JP7697649B2 - Correction parameter setting method and data correction method - Google Patents

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 light intensity and spectrum are calculated. The observer checks the characteristics of the sample by looking at the calculated spectrum. For example, in Patent Document 1, a microscope device is used to measure the spectrum of the autofluorescence of cells, calculate the spectrum, and identify the type of cell, etc., from the spectrum.

Patent No. 6422616

However, even if the microscope devices are the same model, their sensitivity to light may differ due to individual differences between devices. This is due to differences in the sensitivity of individual light-receiving elements, and is manifested as differences in brightness values. When observing cell characteristics based on the intensity of the autofluorescence of cells, the brightness values obtained differ for each microscope device even if the intensity of light emitted by the sample is the same, and in order to use the brightness value data obtained by multiple microscopes for quantitative evaluation, the data must be corrected using appropriate parameters.

The present invention has been made in consideration of the above, and aims to provide a correction parameter setting method and a data correction method that can suppress deviations 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-mentioned problems and achieve the object, the correction parameter setting method of the present invention sets, as a correction parameter, a parameter p that minimizes a relative error C shown in the following formula (1), based on first emission spectrum data obtained by imaging a first sample having a known emission intensity using a first microscope as a reference, 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 the first sample using a second microscope different from the first microscope.

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

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

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

The correction parameter setting method according to the present invention is implemented in a plurality of focal planes different from each other 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, in the above invention, the α _i and β _i are calculated based on the following expressions (2) and (3).

Here, _m1 and _m2 are the total number of measurement points, which are the total number of any range in the optical axis direction of the first or second microscope, the parameter represented by _xj with a superscript bar is the average brightness value of a preset measurement range at the jth position in the optical axis direction of the first microscope, and the parameter represented by _yk with a superscript bar is the average brightness value of a preset measurement range at the kth position in the optical axis direction of the second microscope.

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 a base noise from the α _i and/or β _i .

In the correction parameter setting method according to the present 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.

In the correction parameter setting method according to the present invention, the first and second emission spectrum data are fluorescence data including spectral profile data constituted by irradiating a plurality of excitation light beams having different wavelengths and emitting spectrum data of each fluorescence beam obtained by the plurality of excitation light beams.

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 calculated based on a hyperparameter C _hyper which is a sum of relative errors C obtained by the plurality of excitation light beams, as shown in the following formula (4).

Here, 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 sets, as a correction parameter, a parameter p i that minimizes a relative error C i shown in the following formula (5), based on first emission spectrum data obtained by imaging a first sample having a known emission intensity using a first microscope as a reference, 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 the first sample using a second _microscope different from the first microscope.

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, and β _i is the luminance value of the second emission spectrum data in the i-th channel.

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

The present invention has the effect of suppressing deviations in detection results due to individual differences.

FIG. 1 is a diagram showing a schematic configuration of a data correction system according to an embodiment of the present invention. FIG. 2 is a diagram showing a schematic configuration of a reference microscope unit according to an embodiment of the present invention. FIG. 3 is a diagram for 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 present invention. FIG. 5 is a diagram showing a schematic configuration of a microscope unit according to an embodiment of the present invention. FIG. 6 is a diagram showing a schematic configuration of a correction parameter setting device according to an embodiment of the present invention. FIG. 7 is a flowchart illustrating an example of a correction parameter setting method according to an embodiment of the present invention. FIG. 8 is a diagram showing an example of a sample holding device according to an embodiment of the present invention. FIG. 9 is a diagram (part 1) showing an example of detection data used in a correction parameter setting method according to an 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 one 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.

Below, a mode for carrying out the present invention (hereinafter referred to as "embodiment") will be described with reference to the attached drawings.

(Embodiment)
Fig. 1 is a diagram showing a schematic configuration of a data correction system according to an embodiment of the present invention. The data correction system 1 includes a reference microscope unit 2A having 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. In the data correction system 1, the correction parameter setting device 3 sets correction parameters based on data acquired by the reference microscope unit 2A and data acquired by a microscope unit (any of the microscope units 2B to 2D in Fig. 1), and corrects data of the microscope unit using the correction parameters.

