ｂ～２Ｄのいずれかから取得した蛍光スペクトルデータ（第２の発光スペクトルデータ）の輝度値である。
例えば、α_i、β_iの値は、下式（２）、（３）に基づいて算出される。

ここで、ｍ₁、ｍ₂は測定点の総数であって、Ｚ方向の任意の範囲の総数（スライス数））、ｘ_jに上付きバーを付して表すパラメータはｊ番目のＺスライスにおける、ＸＹ平面の一部又は全部の平均輝度値であり、ｙ_kに上付きバーを付して表すパラメータはｋ番目のＺスライスにおける、ＸＹ平面の一部又は全部の平均輝度値である。なお、Ｚ方向の任意の範囲は、例えば最大平均輝度値を含む。また、ＸＹ平面における測定範囲は一例であり、任意に設定できる。例えば、走査によって１０２４×１０２４ピクセルの画像が生成される場合、輝度村ムラを抑制して平均輝度値を算出するという観点で、画像中央の３００×６００ピクセルを測定範囲とし、該測定範囲の平均輝度値を用いることが好ましい。 The calculation unit 32 refers to the calculation formula recorded in the recording unit 35 to calculate parameters. Specifically, the calculator 32 calculates the parameter p with reference to the following formula (1). The calculation unit 32 calculates the parameter p that minimizes the relative error C based on Equation (1).

Here, n is the total number of channels (here, n=32), α _i is the luminance value of the fluorescence spectrum data (first emission spectrum data) obtained from the reference microscope unit 2A in the i-th channel, and β _i is the i-th channel. is the luminance value of the fluorescence spectrum data (second emission spectrum data) acquired from any one of the microscope units 2B to 2D in the channel of .
For example, the values of α _i and β _i are calculated based on the following equations (2) and (3).

Here, m ₁ and m ₂ are the total number of measurement points, the total number of arbitrary ranges in the Z direction (number of slices), and the parameter indicated by a superscript bar on x _j is the j-th Z slice , is the average brightness value of part or all of the XY plane, and the parameter represented by a superscript bar on y _k is the average brightness value of part or all of the XY plane at the k-th Z slice. Note that the arbitrary range in the Z direction includes, for example, the maximum average brightness value. Also, the measurement range on the XY plane is an example and can be set arbitrarily. For example, when an image of 1024×1024 pixels is generated by scanning, the measurement range is 300×600 pixels in the center of the image from the viewpoint of suppressing luminance unevenness and calculating the average luminance value, and the average of the measurement range is Preferably, luminance values are used.

The setting unit 33 sets the parameter p calculated by the calculation unit 32 as a correction parameter of the microscope unit (one of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31. FIG.

The control unit 34 controls the correction parameter setting device 3 by reading information stored in the recording unit 35 and executing various kinds of arithmetic processing.

The recording unit 35 records various programs including programs for executing the operation of the correction parameter setting device 3 . The recording unit 35 has a setting information recording unit 351 that records setting information such as calculation formulas used for setting correction parameters. The recording unit 35 is configured using a ROM (Read Only Memory) in which various programs and the like are installed in advance, a RAM (Random Access Memory) for recording calculation parameters and the like, and the like.

The control device 200, the data processing devices 300 and 300A, and the correction parameter setting device 3 include a CPU (Central Processing Unit) having arithmetic and control functions, various arithmetic circuits such as FPGA (Field Programmable Gate Array), ROM, RAM, and the like. It is configured using one or more computers including

Next, a correction parameter setting method by the correction parameter setting device 3 will be described with reference to FIG. FIG. 7 is a flow chart explaining an example of a correction parameter setting method according to one embodiment of the present invention. The flow of setting correction parameters based on the obtained autofluorescence will be described below.

