Correction Parameter Setting Method and Data Correction Method

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

Technical Field

[0001] The present invention relates to a correction parameter setting method and a data correction method for correcting data between microscope apparatuses.

Background Art

[0002] Conventionally, when observing characteristics of cells or the like contained in a sample, the intensity and spectrum of light are calculated. An observer checks the characteristics of the sample by looking at the calculated spectrum or the like. For example, Patent Document 1 uses a microscope apparatus to measure the spectrum of autofluorescence of cells, calculates the spectrum, and identifies the type of cells or the like from the spectrum.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] By the way, even for microscope apparatuses of the same model, the sensitivity to light may differ due to individual differences between the apparatuses. This is due to differences in sensitivity for each light-receiving element and appears as a difference in luminance values. When observing the characteristics of cells based on the intensity of autofluorescence of the cells, even if the intensity of the light emitted from the sample is the same, the luminance values obtained for each microscope apparatus are different, and it is necessary to correct the data with appropriate parameters in order to use the luminance value data obtained by a plurality of microscopes for quantitative evaluation.

[0005] The present invention has been made in view of the above, and an object thereof is to provide a correction parameter setting method and a data correction method capable of suppressing a deviation in detection results due to individual differences.

Means for Solving the Problems

[0006] In order to solve the above-described problems and achieve the object, the correction parameter setting method according to 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 with a known emission intensity using a first microscope serving as a reference, and second emission spectrum data obtained by imaging a second sample, which is 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.

Equation

[0007] In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are respectively generated based on light obtained from the first and second samples arranged at one coordinate on a predetermined focal plane.

[0008] In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are respectively acquired at a plurality of different coordinates on the predetermined focal plane.

[0009] In the correction parameter setting method according to the present invention, in the above invention, it is carried out at a plurality of different focal planes.

[0010] In the correction parameter setting method according to the present invention, in the above invention, the α i , β i are calculated based on the following formulas (2) and (3).

Equation

[0011] In the correction parameter setting method according to the present invention, in the above invention, the relative error C is based on the value obtained by removing the base noise from the α i and / or β i and is calculated.

[0012] 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.

[0013] In the correction parameter setting method according to the present invention, in the above invention, the first and second emission spectrum data are fluorescence data including spectrum profile data constituted by emission spectrum data of each fluorescence obtained by irradiating a plurality of excitation lights having different wavelengths and obtained by the plurality of excitation lights.

[0014] In the correction parameter setting method according to the present invention, in the above invention, the parameter p is calculated based on the hyperparameter C which is the sum of each relative error C obtained by the plurality of excitation lights and is shown in the following formula (4). hyper Here, o is the total number of excitation lights used (0 < q ≤ o).

Equation

[0015] The correction parameter setting method according to the present invention is based on the first emission spectrum data obtained by imaging a first sample with a known emission intensity using a first reference microscope, and a second microscope different from the first microscope. Based on the second emission spectrum data obtained by imaging a second sample known to have the same emission intensity distribution as the first sample in the wavelength axis direction, the relative error C shown in the following formula (5) i The parameter p at which is minimized i Is set as the correction parameter.

Equation

[0016] The 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 above invention.

Advantages of the Invention

[0017] According to the present invention, there is an effect that it is possible to suppress the deviation of the detection result due to individual differences.

Brief Description of the Drawings

[0018]

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Embodiments for Carrying Out the Invention

[0019] Hereinafter, embodiments for carrying out the present invention (hereinafter referred to as "embodiments") will be described with reference to the accompanying drawings.

[0020] (Embodiment) FIG. 1 is a diagram schematically 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 including a reference microscope (a confocal laser scanning microscope in the present embodiment), a plurality of microscope units (microscope units 2B, 2C, 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 the data acquired by the reference microscope unit 2A and the data acquired by a microscope unit (any one of the microscope units 2B to 2D in FIG. 1), and corrects the data of the microscope unit using these correction parameters.

[0021] FIG. 2 is a diagram schematically 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 serves as a reference 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 microorganisms, based on the image data acquired by the confocal laser scanning microscope 100, and displays the identification result and the acquired image. Here, microorganisms include bacteria, fungi, viruses, microalgae, protozoa, and the like. In the present embodiment, the sample to be imaged is any one of a tissue section, a tissue, an animal cell, a plant cell, a yeast cell, a fungal cell, a microalgal cell, bacteria, archaea, a virus, a phage, and spores, endospores, and membrane vesicles produced by them.

[0022] As shown in FIG. 2, the reference microscope unit 2A includes a confocal laser scanning microscope 100 that irradiates laser light to acquire autofluorescence of a specimen or transmitted / reflected light from the specimen, a control device 200 that comprehensively controls 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 the present embodiment, the confocal laser scanning microscope 100 included in the reference microscope unit 2A corresponds to the first microscope.

