Signal processing method, signal processing device, and signal processing system

Abstract

A data processing device 12: acquires intensity signals of channels for an experiment target that is provided with a staining phosphor U; acquires intensity signals of the channels for the experiment target that is provided with a staining phosphor V; calculates a variation amount between the intensity signals; calculates, on the basis of the variation amount, an independent variation parameter representing an independent variation degree of mutual non-interference between the channels, and a correlation variation parameter representing a correlation variation degree of mutual interference between the channels; calculates, on the basis of the independent variation parameter, of the correlation variation parameter, and of the intensity signals of two channels for measurement targets, an evaluation value by evaluating the variation in the number of photons of signal light rays entering the channels, or the variation in the number of photoelectrons discharged from photoelectric conversion units of the channels; and executes data analysis on the basis of the evaluation value.

Description

Signal processing method, signal processing device, and signal processing system

[0001] One aspect of the embodiment relates to a signal processing method, a signal processing device, and a signal processing system.

[0002] Flow cytometry has been known as a technique for counting, selecting, and analyzing the characteristics of samples such as cells using laser light. For example, Patent Document 1 listed below discloses a flow cytometry system that includes a first sensor that is arranged axially with respect to a light source and senses forward scattered components, and a second sensor that is arranged at a certain angle with respect to the first sensor and senses side scattered components and / or fluorescent components.

[0003] Special Publication No. 2013-504051

[0004] In the conventional device described above, when multiple sensors for detecting multiple fluorescent components are used, the output signals of the multiple sensors may affect each other in terms of variations in their output signals. In this case, it is difficult to know in advance the influence of the output signals of the sensors on each other when evaluating the variations in their output signals. As a result, the accuracy of the analysis results based on the evaluation of the variations may be reduced.

[0005] Therefore, one aspect of the embodiments has been made in consideration of such problems, and aims to provide a signal processing method, a signal processing device, and a signal processing system that are capable of accurately evaluating the variation in output signals targeting multiple fluorescent components.

[0006] A signal processing method according to a first aspect of an embodiment is a signal processing method for processing outputs from a first photomultiplier tube and a second photomultiplier tube that detect fluorescence from two phosphors, respectively, of a test object provided with two phosphors, the signal processing method comprising the steps of: acquiring a first intensity signal output from the first photomultiplier tube and a second intensity signal output from the second photomultiplier tube, for a test object provided with one of the two phosphors; acquiring a third intensity signal output from the first photomultiplier tube and a fourth intensity signal output from the second photomultiplier tube, for a test object provided with the other of the two phosphors; calculating a first variation amount, a second variation amount, a third variation amount, and a fourth variation amount as variations of the first intensity signal, the second intensity signal, the third intensity signal, and the fourth intensity signal; Based on the amount of light emitted, an independent variation parameter representing the degree of independent variation in which the output of the first photomultiplier tube and the output of the second photomultiplier tube do not affect each other in measuring the fluorescence from each of the two phosphors, and a correlated variation parameter representing the degree of correlated variation in which the output of the first photomultiplier tube and the output of the second photomultiplier tube affect each other in measuring the fluorescence from each of the two phosphors, are calculated, and an evaluation value evaluating the variation in the number of photons of the signal light incident on each of the first photomultiplier tube and the second photomultiplier tube, or the variation in the number of photoelectrons emitted from the photoelectric conversion units of each of the first photomultiplier tube and the second photomultiplier tube, is calculated based on the independent variation parameter, the correlated variation parameter, and the intensity signal output by the first photomultiplier tube and the intensity signal output by the second photomultiplier tube, with the object being measured, is calculated, and data analysis is performed based on the evaluation value.