First, the fluorescence data acquisition unit 31 acquires reference fluorescent material data from the reference microscope unit 2A (step S101). Further, the fluorescence data acquisition unit 31 acquires reference fluorescent substance data from the microscope unit for which correction parameters are to be set (for example, the microscope unit 2B) (step S102). At this time, if the reference fluorescent substance data of the reference microscope unit 2A has already been acquired, the processing of step S101 may be omitted.

The reference fluorescent substance data acquired in steps S101 and S102 have the same emission intensity distribution in the wavelength axis direction, and are based on fluorescence obtained by exciting a sample (reference fluorescent substance) whose concentration and conditions are known. be. Specifically, a sample of the same kind and the same concentration that is poured into the fluidic device is excited by excitation light of the same wavelength band, and the fluorescence from the sample is acquired.

FIG. 8 is a diagram showing an example of a sample holding device according to one embodiment of the present invention. A sample holding device 500 is formed with a channel 501 . The channel 501 has a first opening 502 , a second opening 503 , and a circulation portion 504 connecting the first opening 502 and the second opening 503 . The circulation portion 504 forms a closed space except for the connecting portion with the first opening portion 502 and the second opening portion 503 . The channel shown in FIG. 8 is an example, and a meandering or branching channel can be applied.

A sample is introduced into the channel 501 through, for example, the first opening 502, passes through the circulation portion 504, and is discharged to the outside through the second opening 503 (see the arrow in FIG. 8). The confocal laser scanning microscope 100 scans a region R including the central portion of the circulation portion 504 and performs photometry of the region R. FIG. At this time, the optical axis N of the confocal laser scanning microscope 100 scans the region R, and measures the light from the sample (hereinafter also referred to as “fluid sample”) flowing through the circulation portion 504 . Note that the scanning within the region R may be performed a plurality of times, and the photometric value averaging process or integration process may be performed for each scanning position or the entire scanning range.
By continuing to flow the fluid sample through the channel 501 and measuring its fluorescence, it is possible to perform photometry while suppressing fading of sample fluorescence due to laser light irradiation. It should be noted that the flow velocity of the liquid sample is preferably set to such a velocity that the residence time in the region R is sufficiently short to prevent fluorescence fading.

Also, this reference fluorescent substance data is reference fluorescent substance data obtained by repeatedly scanning the XY plane while moving in the Z direction. That is, the reference fluorescent material data is point group data in which a plurality of measurement points are arranged along the Z direction in a three-dimensional space, and each point has spectral data.

After that, the calculator 32 calculates the parameter p that minimizes the relative error C using the above equation (1) (step S103). The calculation unit 32 calculates each relative error C by changing the parameter p by 0.01, for example, and extracts the smallest relative error C. FIG. The calculation unit 32 outputs the extracted parameter p corresponding to the minimum relative error C to the setting unit 33 .

The setting unit 33 sets the parameter p acquired from the calculation unit 32 as the correction parameter used by the microscope unit for which the correction parameter is to be set (step S104).

Here, reference fluorescent material data and corrected intensity data corrected by correction parameters will be described with reference to FIGS. 9 to 12. FIG. FIG. 9 is a diagram (No. 1) showing an example of detection data used in the correction parameter setting method according to the embodiment of the present invention. The fluorescence data shown in FIG. 9 are based on the fluorescence of a sample using Coumarin 6H as a fluorescent substance at a concentration of 1×10 ⁻⁴ % (in 99% ethanol). In FIG. 9, intensity data S1 (Microscope 1) is intensity data acquired by the reference microscope unit 2A, and intensity data S2 (Microscope 2) is intensity data acquired by the microscope unit 2B. Each microscope unit scans the XY plane and acquires fluorescence on a plurality of XY planes with different Z positions while moving at a predetermined pitch in the Z direction. The intensity data shown in FIG. 9 are data acquired at 18 points along the Z direction. Intensity data S1, S2 represent the average brightness of the central region of the image based on each XY plane scan at the Z position (z slice). Note that deviations in the horizontal axis (wavelength axis) direction of the intensity data S1 and S2 correspond to structural deviations caused by individual differences in microscope units. Further, deviations in the vertical axis direction of the intensity data S1 and S2 correspond to performance deviations caused by individual differences of the detectors 109 . The intensity data S1 and S2 have the same waveform pattern (emission intensity distribution) in the wavelength axis direction, although there are deviations in the wavelength axis direction and intensity.