[0023] The 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, 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 mounting surface of the stage 101 are defined as the X-axis and the Y-axis, and an axis orthogonal 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.

[0024] The stage 101 mounts a specimen. The stage 101 is configured to be movable in the Z-axis direction under the control of the control device 200, using a drive source such as a motor, for example. The specimen is a solution or 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.

[0025] The objective lens 102 condenses the laser light reflected by the beam splitter 106 toward the stage 101, and makes the light from the specimen on the stage 101 parallel and incident on the beam splitter 106.

[0026] 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 a plurality of light sources each capable of emitting laser light of a different wavelength, or may irradiate white laser light so that the wavelength of the emitted light can be selected by a filter.

[0027] The lens 104 emits the laser light emitted by the laser light source 103 as radial laser light.

[0028] 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.

[0029] The beam splitter 106 allows a part of the incident light to pass through and reflects the remaining light. Specifically, the beam splitter 106 bends a part of the light emitted from the laser light source 103 toward the objective lens 102 and allows a part of the light incident from the objective lens 102 to pass through, thereby making it incident on the imaging lens 107. The beam splitter 106 is configured using, for example, a half mirror, and allows half of the incident laser light to pass through and reflects the remaining half of the laser light.

[0030] The imaging lens 107 forms an image of the light that has passed through the beam splitter 106.

[0031] The confocal pinhole 108 allows at least a part of the light imaged by the imaging lens 107 to pass through. The confocal pinhole 108 is formed with a pinhole 108a which is a hole through which light can pass. Further, the confocal pinhole 108 is provided at a position conjugate to the objective lens 102. For this reason, 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 a position that is not in focus is blocked. For example, when the spot diameter of the laser light imaged by the imaging lens 107 is 0.2 μm, at the imaging position, light from a range of about 0.03 μm 2 passes through the pinhole 108a. Note that the diameter of the pinhole 108a and the size of the focal space can be changed by setting.

[0032] Detector 109 is composed of a reflective diffraction grating that separates the incident light into a set wavelength band, and a plurality of photomultiplier tubes (PMT, hereinafter sometimes referred to as channels) that perform photoelectric conversion on the obtained light and amplify the current of the converted electrical signal. Detector 109 separates, for example, into 32 lights with different wavelength bands by a reflective diffraction grating, and the separated lights are incident on 32 photomultiplier tubes respectively. Each photomultiplier tube performs photoelectric conversion on the incident light and outputs an electrical signal. In this embodiment, an example having 32 photomultiplier tubes will be described, but the number of photomultiplier tubes is not limited to this.

[0033] Scanning mirror 110 controls the irradiation position of the laser light on the focal plane P of the specimen under the control of control device 200. Scanning mirror 110 is composed of, 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. Scanning mirror 110 moves the irradiation position of the laser light along a preset scanning path by changing the angles of the respective position control mirrors under the control of control device 200. F Under the control of control device 200, scanning mirror 110 controls the irradiation position of the laser light on the focal plane P of the specimen. Scanning mirror 110 is composed of, 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. Scanning mirror 110 moves the irradiation position of the laser light along a preset scanning path by changing the angles of the respective position control mirrors under the control of control device 200.

[0034] Transmission light source 111 emits transmission light that passes through stage 101 under the control of control device 200. Transmission light source 111 is composed of, for example, a halogen lamp, a laser light source, etc.

[0035] Collimating lens 112 converts the radial light emitted by transmission light source 111 into parallel light and emits it to scanning mirror 113.

[0036] Scanning mirror 113 controls the irradiation position of the transmission light on the focal plane P of the specimen under the control of control device 200. Scanning mirror 113 is composed of an X-position control mirror and a Y-position control mirror, for example, in the same way as scanning mirror 110. F Under the control of control device 200, scanning mirror 113 controls the irradiation position of the transmission light on the focal plane P of the specimen. Scanning mirror 113 is composed of an X-position control mirror and a Y-position control mirror, for example, in the same way as scanning mirror 110.

[0037] Imaging lens 114 forms an image of the light that has passed through scanning mirror 113.

[0038] 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. 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.

[0039] The control unit 201 reads the information stored in the recording unit and executes various arithmetic processes to comprehensively control the microscope unit 2. The control unit 201 includes a laser control unit 203, a scanning control unit 204, and a transmitted light control unit 205.

[0040] The laser control unit 203 controls the emission of laser light from the laser light source 103 based on a control program and the 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 the laser light, for example, by pulse control.