[0007] Alternatively, a signal processing device according to a second aspect of the embodiment is a signal processing device including a processor, and processes outputs from a first photomultiplier tube and a second photomultiplier tube that detect fluorescence from two phosphors provided for a measurement object, respectively, the first photomultiplier tube and the second photomultiplier tube being configured to detect fluorescence from the two phosphors provided for a measurement object, the processor acquiring a first intensity signal output from the first photomultiplier tube and a second intensity signal output from the second photomultiplier tube being configured to detect fluorescence from the two phosphors provided for a measurement object, the processor acquiring a third intensity signal output from the first photomultiplier tube and a fourth intensity signal output from the second photomultiplier tube being configured to detect fluorescence from the other of the two phosphors, and calculating a first variation amount, a second variation amount, a third variation amount, and a fourth variation amount as variations of the first intensity signal, the second intensity signal, the third intensity signal, and the fourth intensity signal, respectively, and calculating the first variation amount, the second variation amount, the third variation amount, and the fourth variation amount. and a fourth amount of variation, calculates an independent variation parameter that indicates the degree of independent variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube do not affect each other when measuring the fluorescence from each of the two phosphors, and a correlated variation parameter that indicates the degree of correlated variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube affect each other when measuring the fluorescence from each of the two phosphors; calculates an evaluation value that evaluates the variation in the number of photons of the signal light incident on each of the first photomultiplier tube and the second photomultiplier tube, or the variation in the number of photoelectrons emitted from the photoelectric conversion units of each of the first photomultiplier tube and the second photomultiplier tube, based on the independent variation parameter, the correlated variation parameter, and the intensity signal output by the first photomultiplier tube and the intensity signal output by the second photomultiplier tube, with the object to be measured, and performs data analysis based on the evaluation value.

[0008] Alternatively, a signal processing system according to a third aspect of the embodiment includes the above-described signal processing device, a first photomultiplier tube, a second photomultiplier tube, and an optical system that guides signal light to the first photomultiplier tube and the second photomultiplier tube.

[0009] According to the first, second, or third aspect, outputs of two photomultiplier tubes detecting fluorescence from two phosphors are obtained for a test object provided with one phosphor, and outputs of the two photomultiplier tubes are obtained for a test object provided with the other phosphor. Based on the respective variations in these outputs, an independent variation parameter representing the degree of variation in the mutual influence of the outputs of the two photomultiplier tubes and a correlated variation parameter representing the mutual influence of the outputs of the two photomultiplier tubes are calculated in advance. Then, based on the outputs of the two photomultiplier tubes for a measurement object provided with two phosphors, the independent variation parameter, and the correlated variation parameter, the variation in the number of photons incident on the two photomultiplier tubes or the variation in the number of photoelectrons emitted from the photoelectric conversion units of the two photomultiplier tubes is evaluated. This allows for accurate evaluation of the variation in the output signals of the two photomultiplier tubes. As a result, data analysis targeting the fluorescence from the two phosphors can be performed accurately.

[0010] According to any one of the aspects of the present invention, it is possible to accurately evaluate the variation in output signals for a plurality of fluorescent components.

[0011] Fig. 4 is a schematic configuration diagram of a flow cytometer system 1 which is a flow cytometer according to an embodiment. Fig. 5 is a block diagram showing an example of the hardware configuration of the data processing device 12 of Fig. 1. Fig. 6 is a block diagram showing the functional configuration of the data processing device 12. Fig. 7 is a graph showing an example of a dot plot generated and output in data analysis by the analysis unit 203 of Fig. 3. Fig. 8 is a graph showing an example of a dot plot generated and output in data analysis by the analysis unit 203 of Fig. 3. Fig. 9 is a flowchart showing the procedure of a signal processing method according to an embodiment. Fig. 10 is a flowchart showing the procedure of a signal processing method according to an embodiment.

[0012] In the first and second aspects, it is preferable that the evaluation value is a variation in the number of photons. In this case, data analysis can be performed with high accuracy on the number of incident photons based on the fluorescence from the two phosphors.

[0013] In the first and second aspects, it is preferable that the data analysis includes a gating process for defining a boundary of a population to be analyzed, whereby the gating process can be performed based on quantitative data on the variation of signal light, thereby improving the accuracy of identifying the population to be analyzed.