FIG. 10 is a diagram (Part 1) showing an example of spectra before and after correction processing using correction parameters according to an embodiment of the present invention. The intensity data S11 (Microscope 1) shown in FIG. 10 indicates the average brightness for each wavelength band based on the intensity data S1 shown in FIG. 9, and the intensity data S12 (Microscope 2) is based on the intensity data S2 shown in FIG. , indicates the average brightness for each wavelength band. The intensity data S11, S12 represent the average brightness of each wavelength band in the image data of a predetermined Z range including the Z position with the maximum average brightness. Each wavelength band corresponds to each channel of detector 109 . That is, the intensity of each wavelength band is the intensity of the signal output by the channel. For the predetermined Z range, for example, the distance in the Z direction (several μm) or the number of images in the Z direction (the number of slices) are set.

For the intensity data S11 and S12, the calculator 32 calculates the relative error C while changing the parameter p in the above equation (1), and the setting unit 33 sets the parameter p. In the example shown in FIG. 9, the parameter p is set to p=1.33.
The data corrector 302e corrects the intensity data S2 of the microscope unit 2B using the set parameter p.

Corrected intensity data SC1 (Corrected Microscope 2) shown in FIG. 10 represents waveform data obtained by correcting the intensity data S12 by a parameter p (here, p=1.33). As shown in FIG. 10, the intensity data SC1 after correction had a waveform substantially equivalent to that of the intensity data S11 of the reference microscope unit 2A, and the average percentage error was 6%.

FIG. 11 is a diagram (part 2) showing an example of detection data used in the correction parameter setting method according to the embodiment of the present invention. The fluorescence data shown in FIG. 11 are based on the autofluorescence of the sample with Fluorescein as the fluorescent substance and a concentration of 1×10 ⁻⁵ %. In FIG. 11, intensity data S3 (Microscope 1) is intensity data acquired by the reference microscope unit 2A, and intensity data S4 (Microscope 2) is intensity data acquired by the microscope unit 2B. The strength data S3 and S4 are data acquired under the same conditions as the strength data S1 and S2 shown in FIG.

FIG. 12 is a diagram (part 2) showing an example of spectra before and after correction processing using correction parameters according to an embodiment of the present invention. The intensity data S13 (Microscope 1) shown in FIG. 12 indicates the average brightness for each wavelength band based on the intensity data S3 shown in FIG. 11, and the intensity data S14 (Microscope 2) is based on the intensity data S4 shown in FIG. , indicates the average brightness for each wavelength band. The intensity data S13, S14 represent the average brightness of each wavelength band in the image data of the predetermined Z range including the Z position with the maximum average brightness.

For the intensity data S13 and S14, the calculation unit 32 calculates the relative error C while changing the parameter p in the above equation (1), and the setting unit 33 sets the parameter p. In the example shown in FIG. 11, the parameter p is set to p=1.76.
The data corrector 302e corrects the intensity data S4 of the microscope unit 2B using the set parameter p.

Corrected intensity data SC2 (Corrected Microscope 2) shown in FIG. 12 represents waveform data obtained by correcting the intensity data S14 by a parameter p (here, p=1.76). As shown in FIG. 12, the intensity data SC2 after correction had a waveform substantially equivalent to that of the intensity data S13 of the reference microscope unit 2A, and the average percentage error was 7.1%.

In the embodiment of the present invention described above, the reference microscope unit 2A and the microscope units 2B to 2D use the intensity data obtained from the sample under the same conditions and the above equation (1) to obtain the microscope units 2B to 2D By setting the correction parameters of , corrected intensity data is obtained that suppresses deviations in detection results due to individual differences. According to the present embodiment, by performing various analyzes using the corrected intensity data corrected by the correction parameters, it is possible to obtain analysis results in which the influence of deviations due to individual differences between microscope units is suppressed.