[0041] The scanning control unit 204 controls the position of the stage 101 in the Z direction and the irradiation position of the laser light by the scanning mirror 110 based on a control program and the instruction information received by the input unit 202.

[0042] The transmitted light control unit 205 performs drive control of the transmitted light source 111 based on a control program and the instruction information received by the input unit 202.

[0043] 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 an embodiment of the present invention. In the confocal laser scanning microscope 100, after scanning the XY plane at a 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. For example, the Z scanning range R shown in FIG. 2 ZScanning is performed for each Z position set therein to obtain light (reflected light or autofluorescence) 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.

[0044] For example, as shown in FIG. 3, after scanning the laser beam on the focal plane P F 1, the stage 101 is moved in the Z-axis direction, and after the movement, the laser beam is scanned on the focal plane P F 2 where the focus of the laser beam is arranged. This is repeated for the preset Z scanning range R Z for the focal planes P F 3, P F 4, P F 5, P F 6, P F 7, ··· in sequence.

[0045] Regarding the scanning method in the XY plane, for example, as shown in FIG. 3, the laser beam is irradiated from one corner of the rectangular focal plane (focal plane P in FIG. 3 F 7), and the light from the spot SP which is the irradiation area is received. By scanning this spot SP in a zigzag manner, it is possible to obtain the light corresponding to the number of data for generating one two-dimensional image (in-focus image) on the focal plane P F 7. If the diameter of this spot SP is made approximately equal to the size of one pixel (corresponding to one dot displayed on the monitor), the colors of the two-dimensional image and the three-dimensional image can be expressed in pixel units, and furthermore, the visual information corresponding to the identification information can be color-coded in pixel units. "Approximately equal to the size of the pixel" means, for example, when the spot SP is a circle, it means a size that is almost the same as the size inscribed in the pixel. Note that the above-described scanning path is an example, and as long as the focal plane can be scanned, it is not limited to this path. 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 approximately 0.2 μm which is the limit of the resolution of the optical microscope.

[0046] The input unit 202 receives the input of various types of information. The input unit 202 is configured using a user interface such as a keyboard, a mouse, a touch panel, etc.

[0047] Next, the configuration of the image processing apparatus 300 will be described. The image processing apparatus 300 includes a detection signal receiving unit 301, a data generation unit 302, and a recording unit 303.

[0048] The detection signal receiving unit 301 receives the electrical signals of each channel from the detector 109. The detection signal receiving unit 301 associates the received electrical signals of each channel with the position information (laser light irradiation position) on the scanning surface and outputs the result to the data generation unit 302. Note that the detection signal receiving unit 301 may be provided separately for reflected light detection and autofluorescence detection.

[0049] The data generation unit 302 generates data associating the intensity of light based on the electrical signals received from the detection signal receiving unit 301 with the position information on the scanning surface. The data generation unit 302 includes an autofluorescence data generation unit 302a, a reflected light data generation unit 302b, a corresponding data generation unit 302c, and a reference fluorescent substance data generation unit 302d.

[0050] The autofluorescence data generation unit 302a acquires electrical signals related to autofluorescence received by the detection signal receiving unit 301, obtains the electrical signals of each channel, and generates intensity data and / or fluorescence spectra (spectral data) for each coordinate on a predetermined focal plane (the XY plane in FIG. 2). For one position on the scanning surface, when one excitation light is irradiated, the autofluorescence data generation unit 302a generates one fluorescence spectrum; when excitation lights of a plurality of different wavelengths are irradiated at different timings, the autofluorescence data generation unit 302a generates a plurality of fluorescence spectra according to the excitation lights. Here, the "fluorescence spectrum" means the "intensity distribution with respect to wavelength" of the autofluorescence generated when laser light of a predetermined wavelength is irradiated as the excitation light. Also, the "intensity" here refers to, for example, the signal value obtained by photoelectrically converting the obtained autofluorescence. The fluorescence spectrum consists of, for example, a waveform obtained by complementing between plots and performing smoothing processing. In this specification, data consisting of a plurality of fluorescence spectra may be referred to as spectral profile data. In this specification, "autofluorescence data" includes any or all of the intensity data, spectral data, and spectral profile data of autofluorescence. The autofluorescence data generation unit 302a generates autofluorescence data in which the fluorescence spectra generated according to the excitation wavelength are associated with each position (a plurality of coordinates on a predetermined focal plane) on the scanning surface.

[0051] The reflected light data generation unit 302b acquires a detection signal related to the reflected light reflected by the specimen, which is received by the detection signal receiving unit 301, and generates reflected light data in which the intensity of the reflected light based on the acquired detection signal is associated with the position information on the scanning surface. The reflected light data generation unit 302b, for example, sums up the intensities of the light based on the electrical signals of each channel to obtain the intensity of the reflected light at that position on the scanning surface.