FIG. 2 is a diagram showing a schematic configuration of a reference microscope unit according to an embodiment of the present invention. The reference microscope unit 2A shown in the figure generates data that is the basis for parameter setting by the correction parameter setting device 3. The reference microscope unit 2A identifies the type of object captured in the image, for example, the microbial species of a microorganism, based on image data acquired by the confocal laser scanning microscope 100, and displays the identification result and the acquired image. Here, the microorganisms include bacteria, fungi, viruses, microalgae, protozoa, etc. In addition, in this embodiment, the sample to be imaged is any of tissue slices, tissues, animal cells, plant cells, yeast cells, fungal cells, microalgae cells, bacteria, archaea, viruses, and phages, and any of the spores, spores, and membrane vesicles produced by them.

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

The confocal laser scanning microscope 100 comprises 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, a confocal pinhole 108, a detector 109, scanning mirrors 110 and 113, and a transmission light source 111. Hereinafter, two orthogonal axes on a plane parallel to the specimen placement surface of the stage 101 are defined as the X-axis and the Y-axis, and an axis perpendicular to this plane is defined as the Z-axis. Note that the Z-axis will be described as being parallel to the optical axis of the objective lens 102.

The specimen is placed on the stage 101. The stage 101 is configured to be movable in the Z-axis direction using a drive 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 focuses the laser light reflected by the beam splitter 106 toward the stage 101, and also collimates the light from the specimen on the stage 101 so that it is incident on the beam splitter 106.

The 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 multiple light sources each capable of emitting laser light of the wavelength to be used, or may emit white laser light and allow the wavelength of the emitted light to be selected by a filter.

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

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

The beam splitter 106 passes a portion of the incident light and reflects the remaining light. Specifically, the beam splitter 106 bends a portion of the light emitted from the laser light source 103 toward the objective lens 102, and passes a portion of the light incident from the objective lens 102, causing it to enter the imaging lens 107. The beam splitter 106 is configured using, for example, a half mirror, and passes half of the incident laser light and reflects the remaining half.

The imaging lens 107 forms an image of the light that passes through the beam splitter 106.

The confocal pinhole 108 passes at least a part of the light imaged by the imaging lens 107. The confocal pinhole 108 has a pinhole 108a, which is a hole through which light can pass. The confocal pinhole 108 is provided at a position conjugate with the objective lens 102. Therefore, in the confocal pinhole 108, light from the focal plane of the objective lens 102 passes through the pinhole 108a, and light from an unfocused 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 is configured using a reflective diffraction grating that separates the incident light into set wavelength bands, and multiple photomultiplier tubes (PMTs, hereinafter sometimes referred to as channels) that photoelectrically convert the obtained light and current-amplify the converted electrical signal. The detector 109 separates the light into 32 light beams with different wavelength bands, for example, using a reflective diffraction grating, and the separated light beams are incident on each of the 32 photomultiplier tubes. Each photomultiplier tube photoelectrically converts the incident light and outputs an electrical signal. Note that 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 configured using, for example, an X-position control mirror and a Y-position control mirror, and guides the laser light to a predetermined position on the XY plane. Under the control of the control device 200, the scanning mirror 110 changes the angle of each position control mirror to move the irradiation position of the laser light along a preset scanning path.

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, etc.

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

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

The imaging lens 114 forms an image from the light that passes through the scanning mirror 113.

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

The control unit 201 controls the microscope unit 2 by reading out the information stored in the recording unit and executing various 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 the emission of laser light by 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 controls the intermittent emission of the laser light by, for example, pulse control.

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

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

Here, the scanning method by the microscope unit 2A will be described with reference to FIG. 3. FIG. 3 is a diagram for explaining the scanning method of the microscope unit according to one embodiment of the present invention. In the confocal laser scanning microscope 100, after scanning on the XY plane at a focal plane of a certain Z position to receive light from a specimen, the Z position is changed and scanning on the XY plane at the changed Z position is performed. 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 multiple positions on the focal plane at each Z position. In the confocal laser scanning microscope 100, the configuration of the beam splitter 106 and the detector 109 can be appropriately changed according to the image to be generated.