(Modification 1)
Next, Modification 1 of the embodiment will be described. Although the example in which the correction parameter is set for each excitation light has been described in the embodiment, the correction parameter unique to the specimen may be set regardless of the excitation light.

ここで、ｏは使用した励起光の総数（励起波長の種類の総数）である（０＜ｑ≦ｏ）。例えば、使用した励起光が、４０５ｎｍ、４８８ｎｍ、５６１ｎｍ、６４０ｎｍの場合、ｏ＝４となる。 In Modified Example 1, the calculation unit 32 calculates the parameter p that minimizes the hyperspectrum C _hyper corresponding to the relative error based on the following formula (4).

Here, o is the total number of excitation lights used (the total number of types of excitation wavelengths) (0<q≦o). For example, when the excitation light used is 405 nm, 488 nm, 561 nm and 640 nm, o=4.

The setting unit 33 sets the parameter p calculated by the calculation unit 32 as a correction parameter of the microscope unit (one of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31. FIG.

According to the modified example 1 described above, as in the above-described embodiment, various analyzes are performed using the corrected intensity data corrected by the correction parameter, thereby suppressing the influence of deviation due to individual differences between microscope units. It is possible to obtain the analysis results of Furthermore, according to Modification 1, it is possible to set a specimen-specific correction parameter that does not depend on the excitation light, and as a result, it is possible to suppress the complication of the correction parameter due to the excitation light.

(Modification 2)
Next, Modification 2 of the embodiment will be described. Although the example in which the correction parameter is set for each excitation light has been described in the embodiment, the correction parameter may be set for each channel of the detector 109 .

ここで、上式（５）は１≦ｉ≦ｎとした場合について示す式である。ｉ、ｎ、α_i、β_iについては、上式（１）と同様である。 In Modified Example 2, the calculator 32 calculates the parameter p _i that minimizes the relative error C _i based on the following equation (5).

Here, the above formula (5) is a formula showing the case where 1≤i≤n. i, n, α _i and β _i are the same as in the above equation (1).

The setting unit 33 sets the parameter p _i calculated by the calculation unit 32 as a correction parameter for each channel of the microscope unit (one of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31. FIG. Correction parameters set according to the channel are used in the correction process.

According to the modified example 2 described above, as in the above-described embodiment, various analyzes are performed using the corrected intensity data corrected by the correction parameter, thereby suppressing the influence of deviation due to individual differences between microscope units. It is possible to obtain the analysis results of Furthermore, according to Modification 2, a correction parameter can be set for each channel, and as a result, individual differences between microscope channels can be suppressed.

(Modification 3)
Next, Modification 3 of the embodiment will be described. Base noise may be present in the electrical signal (detection value) output from the detector 109 . Since the base noise causes deviations in the intensity data between microscopes, it is preferable to calculate the correction parameters using the intensity data excluding the base noise. Here, the base noise is defined by either definition 1 or 2 below.
Definition 1. The average luminance value of the channel that detects light outside the fluorescence wavelength band of the sample-derived reference fluorescent substance. For example, the channel corresponds to a channel whose luminance value is an arbitrary value or less, for example, 20 or less when the reference fluorescent substance is observed under a microscope without base noise.
Definition 2. Average luminance value detected when the detector voltage is zero.

The calculator 32 calculates correction parameters according to the microscope in which the base noise exists. Specifically, the correction parameter is calculated using one of the following formulas (6) to (8).

ここで、γ_βは顕微鏡ユニット（顕微鏡ユニット２Ｂ～２Ｄのいずれか）のベースノイズの値である。 [When base noise exists in microscope units 2B to 2D]
The calculator 32 calculates the parameter p that minimizes the relative error C based on the following equation (6).

Here, γ _β is the base noise value of the microscope unit (one of the microscope units 2B to 2D).