[0052] The corresponding data generation unit 302c generates corresponding data consisting of autofluorescence data and reflected light data at one coordinate on a predetermined focal plane. If autofluorescence data and 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. Further, if autofluorescence data by a plurality of excitation lights is generated at the same coordinate, the autofluorescence data is associated with each other at that coordinate.

[0053] Here, the significance of associating the reflected light data and the autofluorescence data at one coordinate on a predetermined focal plane will be described. The intensity of the reflected light reflects the presence of a specimen such as a cell at one coordinate on a predetermined focal plane. If there is no specimen (cell) at the coordinate, the intensity of the reflected light is low, and if there is a specimen (cell), reflected light with a high intensity can be obtained. If the reflected light at a high magnification is acquired, it is also possible to obtain the reflected light from the contour portion of the cell, the reflected light from inside the cell, and further the reflected light from intracellular organelles such as the nucleus inside. In this way, information on the presence or absence of a specimen (cell) at a certain coordinate or which part of the specimen (cell) a certain coordinate corresponds to is acquired, and by using this and the autofluorescence data at the coordinate, analysis (for example, identification and evaluation) at the individual cell level and further at the intracellular organelle level, which has been impossible until now, becomes possible.

[0054] The corresponding data generation unit 302c generates image data corresponding to the display image for one frame based on the various associated data. For example, when generating in-focus image data based on reflected light, the corresponding data generation unit 302c generates one or more pieces of in-focus image data with luminance information assigned to each pixel position based on the reflected light data generated by the reflected light data generation unit 302b, according to the number of scanned scanning surfaces. Also, when generating fluorescence image data due to autofluorescence caused by the irradiated excitation light, the corresponding data generation unit 302c generates one or more pieces of fluorescence image data with luminance information assigned to each pixel position based on the fluorescence spectrum, according to the number of scanned scanning surfaces. In addition to the luminance information, the corresponding data generation unit 302c may superimpose color information according to, for example, the microbial species identified by the fluorescence spectrum. The corresponding data generation 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 for one frame, and also performs processing according to the display specifications of the display device 400 to generate image data for display.

[0055] 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 assigning the luminance information in each frame to three-dimensional space.

[0056] Here, the laser light 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 the case of two dimensions, and position information consisting of the X position, Y position, and the position of the pixel on the Z-axis (Z position) in the case of three dimensions. For example, the scanning surface corresponds to a plane orthogonal to the Z-axis, and the position on the scanning surface is represented by the X position and Y position on that scanning surface.

[0057] In addition, the corresponding data generation unit 302c performs analysis processing of the sample based on the spectral profile data. In the analysis processing, depending on the processing content, it identifies a biological kingdom, phylum, class, order, family, genus, species, variety, pathogen type, or antigen type, identifies a microbiological strain or sub-strain, or evaluates the state of the sample regarding the metabolic state or physiological state of an unknown sample or a known sample.

[0058] FIG. 4 is a diagram for explaining a focused image generated by scanning in the microscope unit according to an embodiment of the present invention. The corresponding data generation unit 302c performs image processing based on the intensity of the reflected light among the corresponding data, and thereby generates N focused images D1, D2, ···, D N as shown in FIG. 4, based on the light reflected on each focal plane (N is a natural number of 3 or more). The corresponding 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 the image generated by the reflected light and position information (for example, Z position) regarding the irradiation position of the laser light.

[0059] Furthermore, the corresponding data generation unit 302c generates three-dimensional image data representing a specimen image according to the luminance on the three-dimensional space by associating the luminance information of each focused image in the orthogonal coordinate system of the three-dimensional space based on a plurality of focused image data (focused images D1, D2, ···, D N ).

[0060] The reference fluorescent substance data generation unit 302d generates light intensity data of a reference fluorescent substance used for setting correction parameters.

[0061] The recording unit 303 records various programs including a program for executing the operation 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.

[0062] 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 display various information generated by the control device 200.

[0063] Next, the configuration of the microscope units 2B to 2D will be described with reference to FIG. 5. FIG. 5 is a diagram schematically showing the schematic configuration of the 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 comprehensively controls the microscope unit 2B, 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 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 includes the image processing device 300A instead of the image processing device 300 of the reference microscope unit 2A. In the present embodiment, the confocal laser scanning microscope 100 included in the reference microscope units 2B to 2D corresponds to the second microscope.

[0064] Next, the configuration of the image processing device 300 will be described. The image processing device 300 includes a detection signal receiving unit 301, a data generation unit 302A, and a recording unit 303A.