3, after scanning the laser light on focal plane P _F 1, the stage 101 is moved in the Z-axis direction, and the laser light is scanned on focal plane P _F 2, where the focus of the laser light will be located after the movement. This is then performed sequentially on 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) and light from a spot SP, which is an irradiation area, is received. By scanning this spot SP in a zigzag manner, light corresponding to the number of data for generating one two-dimensional image (focused image) can be obtained on the focal plane P _F 7. If the diameter of this spot SP is approximately equal to the size of one pixel (corresponding to one dot displayed on a monitor), for example, the color of the two-dimensional image and the three-dimensional image can be expressed in pixel units, and further, visual information corresponding to the identification information can be colored in pixel units. "Approximately equal to the size of a pixel" means, for example, that when the spot SP is a circle, the size is approximately the same as the size inscribed in the pixel. Note that the above-mentioned scanning path is an example, and is not limited to this path as long as the focal plane can be scanned. Note that the diameter of the spot SP can be appropriately adjusted by changing the diameter of the confocal pinhole, with the lower limit being about 0.2 μm, which is the limit of resolution of the optical microscope.

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

Next, the configuration of the image processing device 300 will be described. The image processing device 300 has a detection signal receiving unit 301, a data generating unit 302, and a recording unit 303.

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

The data generating unit 302 generates data that associates the light intensity based on the electrical signal received from the detection signal receiving unit 301 with position information on the scanning surface. The data generating unit 302 has an autofluorescence data generating unit 302a, a reflected light data generating unit 302b, a corresponding data generating unit 302c, and a reference fluorescent material data generating unit 302d.

The autofluorescence data generating unit 302a acquires the electrical signals of each channel, which are electrical signals related to the autofluorescence received by the detection signal receiving unit 301, and generates intensity data and/or a fluorescence spectrum (spectral data) for each coordinate on a predetermined focal plane (XY plane in FIG. 2). For one position on the scanning plane, the autofluorescence data generating unit 302a generates one fluorescence spectrum when one excitation light is irradiated, and generates multiple fluorescence spectra according to the excitation light when multiple excitation lights of different wavelengths are irradiated at different times. The "fluorescence spectrum" here means the "intensity distribution with respect to wavelength" of the autofluorescence generated when a laser light of a predetermined wavelength is irradiated as the excitation light. In addition, the "intensity" here refers to, for example, a signal value obtained by photoelectric conversion of the obtained autofluorescence. The fluorescence spectrum is, for example, a waveform that has been subjected to a smoothing process by complementing the plots. In this specification, data consisting of multiple fluorescence spectra may be called spectral profile data. In this specification, "autofluorescence data" includes any one or all of the intensity data, spectral data, and spectral profile data of the autofluorescence. The autofluorescence data generating unit 302a generates autofluorescence data that associates the fluorescence spectrum generated according to the excitation wavelength with each position on the scanning plane (multiple coordinates on a specified focal plane).

The reflected light data generating unit 302b acquires the detection signal related to the reflected light reflected by the specimen received by the detection signal receiving unit 301, and generates reflected light data that associates the intensity of the reflected light based on the acquired detection signal with position information on the scanning surface. For example, the reflected light data generating unit 302b sums up the light intensities based on the electrical signals of each channel to obtain the intensity of the reflected light at that position on the scanning surface.

The corresponding data generating unit 302c generates corresponding data consisting of autofluorescence data and reflected light data at one coordinate on a specified focal plane. If autofluorescence data and reflected light data have been generated at multiple coordinates, the corresponding data generating unit 302c generates corresponding data that associates the autofluorescence data with the reflected light data for each coordinate. Furthermore, if autofluorescence data due to multiple excitation lights has been generated at the same coordinate, each autofluorescence data is associated with the coordinate.

Here, we will discuss the significance of associating reflected light data with autofluorescence data at a coordinate on a specified focal plane. The intensity of reflected light reflects the presence of a specimen such as a cell at a coordinate on a specified focal plane. If a specimen (cell) is not present at the coordinate, the intensity of reflected light is low, and if a specimen (cell) is present, high intensity reflected light is obtained. If reflected light is obtained at a high magnification, it is possible to obtain reflected light from the outline of a cell, reflected light from inside the cell, and even reflected light from internal organelles such as the nucleus. In this way, information on the presence or absence of a specimen (cell) at a certain coordinate, or which part of a specimen (cell) a certain coordinate corresponds to, can be obtained, and by using this information and the autofluorescence data at the coordinate, it becomes possible to perform analysis (e.g., identification and evaluation) at the individual cell level and even the intracellular organelle level, which was previously impossible.