ここで、γ_αは基準顕微鏡ユニット（基準顕微鏡ユニット２Ａ）のベースノイズの値である。 [When Base Noise Exists in Reference Microscope Unit 2A]
The calculator 32 calculates the parameter p that minimizes the relative error C based on the following equation (7).

Here, γ _α is the base noise value of the reference microscope unit (reference microscope unit 2A).

[When Base Noise Exists in Reference Microscope Unit 2A and Microscope Units 2B to 2D]
The calculator 32 calculates the parameter p that minimizes the relative error C based on the following equation (8).

According to the modified example 3 described above, as in the above-described embodiment, various analyzes are performed using the corrected intensity data corrected by the correction parameter, thereby suppressing the influence of deviation due to individual differences between microscope units. It is possible to obtain the analysis results of Furthermore, according to Modification 3, the influence of base noise can be eliminated, and the correction parameter can be set from the value of the reference fluorescent material itself. As a result, individual differences between microscopes can be further suppressed. .

Although the embodiments for carrying out the present invention have been described so far, the present invention should not be limited only to the above-described embodiments.

In the above-described embodiment, the autofluorescence data, the reflected light data, and the corresponding data are generated by scanning the three-dimensional space. YZ plane) to generate reference fluorophore data, autofluorescence data, reflected light data and corresponding data, or any of the X, Y and Z directions shown in FIG. Scanning may be performed in one direction, or fluorescence, autofluorescence, and reflected light at a point in space may be acquired to generate reference fluorescent substance data, autofluorescence data, reflected light data, and corresponding data. good. In particular, as the reference fluorescent substance data for setting the correction parameters, it is only necessary to obtain spectral data, so only one point of reference fluorescent substance data is used, and the correction data setting device 3 sets the one point of reference fluorescent substance data. may be obtained from each microscope unit.

Further, in the above-described embodiment, a three-dimensional image is generated and displayed. However, a two-dimensional image may be displayed or visual information may be superimposed. An image to be displayed may be selected.

Further, in the above-described embodiment, the detector 109 is configured using a reflective diffraction grating and a photomultiplier tube (PMT). Leica's AOBS (registered trademark)), a high-sensitivity detector (HyD detector), and a detector consisting of a movable slit structure provided in front of the detector may be used. With the configuration described above, this detector can acquire data separated into wavelengths of, for example, 1 nm.

Further, in the above-described embodiments, laser light is used to obtain reflected light or autofluorescence. Reflected light or autofluorescence may be obtained by condensing and irradiating the sample. For example, autofluorescence is obtained using a laser beam and reflected light is obtained using a halogen lamp, autofluorescence is obtained using a halogen lamp and reflected light is obtained using a laser beam, or autofluorescence is obtained using a halogen lamp. Fluorescence and reflected light may be acquired. Also, the wavelength of the light may be one that has passed through a filter or one that has been separated by a prism. In addition, it is applicable not only to reflected light from a specimen, but also to transmitted light, fluorescence, etc., as long as it is light detection data that can extract the outline of a cell or the like.

Further, in the above-described embodiment, an example in which fluorescence is measured while the sample is flowing in the channel 501 has been described. Alternatively, light having a known emission intensity (autofluorescence, laser light, etc.) may be measured.

Further, in the above-described embodiment, the configuration in which the microscope unit and the correction parameter setting device 3 are provided separately has been described as an example, but the configuration of the correction parameter setting device 3 may be integrated with the microscope unit. For example, the microscope units 2B to 2D may have the configuration of the correction parameter setting device 3, and the correction parameters may be set in the microscope units 2B to 2D, or the reference microscope unit 2A may have the configuration of the correction parameter setting device 3. The correction parameters may be set in the reference microscope unit 2A.

Thus, the present invention can include various embodiments within the scope of the technical idea described in the claims.

As described above, the correction parameter setting method and data correction method according to the present invention are useful for suppressing deviations in detection results due to individual differences.