[0065] The data generation unit 302A includes a self-fluorescence data generation unit 302a, a reflected light data generation unit 302b, a correspondence data generation unit 302c, a reference fluorescent substance data generation unit 302d, and a data correction unit 302e.

[0066] The data correction unit 302e corrects the intensity data of the self-fluorescence generated by the self-fluorescence data generation unit 302a according to the correction parameters set by the correction parameter setting device 3.

[0067] The recording unit 303A records various programs including a program for executing the 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 arithmetic parameters and the like, and the like.

[0068] The recording unit 303A has a correction information recording unit 303a that records the correction parameters set by the correction parameter setting device 3.

[0069] Subsequently, the configuration of the correction parameter setting device 3 will be described with reference to FIG. 6. FIG. 6 is a diagram schematically showing the schematic configuration of the correction parameter setting device according to an embodiment of the present invention. The correction parameter setting device 3 includes a fluorescence data acquisition unit 31, a calculation unit 32, a setting unit 33, a control unit 34, and a recording unit 35.

[0070] The fluorescence data acquisition unit 31 acquires the reference fluorescence substance data generated by the reference fluorescence substance data generation unit 302d of the reference microscope unit 2A and the reference fluorescence substance data generated by the reference fluorescence substance 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.

[0071] The calculation unit 32 calculates parameters with reference to the calculation formula recorded in the recording unit 35. Specifically, the calculation unit 32 calculates the parameter p with reference to the following formula (1). The calculation unit 32 calculates the parameter p for which the relative error C becomes the smallest based on formula (1).

Equation

Equation

[0072] The setting unit 33 sets the parameter p calculated by the calculation unit 32 as the correction parameter of the microscope unit (any one of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31.

[0073] The control unit 34 reads out the information stored in the recording unit 35 and executes various arithmetic processes to comprehensively control the correction parameter setting device 3.

[0074] 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 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 pre-installed, a RAM (Random Access Memory) that records calculation parameters and the like, and the like.

[0075] 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 including a CPU (Central Processing Unit) having arithmetic and control functions, various arithmetic circuits such as an FPGA (Field Programmable Gate Array), a ROM, a RAM, and the like.

[0076] Next, a correction parameter setting method by the correction parameter setting device 3 will be described with reference to FIG. 7. FIG. 7 is a flowchart for explaining an example of a correction parameter setting method according to an embodiment of the present invention. Hereinafter, the flow of setting correction parameters based on the obtained autofluorescence will be described.

[0077] First, the fluorescence data acquisition unit 31 acquires reference fluorescence substance data from the reference microscope unit 2A (step S101). Further, the fluorescence data acquisition unit 31 acquires reference fluorescence substance data from the microscope unit (for example, microscope unit 2B) for which correction parameters are to be set (step S102). At this time, if the reference fluorescence substance data of the reference microscope unit 2A has already been acquired, the process of step S101 may be omitted.

[0078] The reference fluorescence substance data acquired in steps S101 and S102 is based on fluorescence obtained by exciting a sample (reference fluorescence substance) in which the emission intensity distributions in the wavelength axis direction are the same and whose concentration and conditions are known. Specifically, fluorescence from a sample in which a sample of the same type and the same concentration flowing into the fluid device is excited by excitation light in the same wavelength band is acquired.

[0079] FIG. 8 is a diagram showing an example of a sample holding device according to an embodiment of the present invention. A sample holding device 500 has a flow path 501 formed therein. The flow path 501 has a first opening 502, a second opening 503, and a flow-through portion 504 connecting the first opening 502 and the second opening 503. The flow-through portion 504 forms a closed space except at the connecting portions with the first opening 502 and the second opening 503. Note that the flow path shown in FIG. 8 is an example, and a meandering or branched flow path can be applied.

[0080] A sample is introduced into the flow path 501 from the first opening 502, for example, and is discharged to the outside from the second opening 503 through the flow-through portion 504 (see the arrow in FIG. 8). The confocal laser scanning microscope 100 scans a region R including the central portion of the flow-through portion 504 and performs photometry of the region R. At this time, the optical axis N of the confocal laser scanning microscope 100 scans within the region R and measures light from a sample (hereinafter, also referred to as a "fluid sample") flowing through the flow-through portion 504. Note that the scanning within the region R may be performed a plurality of times, and an averaging process or an integration process of the photometric 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 its fluorescence, photometry can be performed while suppressing the fading of the sample fluorescence due to laser light irradiation. Note that the flow rate of the liquid sample is preferably set to a speed such that the residence time within the region R is sufficiently short to prevent the fading of fluorescence.