The corresponding data generating unit 302c generates image data corresponding to a display image of one frame based on the various associated data. For example, when generating focused image data based on reflected light, the corresponding data generating unit 302c generates one or more focused image data to which luminance information is added for each pixel position based on the reflected light data generated by the reflected light data generating unit 302b according to the number of scanned scanning surfaces. When generating fluorescent image data based on autofluorescence generated by the irradiated excitation light, the corresponding data generating unit 302c generates one or more fluorescent image data to which luminance information is added for each pixel position based on the fluorescent spectrum according to the number of scanned scanning surfaces. In addition to the luminance information, the corresponding data generating unit 302c may superimpose color information according to the microbial species identified by the fluorescent spectrum. The corresponding data generating unit 302c performs image processing using known techniques such as gain processing, contrast processing, and gamma correction processing on the generated two-dimensional image data of one frame, and generates image data for display by performing processing according to the display specifications of the display device 400.

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

Here, the laser light irradiation position corresponds to the spatial information of the image data generated by the corresponding data generating unit 302c. If the spatial position is two-dimensional, it is position information consisting of the pixel position on the X axis (X position) and the pixel position on the Y axis (Y position), and if it is three-dimensional, it is position information consisting of the X position, Y position, and pixel position on the Z axis (Z position). For example, the scanning plane corresponds to a plane perpendicular to the Z axis, and the position on the scanning plane is expressed by the X position and Y position on the scanning plane.

The corresponding data generating unit 302c also performs analysis processing of the sample based on the spectral profile data. In the analysis processing, depending on the processing content, the biological kingdom, phylum, class, order, family, genus, species, variety, pathotype, or antigenic type is identified, the microbiological strain or substrain is identified, or the state of the sample with respect to the metabolic or physiological state of an unknown or known sample is evaluated.

4 is a diagram for explaining focused images generated by scanning in a microscope unit according to an embodiment of the present invention. The corresponding data generating unit 302c performs image processing based on the intensity of reflected light in the corresponding data to generate N focused images D ₁ , D ₂ , ..., D _N based on the light reflected at each focal plane as shown in FIG. 4 (N is a natural number of 3 or more). The corresponding data generating unit 302c converts the intensity of 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 generating unit 302c generates two-dimensional image data including an image generated by reflected light and position information (e.g., Z position) related to the irradiation position of the laser light.

Furthermore, _the correspondence data generation unit 302c generates three-dimensional image data that represents _the specimen image according to the brightness in three-dimensional space by corresponding the brightness information of each focused image in a Cartesian coordinate system in three-dimensional space based on multiple focused image data (focused images D1, D2, ..., _DN ).

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

The recording unit 303 records various programs including a program for executing the operation of the image processing device 300. The recording unit 303 is configured using a ROM (Read Only Memory) in which various programs, etc. are pre-installed, a RAM (Random Access Memory) in which calculation parameters, etc. are recorded, etc.

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

Next, the configuration of the microscope units 2B to 2D will be described with reference to FIG. 5. FIG. 5 is a diagram showing a schematic configuration of a microscope unit according to an embodiment of the present invention. In FIG. 5, the configuration of the microscope unit 2B is shown as a representative, but 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 that controls the microscope unit 2B in an integrated manner, an image processing device 300A that generates various data such as intensity data and image data based on the light acquired by the confocal laser scanning microscope 100, and a display device 400 that displays an image based on the image data for display generated by the image processing device 300A. The microscope unit 2B has the same configuration as the reference microscope unit 2A, except that the microscope unit 2B includes the image processing device 300A instead of the image processing device 300 of the reference microscope unit 2A. In this 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 device 300 will be described. The image processing device 300 has a detection signal receiving unit 301, a data generating unit 302A, and a recording unit 303A.

The data generation unit 302A has an autofluorescence data generation unit 302a, a reflected light data generation unit 302b, a corresponding data generation unit 302c, a reference fluorescent material data generation unit 302d, and a data correction unit 302e.

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

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

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

Next, the configuration of the correction parameter setting device 3 will be described with reference to FIG. 6. FIG. 6 is a diagram 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 unit 31, a calculation unit 32, a setting unit 33, a control unit 34, and a recording unit 35.