1 data correction system 2A reference microscope unit 2B-2D microscope unit 3 correction parameter setting device 31 fluorescence data acquisition unit 32 calculation unit 33 setting unit 34, 201 control unit 35, 303 recording unit 100 confocal laser scanning microscope 101 stage 102 objective lens 103 laser light source 104 lens 105, 112 collimating lens 106 beam splitter 107, 114 imaging lens 108 confocal pinhole 109 detector 110, 113 scanning mirror 111 transmission light source 200 control device 202 input section 203 laser control section 204 scanning control section 205 Transmitted light controller 300 Image processor 301 Detection signal receiver 302 Data generator 302a Autofluorescence data generator 302b Reflected light data generator 302c Corresponding data generator 302d Reference fluorescent material data generator 302e Data corrector 400 Display device 500 Sample holding device

Claims (11)

First emission spectrum data obtained by imaging a first sample with a known emission intensity using a first microscope as a reference, and using a second microscope different from the first microscope , and second emission spectrum data obtained by imaging a second sample known to have the same emission intensity distribution in the wavelength axis direction as that of the first sample, the following formula (1) A correction parameter setting method for setting a parameter p that minimizes the relative error C shown in FIG.

Here, n is the total number of channels, α _i is the brightness value of the first emission spectrum data in the i-th channel, and β _i is the brightness value of the second emission spectrum data in the i-th channel. wherein the first and second emission spectrum data are generated based on light obtained from the first and second samples positioned at one coordinate on a predetermined focal plane, respectively;
The correction parameter setting method according to claim 1. The first and second emission spectrum data are obtained at a plurality of mutually different coordinates on the predetermined focal plane,
The correction parameter setting method according to claim 2. is carried out in different focal planes,
The correction parameter setting method according to any one of claims 1 to 3. The α _i and β _i are calculated based on the following formulas (2) and (3),
The correction parameter setting method according to claim 1.

Here, m ₁ and m ₂ are the total number of measurement points, the total number of arbitrary ranges in the direction of the optical axis of the first or second microscope, and the parameter indicated by adding a superscript bar to x _j is the above is the average luminance value in a preset measurement range at the j-th position in the optical axis direction of the first microscope, and the parameter represented by y _k with a superscript bar is the parameter in the optical axis direction of the second microscope is the average brightness value in a preset measurement range at the k-th position of . The relative error C is calculated based on the value obtained by subtracting the base noise from the α _i and / or β _i ,
The correction parameter setting method according to any one of claims 1 to 5. The first and second emission spectrum data are generated based on fluorescence obtained by irradiating the first and second samples with excitation light, respectively.
The correction parameter setting method according to any one of claims 1 to 6. The first and second emission spectrum data are obtained by irradiating a plurality of excitation lights with different wavelengths, and fluorescence including spectrum profile data composed of emission spectrum data of each fluorescence obtained by the plurality of excitation lights. is the data,
The correction parameter setting method according to claim 7. The parameter p is calculated based on the hyper parameter C _hyper , which is the sum of the relative errors C obtained by the plurality of excitation lights, as shown in the following formula (4):
The correction parameter setting method according to claim 8.

where o is the total number of excitation lights used (0<q≦o). First emission spectrum data obtained by imaging a first sample with a known emission intensity using a first microscope as a reference, and using a second microscope different from the first microscope , and second emission spectrum data obtained by imaging a second sample known to have the same emission intensity distribution in the wavelength axis direction as that of the first sample, the following formula (5) A correction parameter setting method for setting a parameter p _i that minimizes the relative error C _i shown in .

Here, 1 ≤ i ≤ n, n is the total number of channels, α _i is the luminance value of the first emission spectrum data in the i-th channel, β _i is the second emission spectrum data in the i-th channel is the luminance value of A data correction method for correcting the second emission spectrum data using the correction parameter set by the correction parameter setting method according to any one of claims 1 to 10.

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