[0081] Further, 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 substance data is data of a point group in which a plurality of measurement points are arranged along the Z direction in a three-dimensional space, and has spectral data at each point.

[0082] After that, the calculation unit 32 calculates a parameter p that minimizes the relative error C using the above equation (1) (step S103). For example, the calculation unit 32 calculates the relative error C by changing the parameter p in increments of 0.01, and extracts the smallest relative error C. The calculation unit 32 outputs the parameter p corresponding to the extracted minimum relative error C to the setting unit 33.

[0083] 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).

[0084] Here, the reference fluorescent substance data and the corrected intensity data corrected by the correction parameter 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 the 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 substance is Coumarin 6H and its concentration is 1×10 -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 the fluorescence of 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 locations along the Z direction. The intensity data S1 and S2 indicate the average luminance of the central region of the image based on the scanning of each XY plane at the Z position (z slice). Note that the horizontal axis Side shift of the intensity data S1 and S2 corresponds to a structural shift caused by individual differences in the microscope units. and this deviation causes a deviation in the wavelength axis direction in the intensity data Also, the vertical axis shift of the intensity data S1 and S2 corresponds to a performance shift caused by individual differences in the detector 109. Although the intensity data S1 and S2 are shifted in the wavelength axis direction and intensity, the waveform pattern (emission intensity distribution) in the wavelength axis direction is the same.

[0085] 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 luminance for each wavelength band based on the intensity data S1 shown in FIG. 9, and the intensity data S12 (Microscope 2) indicates the average luminance for each wavelength band based on the intensity data S2 shown in FIG. 9. The intensity data S11 and S12 indicate the average luminance of each wavelength band in the image data within a predetermined Z range including the Z position having the maximum average luminance. Each wavelength band corresponds to each channel of the detector 109. That is, the intensity of each wavelength band is the intensity of the signal output by the channel. Also, for the predetermined Z range, for example, the distance in the Z direction (several μm) or the number of image sheets (number of slices) in the Z direction is set.

[0086] Regarding this 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.

[0087] The corrected intensity data SC1 (Corrected Microscope 2) shown in FIG. 10 indicates waveform data obtained by correcting the intensity data S12 with the parameter p (here p = 1.33). As shown in FIG. 10, the corrected intensity data SC1 has a waveform substantially equivalent to the intensity data S11 of the reference microscope unit 2A, and its average percentage error was 6%.

[0088] FIG. 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 shows that the fluorescent substance is Fluorescein and its concentration is 1×10 -5It is based on the autofluorescence of the sample expressed as %. In Fig. 11, the intensity data S3 (Microscope 1) is the intensity data acquired by the reference microscope unit 2A, and the intensity data S4 (Microscope 2) is the 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.

[0089] Fig. 12 is a diagram (part 2) showing an example of spectra before and after correction processing using the correction parameter according to an embodiment of the present invention. The intensity data S13 (Microscope 1) shown in Fig. 12 indicates the average luminance for each wavelength band based on the intensity data S3 shown in Fig. 11, and the intensity data S14 (Microscope 2) indicates the average luminance for each wavelength band based on the intensity data S4 shown in Fig. 11. The intensity data S13 and S14 indicate the average luminance of each wavelength band in the image data within a predetermined Z range including the Z position having the maximum average luminance.

[0090] For this 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.

[0091] The corrected intensity data SC2 (Corrected Microscope 2) shown in Fig. 12 indicates waveform data obtained by correcting the intensity data S14 with the parameter p (here p = 1.76). As shown in Fig. 12, the corrected intensity data SC2 has a waveform substantially equivalent to the intensity data S13 of the reference microscope unit 2A, and its average percentage error was 7.1%.

[0092] In one embodiment of the present invention described above, correction parameters of the microscope units 2B to 2D are set by using the intensity data obtained from the 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 the deviation of the detection result due to individual differences is suppressed. According to the present embodiment, by performing various analyses using the corrected intensity data corrected by the correction parameters, it is possible to obtain an analysis result in which the influence of the deviation due to individual differences between the microscope units is suppressed.

[0093] (Modification Example 1) Next, Modification Example 1 of the embodiment will be described. In the embodiment, an example of setting correction parameters for each excitation light has been described, but correction parameters specific to the specimen that do not depend on the excitation light may be set.

[0094] In Modification Example 1, the calculation unit 32 calculates a parameter p for which the hyperspectrum C corresponding to the relative error is minimized based on the following formula (4). hyper becomes the smallest.

Equation

[0095] The setting unit 33 sets the parameter p calculated by the calculation unit 32 as the correction parameter of the microscope unit (any one of the microscope units 2B to 2D) acquired by the fluorescence data acquisition unit 31.