The fluorescence data acquisition unit 31 acquires the reference fluorescent material data generated by the reference fluorescent material data generation unit 302d of the reference microscope unit 2A and the reference fluorescent material data generated by the reference fluorescent material data generation unit 302d of the microscopes 2B to 2D. The fluorescence data acquisition unit 31 acquires data from each microscope unit via a communication cable or a 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 calculates the parameters by referring to the calculation formula recorded in the recording unit 35. Specifically, the calculation unit 32 calculates the parameter p by referring to the following formula (1). The calculation unit 32 calculates the parameter p that minimizes the relative error C based on the formula (1).

Here, n is the total number of channels (here, n = 32), α _i is the brightness value of the fluorescence spectrum data (first emission spectrum data) acquired from the reference microscope unit 2A in the i-th channel, and β _i is the brightness value of the fluorescence spectrum data (second emission spectrum data) acquired from any of the microscope units 2B to 2D in the i-th channel.
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, and the total number of arbitrary ranges in the Z direction (number of slices), the parameter represented by adding a superscript bar to x _j is the average luminance value of a part or all of the XY plane in the j-th Z slice, and the parameter represented by adding a superscript bar to y _k is the average luminance value of a part or all of the XY plane in the k-th Z slice. Note that the arbitrary range in the Z direction includes, for example, the maximum average luminance value. Also, the measurement range in the XY plane is one example and can be set arbitrarily. For example, when an image of 1024×1024 pixels is generated by scanning, it is preferable to use the 300×600 pixels in the center of the image as the measurement range and the average luminance value of the measurement range in terms of suppressing luminance unevenness and calculating the average luminance value.

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

The control unit 34 controls the correction parameter setting device 3 by reading the information stored in the recording unit 35 and executing various calculation processes.

The recording unit 35 records various programs including a program 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 a calculation formula used to set the correction parameters. The recording unit 35 is configured using a ROM (Read Only Memory) in which various programs etc. are pre-installed, a RAM (Random Access Memory) that records calculation parameters etc., etc.

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

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

First, the fluorescence data acquisition unit 31 acquires reference fluorescent material data from the reference microscope unit 2A (step S101). The fluorescence data acquisition unit 31 also acquires reference fluorescent material data from the microscope unit (e.g., microscope unit 2B) for which correction parameters are to be set (step S102). At this time, if the reference fluorescent material 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 are based on the fluorescence obtained by exciting a sample (reference fluorescent substance) that has the same emission intensity distribution in the wavelength axis direction and whose concentration and conditions are known. Specifically, samples of the same type and concentration are poured into a fluid device, and the fluorescence from the samples is acquired by exciting them with excitation light of the same wavelength band.

Figure 8 is a diagram showing an example of a sample retention device according to an embodiment of the present invention. A flow path 501 is formed in the sample retention device 500. The flow path 501 has a first opening 502, a second opening 503, and a flow path 504 connecting the first opening 502 and the second opening 503. The flow path 504 forms a closed space except for the connection part with the first opening 502 and the second opening 503. Note that the flow path shown in Figure 8 is only an example, and a meandering or branching flow path can also be applied.

For example, the sample is introduced into the flow channel 501 from the first opening 502, passes through the flow section 504, and is released to the outside from the second opening 503 (see the arrows in FIG. 8 ). The confocal laser scanning microscope 100 scans a region R including the center of the flow section 504 to measure the light of the region R. At this time, the optical axis N of the confocal laser scanning microscope 100 scans the region R to measure the light from the sample (hereinafter, also referred to as a "fluid sample") flowing through the flow section 504. Note that scanning of the region R may be performed multiple times, and averaging or integrating the light measurement values may be performed for each scanning position or the entire scanning range.
By continuously flowing the fluid sample through the flow path 501 and measuring the fluorescence, it is possible to perform photometry while suppressing fading of the sample fluorescence caused by laser light irradiation. Note that the flow rate of the liquid sample is preferably set to a rate that makes the residence time of the liquid sample in the region R sufficiently short to prevent fading of the fluorescence.

This reference fluorescent material data is obtained by repeatedly scanning the XY plane while moving in the Z direction. In other words, the reference fluorescent material data is point cloud data in which multiple measurement points are arranged along the Z direction in three-dimensional space, and each point has spectral data.