[0014] In the first aspect, it is also preferable that, before calculating the first and third variation amounts, the first intensity signal and the third intensity signal are subjected to a correction process in which a signal component leaking from the channel of the second photomultiplier tube into the channel of the first photomultiplier tube is subtracted, and, before calculating the second and fourth variation amounts, the second intensity signal and the fourth intensity signal are subjected to a correction process in which a signal component leaking from the channel of the first photomultiplier tube into the channel of the second photomultiplier tube is subtracted. In the second aspect, it is also preferable that the processor, before calculating the first and third variations, performs a correction process on the first and third intensity signals to subtract a signal component that has leaked into the channel of the first photomultiplier tube from the channel of the second photomultiplier tube, and, before calculating the second and fourth variations, performs a correction process on the second and fourth intensity signals to subtract a signal component that has leaked into the channel of the second photomultiplier tube from the channel of the first photomultiplier tube. In this case, the signal component that has leaked into the channel of one photomultiplier tube is subtracted from the intensity signal output by the other photomultiplier tube, enabling data analysis that excludes the influence of leak-in between channels and further improving the accuracy of identification of the population to be analyzed.

[0015] The signal processing method of the embodiment is [1] "A signal processing method for processing outputs of a first photomultiplier tube and a second photomultiplier tube that detect fluorescence from two phosphors, the first photomultiplier tube and the second photomultiplier tube being used for detecting fluorescence from two phosphors, the method comprising the steps of: obtaining a first intensity signal output from the first photomultiplier tube and a second intensity signal output from the second photomultiplier tube for a test object to which one of the two phosphors has been applied; a third intensity signal output from the first photomultiplier tube and a fourth intensity signal output from the second photomultiplier tube are acquired for a test object having a first intensity signal, a second intensity signal, a third intensity signal, and a fourth intensity signal, respectively, are calculated as the amounts of variation of the first intensity signal, the second intensity signal, the third intensity signal, and the fourth intensity signal; and a comparison of the two intensity signals is performed based on the first variation amount, the second variation amount, the third variation amount, and the fourth variation amount. a signal processing method for calculating an independent variation parameter that represents the degree of independent variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube do not affect each other in measuring the fluorescence from each of the two phosphors, and a correlated variation parameter that represents the degree of correlated variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube affect each other in measuring the fluorescence from each of the two phosphors, and calculating an evaluation value that evaluates the variation in the number of photons of signal light incident on each of the first photomultiplier tube and the second photomultiplier tube, or the variation in the number of photoelectrons emitted from each of the photoelectric conversion units of the first photomultiplier tube and the second photomultiplier tube, based on the independent variation parameter, the correlated variation parameter, and the intensity signal output by the first photomultiplier tube and the intensity signal output by the second photomultiplier tube, and performing data analysis based on the evaluation value.

[0016] The signal processing method of the embodiment may be [2] "the signal processing method according to the above [1], in which the evaluation value is the variation in the number of photons."

[0017] The signal processing method of the embodiment may be [3] "the signal processing method according to the above [1] or [2], wherein the data analysis includes a gating process for defining the boundaries of the population to be analyzed."

[0018] The signal processing method of the embodiment may be [4] "the signal processing method according to any one of the above [1] to [3], wherein before calculating the first variation amount and the third variation amount, a correction process is performed on the first intensity signal and the third intensity signal to subtract a signal component leaking from the channel of the second photomultiplier tube into the channel of the first photomultiplier tube, and before calculating the second variation amount and the fourth variation amount, a correction process is performed on the second intensity signal and the fourth intensity signal to subtract a signal component leaking from the channel of the first photomultiplier tube into the channel of the second photomultiplier tube."