[0096] According to Modification Example 1 described above, similar to the above-described embodiment, by performing various analyses using the corrected intensity data corrected by the correction parameters, it is possible to obtain an analysis result in which the influence of the deviation due to individual differences between the microscope units is suppressed. Furthermore, according to the present Modification Example 1, correction parameters specific to the specimen that do not depend on the excitation light can be set, and as a result, the complication of the correction parameters due to the excitation light can be suppressed.

[0097] (Modification Example 2) Next, Modification Example 2 of the embodiment will be described. In the embodiment, an example of setting correction parameters for each excitation light has been described, but correction parameters may be set for each channel of the detector 109.

[0098] In Modification Example 2, the calculation unit 32 calculates the parameter p i for which the relative error C i becomes the smallest. [Equation] Here, the above equation (5) is an equation shown for the case where 1 ≤ i ≤ n. For i, n, α i , β i , it is the same as the above equation (1).

[0099] The setting unit 33 sets the parameter p i calculated by the calculation unit 32 as the correction parameter for each channel of the microscope unit (any one 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.

[0100] According to Modification Example 2 described above, similar to the above-described embodiment, by performing various analyses using the corrected intensity data corrected by the correction parameter, it is possible to obtain an analysis result in which the influence of the deviation due to individual differences between microscope units is suppressed. Furthermore, according to this Modification Example 2, correction parameters for each channel can be set, and as a result, individual differences between channels of the microscope can be suppressed.

[0101] (Modification Example 3) Next, a third modification of the embodiment will be described. There may be base noise in the electrical signal (detection value) output from the detector 109. Since the base noise is a factor in the deviation of the intensity data between microscopes, it is preferable to calculate the correction parameter using the intensity data excluding this base noise. Here, the base noise is defined by either of the following Definitions 1 and 2. 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, in a microscope where there is no base noise, the channels with luminance values of, for example, 20 or less when observing the reference fluorescent substance correspond to this. Definition 2. The average luminance value detected when the voltage of the detector is zero.

[0102] The calculation unit 32 calculates the correction parameter according to the microscope in which the base noise exists. Specifically, the correction parameter is calculated using any of the following formulas (6) to (8).

[0103] [When there is base noise in the microscope units 2B to 2D] The calculation unit 32 calculates the parameter p for which the relative error C is minimized based on the following formula (6). [Equation] Here, γ β is the value of the base noise of the microscope unit (any of the microscope units 2B to 2D).

[0104] [When there is base noise in the reference microscope unit 2A] The calculation unit 32 calculates the parameter p for which the relative error C is minimized based on the following formula (7). [Equation] Here, γ α is the value of the base noise of the reference microscope unit (reference microscope unit 2A).

[0105] [When there is base noise in the reference microscope unit 2A and the microscope units 2B to 2D] Based on the following formula (8), the calculation unit 32 calculates the parameter p that minimizes the relative error C. [Equation]

[0106] According to the modified example 3 described above, similar to the above-described embodiment, by performing various analyses using the correction intensity data corrected by the correction parameter, it is possible to obtain an analysis result in which the influence of the deviation due to individual differences between the microscope units is suppressed. Further, according to this modified example 3, the influence of the base noise can be eliminated, and the correction parameter can be set from the value of the reference fluorescent substance itself. As a result, the individual differences between the microscopes can be further suppressed.

[0107] So far, the embodiments for carrying out the present invention have been described, but the present invention should not be limited only by the above-described embodiments.

[0108] In the above-described embodiment, it has been described as scanning a three-dimensional space to generate autofluorescence data, reflected light data, and correspondence data. However, it is also possible to scan a two-dimensional space (any one of the XY plane, XZ plane, and YZ plane shown in FIG. 3) to generate reference fluorescent substance data, autofluorescence data, reflected light data, and correspondence data, or to scan in any one of the X direction, Y direction, and Z direction shown in FIG. 3, or to obtain fluorescence, autofluorescence, and reflected light at a certain point in space to generate reference fluorescent substance data, autofluorescence data, reflected light data, and correspondence data. In particular, since the reference fluorescent substance data for setting the correction parameter only needs to be able to acquire spectral data, only the reference fluorescent substance data at a certain point is used, and the correction data setting device 3 may acquire the reference fluorescent substance data at this point from each microscope unit.

[0109] Also, in the above-described embodiments, although the description has been made on generating and displaying a three-dimensional image, it may be possible to display a two-dimensional image or superimpose visual information, or to select an image to be displayed by an operation input from the user.