Then, the calculation unit 32 calculates the parameter p that minimizes the relative error C using the above formula (1) (step S103). For example, the calculation unit 32 changes the parameter p in increments of 0.01 to calculate the relative error C, and extracts the smallest relative error C. The calculation unit 32 outputs the parameter p that corresponds to the extracted smallest 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, the reference fluorescent material data and the corrected intensity data corrected by the correction parameters will be described with reference to Figs. 9 to 12. Fig. 9 is a diagram (part 1) showing an example of detection data used in a correction parameter setting method according to an embodiment of the present invention. The fluorescence data shown in Fig. 9 is based on the fluorescence of a sample in which the fluorescent material is Coumarin 6H and its concentration is 1 x ^10-4 % (in 99% ethanol). In Fig. 9, the intensity data S1 (Microscope 1) is the intensity data acquired by the reference microscope unit 2A, and the intensity data S2 (Microscope 2) is the intensity data acquired by the microscope unit 2B. Each microscope unit scans the XY plane and acquires fluorescence in a plurality of XY planes with different Z positions while moving in the Z direction at a predetermined pitch. The intensity data shown in Fig. 9 is data acquired at 18 points along the Z direction. The intensity data S1 and S2 indicate the average brightness of the central region of the image based on the scanning of each XY plane at the Z position (z slice). The deviation in the horizontal direction of the intensity data S1, S2 corresponds to a structural deviation caused by individual differences in the microscope units, and this deviation causes a deviation in the intensity data in the wavelength axis direction . The deviation in the vertical direction of the intensity data S1, S2 corresponds to a performance deviation caused by individual differences in the detector 109. Although the intensity data S1, S2 are offset in the wavelength axis direction and intensities, the waveform patterns (emission intensity distributions) in the wavelength axis direction are the same.

Figure 10 is a diagram (part 1) showing an example of a spectrum before and after correction processing using correction parameters according to an embodiment of the present invention. Intensity data S11 (Microscope 1) shown in Figure 10 indicates the average brightness for each wavelength band based on the intensity data S1 shown in Figure 9, and intensity data S12 (Microscope 2) indicates the average brightness for each wavelength band based on the intensity data S2 shown in Figure 9. The intensity data S11 and S12 indicate the average brightness for 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 the detector 109. In other words, the intensity of each wavelength band is the intensity of the signal output by the channel. In addition, the predetermined Z range is set, for example, as the distance in the Z direction (several μm) or the number of images in the Z direction (number of slices).

For the intensity data S11 and S12, the calculation unit 32 calculates the relative error C while changing the parameter p in the above formula (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 correction unit 302e corrects the intensity data S2 of the microscope unit 2B using the set parameter p.

The corrected intensity data SC1 (Corrected Microscope 2) shown in Figure 10 indicates waveform data obtained by correcting the intensity data S12 using the parameter p (here, p = 1.33). As shown in Figure 10, the corrected intensity data SC1 has a waveform that is almost equivalent to the intensity data S11 of the reference microscope unit 2A, and the average percentage error is 6%.

11 is a diagram (part 2) showing an example of detection data used in a correction parameter setting method according to an embodiment of the present invention. The fluorescence data shown in FIG. 11 is based on the autofluorescence of a sample whose fluorescent substance is fluorescein and whose concentration is 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 intensity data S3 and S4 are data acquired under the same conditions as the intensity data S1 and S2 shown in FIG. 9.

Figure 12 is a diagram (part 2) showing an example of spectra before and after correction processing using correction parameters according to one embodiment of the present invention. Intensity data S13 (Microscope 1) shown in Figure 12 indicates the average brightness for each wavelength band based on intensity data S3 shown in Figure 11, and intensity data S14 (Microscope 2) indicates the average brightness for each wavelength band based on intensity data S4 shown in Figure 11. Intensity data S13 and S14 indicate the average brightness for each wavelength band in image data of a specified Z range that includes 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 formula (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 correction unit 302e corrects the intensity data S4 of the microscope unit 2B using the set parameter p.

The corrected intensity data SC2 (Corrected Microscope 2) shown in Figure 12 indicates waveform data obtained by correcting the intensity data S14 using the parameter p (here, p = 1.76). As shown in Figure 12, the corrected intensity data SC2 has a waveform that is almost equivalent to the intensity data S13 of the reference microscope unit 2A, and the average percentage error is 7.1%.