[0019] The signal processing device of the embodiment is [5] "A signal processing device including a processor that processes outputs of a first photomultiplier tube and a second photomultiplier tube that detect fluorescence from two phosphors provided in a measurement object, the first photomultiplier tube and the second photomultiplier tube each detecting fluorescence from the two phosphors, the processor acquiring a first intensity signal output from the first photomultiplier tube and a second intensity signal output from the second photomultiplier tube in a test object provided with one of the two phosphors, and detecting the fluorescence from one of the two phosphors. a third intensity signal output from the first photomultiplier tube and a fourth intensity signal output from the second photomultiplier tube are acquired for a test object to which the other phosphor is applied, and a first variation amount, a second variation amount, a third variation amount, and a fourth variation amount are calculated as the variations of the first intensity signal, the second intensity signal, the third intensity signal, and the fourth intensity signal, respectively; and a first variation amount, a second variation amount, a third variation amount, and a fourth variation amount are calculated based on the first variation amount, the second variation amount, the third variation amount, and the fourth variation amount. the signal processing device is configured to calculate an independent variation parameter that represents the degree of independent variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube do not affect each other in measuring the fluorescence from each of the two phosphors, and a correlated variation parameter that represents the degree of correlated variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube affect each other in measuring the fluorescence from each of the two phosphors, calculate an evaluation value that evaluates the variation in the number of photons of signal light incident on each of the first photomultiplier tube and the second photomultiplier tube, or the variation in the number of photoelectrons emitted from each photoelectric conversion unit of the first photomultiplier tube and the second photomultiplier tube, based on the independent variation parameter, the correlated variation parameter, and the intensity signal output by the first photomultiplier tube and the intensity signal output by the second photomultiplier tube, and perform data analysis based on the evaluation value.

[0020] The signal processing device of the embodiment may be [6] "the signal processing device according to the above [5], in which the evaluation value is the variation in the number of photons."

[0021] The signal processing device of the embodiment may be [7] "the signal processing method according to the above [5] or [6], wherein the data analysis includes a gating process for defining the boundaries of the population to be analyzed."

[0022] The signal processing device of the embodiment may be [8] "the signal processing device according to any one of the above [5] to [7], wherein the processor performs a correction process on the first intensity signal and the third intensity signal to subtract a signal component leaking from the channel of the second photomultiplier tube into the channel of the first photomultiplier tube before calculating the first variation amount and the third variation amount, and performs a correction process on the second intensity signal and the fourth intensity signal to subtract a signal component leaking from the channel of the first photomultiplier tube into the channel of the second photomultiplier tube before calculating the second variation amount and the fourth variation amount."

[0023] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description, the same elements or elements having the same functions will be denoted by the same reference numerals, and redundant description will be omitted.

[0024] 1 is a schematic diagram of a flow cytometer (signal processing system) according to an embodiment, which is a flow cytometer system 1. The flow cytometer system 1 is a system for performing flow cytometry, and is composed of a fluid system 2, an optical system (optical system) 3, and an electronic system (signal processing device) 4.

[0025] The fluid system 2 includes a flow cell 6 into which a sample fluid containing analytes such as cells or particles is injected, and which allows the analytes (measurement objects) contained in the sample fluid to be aligned and passed through a narrow channel 5. The analytes are given two predetermined dye fluorophores U and V. The flow cell 6 also has a function (not shown) for sorting (classifying and allocating) the gated analytes by electric field control or the like.

[0026] The optical system 3 optically analyzes an analyte passing through a flow cell 6 by flow cytometry. The optical system 3 includes a laser light source 7, a lens 8, filters 9a, 9b, 9c, and 9d, dichroic mirrors 10b and 10c, and photomultiplier tubes 11a, 11b, 11c, and 11d. The optical system 3 guides various types of light generated from the analyte by flow cytometry to the photomultiplier tubes 11a, 11b, 11c, and 11d. The laser light source 7 is a light source device that generates laser light (excitation light) in a single wavelength band at a specific frequency. The lens 8 focuses the laser light emitted from the laser light source 7 onto a channel 5 in the flow cell 6. The filter 9a transmits forward-scattered light generated from the sample fluid by irradiation with the laser light. The dichroic mirror 10b reflects side-scattered light generated from the sample fluid by irradiation with the laser light and transmits fluorescence generated from the sample fluid. The dichroic mirror 10c reflects fluorescence in a first wavelength band corresponding to the fluorescence wavelength of one of the two dye phosphors U and V, namely, dye phosphor U, out of the fluorescence transmitted through the dichroic mirror 10b, and transmits fluorescence in the remaining wavelength bands out of the transmitted fluorescence. The filter 9b transmits the side scattered light reflected by the dichroic mirror 10b, and the filter 9c transmits the first fluorescence in the first wavelength band reflected by the dichroic mirror 10c. The filter 9d transmits the second fluorescence in a second wavelength band corresponding to the fluorescence wavelength of the other of the two dye phosphors U and V, namely, dye phosphor V, out of the fluorescence transmitted through the dichroic mirror 10c. The photomultiplier tubes 11a, 11b, 11c, and 11d are disposed on the optical axes of the forward scattered light, side scattered light, first fluorescence, and second fluorescence, respectively, and measure the intensities of the forward scattered light, side scattered light, first fluorescence, and second fluorescence. That is, the photomultiplier tube 11c is a first photomultiplier tube that detects fluorescence from one phosphor U, and the photomultiplier tube 11d is a second photomultiplier tube that detects fluorescence from the other phosphor V.