[0110] Also, in the above-described embodiments, although the detector 109 has been described as being configured using a reflection-type diffraction grating and a photomultiplier tube (PMT), alternatively, for example, an acousto-optic beam splitter (e.g., AOBS (registered trademark) of Leica), a high-sensitivity detector (HyD detector), and a detector having a movable slit structure provided in front of the detector may be used. With this detector configuration, it is possible to obtain data separated by, for example, 1 nm wavelengths.

[0111] Also, in the above-described embodiments, reflected light or autofluorescence is obtained using laser light. However, not limited to highly directional light such as laser light, reflected light or autofluorescence may be obtained by condensing low-directional light (e.g., light from a halogen lamp) and irradiating the sample. 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. Also, the wavelength of the light may be that which has passed through a filter or that which has been spectroscopically analyzed by a prism. Further, not limited to reflected light from a specimen, it can be applied to light detection data such as transmitted light or fluorescence that can extract the contour of cells or the like.

[0112] Also, in the above-described embodiments, an example of measuring fluorescence in a state where a sample is flowing through the flow path 501 has been described. However, not limited thereto, fluorescence obtained by exciting a sample statically placed in a container may be measured, or light having a known emission intensity (such as autofluorescence or laser light) may be measured.

[0113] In addition, 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. However, 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 include the configuration of the correction parameter setting device 3, and correction parameters may be set in the microscope units 2B to 2D. Alternatively, the reference microscope unit 2A may include the configuration of the correction parameter setting device 3, and correction parameters may be set in the reference microscope unit 2A.

[0114] As described above, the present invention can include various embodiments without departing from the technical idea described in the claims.

[0115] As described above, the correction parameter setting method and the data correction method according to the present invention are useful for suppressing the deviation of detection results due to individual differences.

Explanation of Reference Numerals

[0116] 1 Data correction system 2A Reference microscope unit 2B to 2D Microscope units 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 unit 203 Laser control unit 204 Scanning control unit 205 Transmitted light control unit 300 Image processing device 301 Detection signal reception unit 302 Data generation unit 302a Autofluorescence data generation unit 302b Reflected light data generation unit 302c Corresponding data generation unit 302d Reference fluorescent substance data generation unit 302e Data correction unit 400 Display device 500 Sample holding device

Claims

1. Using a first microscope as a reference, based on first emission spectrum data obtained by imaging a first sample with a known emission intensity and second emission spectrum data obtained by imaging a second sample, which is known to have the same emission intensity distribution as the first sample in the wavelength axis direction, using a second microscope different from the first microscope, a correction parameter setting method for setting, as a correction parameter, a parameter p that minimizes the relative error C shown in the following formula (1). 【Number 1】 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, β i is the luminance value of the second emission spectrum data in the i-th channel.

2. The first and second emission spectrum data are respectively generated based on light obtained from the first and second samples arranged at one coordinate on a predetermined focal plane. The correction parameter setting method according to claim 1.

3. The first and second emission spectrum data are respectively acquired at a plurality of different coordinates on the predetermined focal plane. The correction parameter setting method according to claim 2.

4. It is carried out at a plurality of different focal planes. The correction parameter setting method according to any one of claims 1 to 3.

5. Said α i , β i are calculated based on the following formulas (2) and (3): The correction parameter setting method according to claim 1. 【Number 2】 Here, m 1 , m 2 is the total number of measurement points, which is the total number in an arbitrary range in the optical axis direction of the first or second microscope, and x j with a bar on top represents the average luminance value of a preset measurement range at the j-th position in the optical axis direction of the first microscope, and y k with a bar on top represents the average luminance value of a preset measurement range at the k-th position in the optical axis direction of the second microscope.

6. The relative error C is calculated based on a value obtained by subtracting base noise from the α i and / or β i ​ The correction parameter setting method according to any one of claims 1 to 5.

7. 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.

8. The first and second emission spectrum data are fluorescence data including spectral profile data composed of emission spectrum data of each fluorescence obtained by irradiating a plurality of excitation lights with different wavelengths and obtained by the plurality of excitation lights. The correction parameter setting method according to claim 7.

9. The parameter p is a hyperparameter C that is the sum of each relative error C obtained by the plurality of excitation lights, as shown in the following formula (4). hyper calculated based on The correction parameter setting method according to claim 8. [Number 3] Here, o is the total number of excitation lights used (0 < q ≤ o).

10. Based on the first emission spectrum data obtained by imaging a first sample with a known emission intensity using a reference first microscope, and second emission spectrum data obtained by imaging a second sample, which is 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, a relative error C shown in the following formula (5) i The parameter p at which i is minimized is set as the correction parameter. A correction parameter setting method. [Number 4] Here, 1 ≤ i ≤ n, where n is the total number of channels, and 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.

11. 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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