In the embodiment of the present invention described above, the correction parameters of the microscope units 2B to 2D are set using the intensity data obtained from a sample under the same conditions by the reference microscope unit 2A and the microscope units 2B to 2D and the above formula (1), thereby obtaining corrected intensity data in which deviations in the detection results due to individual differences are suppressed. According to this embodiment, various analyses are performed using the corrected intensity data corrected by the correction parameters, thereby obtaining analysis results in which the effects of deviations due to individual differences between the microscope units are suppressed.

(Variation 1)
Next, a first modified example of the embodiment will be described. In the embodiment, an example in which correction parameters are set for each excitation light has been described, but correction parameters specific to a specimen and not dependent on excitation light may be set.

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 the first modification, the calculation unit 32 calculates a 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 (total number of types of excitation wavelengths) (0<q≦o). For example, when the excitation lights used are 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 for the microscope unit (any of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31.

According to the above-described variant 1, similar to the above-described embodiment, various analyses can be performed using corrected intensity data corrected by the correction parameters, thereby obtaining analysis results that suppress the influence of deviations due to individual differences between microscope units. Furthermore, according to this variant 1, it is possible to set correction parameters that are specific to the specimen and are not dependent on the excitation light, and as a result, it is possible to suppress the complication of the correction parameters due to the excitation light.

(Variation 2)
Next, a second modification of the embodiment will be described. In the embodiment, an example in which the correction parameters are set for each excitation light has been described, but the correction parameters 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 the second modification, the calculation unit 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 shown when 1≦i≦n, and i, n, α _i , and β _i are the same as those in the above formula (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 (any of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31. In the correction process, the correction parameter set according to the channel is used.

According to the above-described variant 2, similar to the above-described embodiment, various analyses can be performed using corrected intensity data corrected by the correction parameters, thereby obtaining analysis results that suppress the effects of deviations due to individual differences between microscope units. Furthermore, according to this variant 2, correction parameters can be set for each channel, and as a result, individual differences between microscope channels can be suppressed.

(Variation 3)
Next, a third modification 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 is a cause of discrepancies in intensity data between microscopes, it is preferable to calculate the correction parameters using intensity data from which this base noise has been removed. Here, the base noise is defined by either Definition 1 or 2 below.
Definition 1. The average value of the luminance values of the channels that detect light outside the wavelength band of the fluorescence of the reference fluorescent substance derived from the sample. For example, when the reference fluorescent substance is observed under a microscope without base noise, the luminance value is equal to or less than a certain value, for example, 20 or less.
Definition 2. The average brightness value detected when the detector voltage is zero.

The calculation unit 32 calculates the correction parameters according to the microscope in which the base noise is present. Specifically, the calculation unit 32 calculates the correction parameters using any 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 the microscope units 2B to 2D]
The calculation unit 32 calculates a 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 (any 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 the reference microscope unit 2A]
The calculation unit 32 calculates a parameter p that minimizes the relative error C based on the following equation (7).

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

[When base noise exists in the reference microscope unit 2A and the microscope units 2B to 2D]
The calculation unit 32 calculates a parameter p that minimizes the relative error C based on the following equation (8).

According to the above-described third modification, similar to the above-described embodiment, various analyses can be performed using corrected intensity data corrected by the correction parameters, thereby obtaining analysis results in which the influence of deviations due to individual differences between microscope units is suppressed. Furthermore, according to the third modification, the influence of base noise can be eliminated and the correction parameters can be set from the values of the reference fluorescent material itself, and as a result, the individual differences between microscopes can be further suppressed.

So far, we have explained the form for implementing the present invention, but the present invention should not be limited to only the above-mentioned embodiment.

In the above embodiment, a three-dimensional space is scanned to generate the autofluorescence data, reflected light data, and corresponding data. However, a two-dimensional space (any of the XY plane, XZ plane, and YZ plane shown in FIG. 3) may be scanned to generate the reference fluorescent material data, autofluorescence data, reflected light data, and corresponding data, or scanning may be performed in any one of the X direction, Y direction, and Z direction shown in FIG. 3, or the fluorescence, autofluorescence, and reflected light of a certain point in the space may be acquired to generate the reference fluorescent material data, autofluorescence data, reflected light data, and corresponding data. In particular, since it is sufficient for the reference fluorescent material data for setting the correction parameters to be able to acquire spectral data, only the reference fluorescent material data of a certain point may be used, and the correction data setting device 3 may acquire the reference fluorescent material data of this point from each microscope unit.