[0027] The electronic system 4 includes a data processing device 12 and is a device for analyzing the light intensity measured by the optical system 3. Specifically, the data processing device 12 is electrically connected to the multiple photomultiplier tubes 11a, 11b, 11c, and 11d, and performs data analysis to create histograms or dot plots (also called cytograms) based on intensity signals indicating the intensities detected in each channel of the multiple photomultiplier tubes 11a, 11b, 11c, and 11d, as well as gating processing. Furthermore, the data processing device 12 sorts (classifies and distributes) analytes contained in the sample fluid based on the gating processing.

[0028] Next, the configuration of the data processing device 12 will be described with reference to Figures 2 and 3. Figure 2 is a block diagram showing an example of the hardware configuration of the data processing device 12, and Figure 3 is a block diagram showing the functional configuration of the data processing device 12.

[0029] 2, the data processing device 12 is physically a computer or the like including a processor such as a CPU (Central Processing Unit) 101, a recording medium such as a RAM (Random Access Memory) 102 or a ROM (Read Only Memory) 103, a communication module 104, and an input / output module 106, all of which are electrically connected to one another. The data processing device 12 may include input / output devices such as a display, a keyboard, a mouse, a touch panel display, or a data recording device such as a hard disk drive or semiconductor memory. The data processing device 12 may also be composed of multiple computers.

[0030] As shown in FIG. 3 , the data processing device 12 includes, as functional components, a signal acquisition unit 201, a calculation unit 202, and an analysis unit 203. Each functional unit of the data processing device 12 shown in FIG. 3 is realized by loading a program onto hardware such as the CPU 101 and RAM 102, which operates the communication module 104 and the input / output module 106 under the control of the CPU 101, and reads and writes data from and to the RAM 102. The CPU 101 of the data processing device 12 executes the program to cause each functional unit of FIG. 3 to function and sequentially executes processing corresponding to the signal processing method described below. The CPU 101 may be a standalone piece of hardware or may be implemented in a programmable logic device such as an FPGA, like a software processor. The RAM and ROM may also be standalone pieces of hardware or may be built into a programmable logic device such as an FPGA. Various data required for executing the program and various data generated by the program are all stored in built-in memory such as the ROM 103 and RAM 102, or in a recording medium such as a hard disk drive. The functionality of the functional components of data processing device 12 will now be described in detail.

[0031] The signal acquisition unit 201 acquires intensity signals (output current signals) output from each channel of the multiple photomultiplier tubes 11a, 11b, 11c, and 11d. The acquired intensity signals are analog signals obtained by detecting currents due to multiplied electrons corresponding to the intensity of signal light, such as forward scattered light, side scattered light, or fluorescence, generated by flow cytometry in each photomultiplier tube. The signal acquisition unit 201 converts the acquired intensity signals of each channel into digital values ​​DN and outputs them to the calculation unit 202. Note that the A / D conversion function of the signal acquisition unit 201 may be realized by an external circuit unit of the data processing device 12.

[0032] The calculation unit 202 performs calculations to convert the digital value DN of each channel output from the signal acquisition unit 201 into the virtual photon number (number of photons), which is an index (analysis evaluation value) corresponding to the number of actual photons incident on each channel, and the variance of the intensity signal.

[0033] First, the calculation unit 202 performs a signal correction process called compensation on the intensity signal X detected in the channel (X axis) of the photomultiplier tube 11c and the intensity signal Y detected in the channel (Y axis) of the photomultiplier tube 11d. Compensation is a process of subtracting the signal component leaking from the other channel from the signal intensity in one channel, thereby correcting the signal intensity so that only the signal light from the target phosphor is reflected.

[0034] Specifically, the calculation unit 202 performs compensation as follows: The calculation unit 202 calculates the intensity signal X acquired in one channel using the following formula: is used to convert the correction value Comp[X]. Here, the coefficient R Ｖ→Ｘ ' is the following formula: In the above formula, G Ｘ is the gain [e / e] of the photomultiplier tube that outputs the intensity signal on the X axis, and G Ｙ is the gain [e / e] of the photomultiplier tube that outputs the intensity signal on the Y axis, and Q Ｘ is the photon-to-electron conversion efficiency [e / photon] of the photomultiplier tube that outputs the intensity signal on the X axis, and Q Ｙ is the photon-to-electron conversion efficiency [e / photon] of the photomultiplier tube that outputs an intensity signal on the Y axis, and these coefficients are set as known values. Ｖ→Ｘ is obtained in advance by a parameter determination function to be described later.

[0035] Similarly, the calculation unit 202 calculates the intensity signal Y acquired in the other channel using the following formula: is used to convert the correction value Comp[Y]. Here, the coefficient R Ｕ→Ｙ ' is the following formula: This coefficient R Ｕ→Ｙ are also acquired in advance by a parameter determination function to be described later.

[0036] Before describing the calculation function of the calculation unit 202, a theoretical model of the correction values ​​Comp[X] and Comp[Y] and their noise (variation) studied by the inventors of the present application will be described.

[0037] According to the theoretical model considered, the average value A of the correction value Comp[X] Ｃｏｍｐ［Ｘ］ [DN: Digital Number] and noise σ of the correction value Comp[X] Ｃｏｍｐ［Ｘ］ The standard deviation [DN rms] is expressed by the following formula (1) and formula (2). Ｃｏｍｐ［Ｙ］ [DN: Digital Number] and noise σ of the correction value Comp[Y] Ｃｏｍｐ［Ｙ］ The standard deviation [DN rms] is expressed by the following formulas (3) and (4).

[0038] Among the parameters in the above formulas (1) to (4), those mainly related to expected values ​​are represented by capital letters, and those mainly related to noise are represented by lowercase Greek letters. ０ is the offset [DN] including the dark current component and background light component on the X axis, and Y ０ is the offset [DN] including the dark current component and background light component on the Y axis, and <S Ｕ > indicates the expected value of the index (expected virtual photon number of the signal light) [photon] corresponding to the wavelength spectrum of the signal light emitted from the dye phosphor U and incident on the photomultiplier tube that outputs an intensity signal on the X axis, and <S Ｖ > denotes the expected value of the index corresponding to the wavelength spectrum of the signal light emitted from the dye phosphor V and incident on the photomultiplier tube that outputs an intensity signal on the Y axis (expected virtual photon number of the signal light) [photon], and R Ｖ→Ｘ represents the ratio [photon / photon] of signal light emitted from the dye phosphor V and leaking into the photomultiplier tube that outputs an intensity signal on the X axis, and R Ｕ→Ｙindicates the ratio [photon / photon] of signal light emitted from the dye phosphor U and leaking into the photomultiplier tube that outputs an intensity signal on the Y axis.