In addition, in the above-mentioned embodiment, a three-dimensional image is generated and displayed, but a two-dimensional image may be displayed or visual information may be superimposed, or the image to be displayed may be selected in response to operational input from the user.

In the above embodiment, the detector 109 is described as being configured using a reflective diffraction grating and a photomultiplier tube (PMT), but it may also be configured as, for example, an acousto-optic beam splitter (e.g., Leica's AOBS (registered trademark)), a high-sensitivity detector (HyD detector), and a detector configured with a movable slit structure provided in front of the detector. With the above-mentioned configuration, this detector is capable of acquiring data separated into wavelengths of, for example, 1 nm each.

In the above-described embodiment, the reflected light or autofluorescence is obtained using laser light, but the reflected light or autofluorescence may be obtained by collecting low-directional light (e.g., light from a halogen lamp) and irradiating it on the sample, not limited to highly directional light such as laser light. For example, autofluorescence may be obtained using laser light and reflected light may be obtained using a halogen lamp, or autofluorescence may be obtained using a halogen lamp and reflected light may be obtained using laser light, or autofluorescence and reflected light may be obtained using a halogen lamp. The wavelength of light may be passed through a filter or may be dispersed by a prism. In addition, the light may be transmitted through a filter, or may be separated by a prism. Any light detection data that can extract the contours of cells, etc., may be used, not limited to reflected light from the specimen, such as transmitted light or fluorescence.

In addition, in the above-mentioned embodiment, an example was described in which fluorescence was measured while a sample was flowing through the flow path 501, but the present invention is not limited to this. It is also possible to measure fluorescence obtained by exciting a sample placed in a container, or to measure light with a known emission intensity (autofluorescence, laser light, etc.).

In the above embodiment, the microscope unit and the correction parameter setting device 3 are provided separately, but the correction parameter setting device 3 may be integrated with the microscope unit. For example, the microscope units 2B to 2D may be provided with 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 be provided with the correction parameter setting device 3, and the correction parameters may be set in the reference microscope unit 2A.

As such, the present invention can include various embodiments without departing from the technical concept described in the claims.

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

1 Data correction system 2A Reference microscope unit 2B to 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 Transmitted light source 200 Control device 202 Input unit 203 Laser control unit 204 Scanning control unit 205 Transmitted light control unit 300 Image processing device 301 Detection signal receiving unit 302 Data generation unit 302a Autofluorescence data generation unit 302b Reflected light data generating section 302c Corresponding data generating section 302d Reference fluorescent material data generating section 302e Data correcting section 400 Display device 500 Sample holding device

Claims (11)

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

Here, n is the total number of channels, α _i is the luminance value of the first emission spectrum data in the i-th channel, and β _i is the luminance value of the second emission spectrum data in the i-th channel. the first and second emission spectrum data are generated based on light obtained from the first and second samples arranged 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 respectively acquired at a plurality of coordinates different from each other on the predetermined focal plane.
The correction parameter setting method according to claim 2 . Performed at 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, _m1 and _m2 are the total number of measurement points, which are the total number of any range in the optical axis direction of the first or second microscope, the parameter represented by _xj with a superscript bar is the average brightness value of a preset measurement range at the jth position in the optical axis direction of the first microscope, and the parameter represented by _yk with a superscript bar is the average brightness value of a preset measurement range at the kth position in the optical axis direction of the second microscope. The relative error C is calculated based on a value obtained by subtracting a 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 fluorescence data including spectral profile data constituted by emission spectrum data of each fluorescence obtained by irradiating a plurality of excitation light beams having different wavelengths, the emission spectrum data being constituted by spectral profile data constituted by emission spectrum data of each fluorescence obtained by irradiating a plurality of excitation light beams having different wavelengths,
The correction parameter setting method according to claim 7 . The parameter p is calculated based on a hyper parameter C _hyper , which is the sum of the relative errors C obtained by the plurality of excitation light beams, as shown in the following formula (4):
The correction parameter setting method according to claim 8.

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

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, and β _i is the luminance value of the second emission spectrum data in the i-th channel. A data correction method for correcting the second emission spectrum data using the correction parameters set by the correction parameter setting method according to any one of claims 1 to 10.

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