[0039] In addition, in the above formulas (1) to (4), σ ｃＸ indicates the readout noise (including noise generated by the circuit, shot noise components of dark current and background light) [DN rms] in the intensity signal on the X axis, and σ ｃＹ indicates the readout noise (including noise generated by the circuit, shot noise components of dark current and background light) [DN rms] in the intensity signal on the Y axis, C indicates the conversion coefficient [DN / e] of the digital value DN to the number of electrons output by the photomultiplier tube in the A / D conversion circuit, and F Ｘ is the excess noise factor of the photomultiplier tube that outputs the intensity signal on the X axis, and F Ｙ is the excess noise coefficient of the photomultiplier tube that outputs the intensity signal on the Y axis, and ρ Ｕｉ indicates the degree of variation in the output of the photomultiplier tube that outputs an intensity signal on the X axis and the output of the photomultiplier tube that outputs an intensity signal on the Y axis, which do not affect each other (independent), in a measurement involving the dye phosphor U, and ρ Ｕｒ indicates the degree of variation in the influence (correlation) between the output of the photomultiplier tube that outputs an intensity signal on the X axis and the output of the photomultiplier tube that outputs an intensity signal on the Y axis in a measurement involving a dye phosphor U, and ρ Ｖｉ indicates the degree of variation in the output of a photomultiplier tube that outputs an intensity signal on the X axis and the output of a photomultiplier tube that outputs an intensity signal on the Y axis, which do not affect each other (independent), in a measurement involving a dye phosphor V, and ρ Ｖｒ indicates the degree of variation in the influence (correlation) between the output of a photomultiplier tube that outputs an intensity signal on the X axis and the output of a photomultiplier tube that outputs an intensity signal on the Y axis in a measurement involving dye phosphor V. Furthermore, the notation "^" in the above formulas (2) and (4) indicates that this is correlation noise resulting from the correlation between channels.

[0040] In the above formula (2), the first term on the right side: means the variance value of the device noise (or circuit noise) on the X-axis side in the optical system 3, and the second term on the right-hand side; means the variance value of the influence of the device noise (or circuit noise) on the Y-axis side on the X-axis side in the optical system 3, and the third term on the right-hand side; means the shot noise element of the dye phosphor U, the first term in the curly brackets of that term corresponds to the shot noise variance value on the X-axis side, and the second term in the curly brackets of that term corresponds to the variance value of the influence of the shot noise on the Y-axis side on the X-axis side. Also, in the same equation, the fourth term on the right side; means the shot noise element of the dye fluorescent substance V, the first term in the curly brackets of that term corresponds to the shot noise variance value on the X-axis side, and the second term in the curly brackets of that term corresponds to the variance value of the influence of the shot noise on the Y-axis side on the X-axis side. Also, in the same equation, the fifth term on the right side; means the independence ρ element of the dye phosphor U (a component of the amount of variation proportional to the number of photons or photoelectrons that has no correlation across axes such as X and Y), and the first term in the curly brackets of that term corresponds to the variance of the independence ρ element on the X axis side, and the second term in the curly brackets of that term corresponds to the variance of the influence of the independence ρ element on the Y axis side on the X axis side. In the same equation, the sixth term on the right side; is the independence ρ element of the dye phosphor V (a component of the amount of variation proportional to the number of photons or photoelectrons that has no correlation across axes such as X and Y), and means the variance value of the influence of the independence ρ element on the Y-axis side on the X-axis side. In the same equation, the seventh term on the right side; means the correlation ρ element of the dyed phosphor U (an element that has correlation across axes such as X and Y among the amount of variation proportional to the number of photons or photoelectrons), and the first term in the curly brackets of that term corresponds to the variance value of the correlation system ρ element on the X-axis side, and the second term in the curly brackets of that term corresponds to the variance value of the influence that the correlation system ρ element on the Y-axis side has on the X-axis side.

[0041] In the above formula (4), the first term on the right side: means the variance value of the device noise (or circuit noise) on the Y-axis side in the optical system 3, and the second term on the right-hand side; means the variance value of the influence of the device noise (or circuit noise) on the X-axis side on the Y-axis side in the optical system 3, and the third term on the right-hand side; means the shot noise element of the dye fluorescent substance V, the first term in the curly brackets of that term corresponds to the shot noise variance value on the Y-axis side, and the second term in the curly brackets of that term corresponds to the variance value of the influence of the shot noise on the X-axis side on the Y-axis side. Also, in the same equation, the fourth term on the right side; means the shot noise element of the dye phosphor U, the first term in the curly brackets of that term corresponds to the shot noise variance on the Y-axis side, and the second term in the curly brackets of that term corresponds to the variance of the effect of the shot noise on the X-axis side on the Y-axis side. means the independence ρ element of the dye phosphor V (a component of the amount of variation proportional to the number of photons or photoelectrons that has no correlation across axes such as X and Y), and the first term in the curly brackets of that term corresponds to the variance of the independence ρ element on the Y axis side, and the second term in the curly brackets of that term corresponds to the variance of the influence of the independence ρ element on the X axis side on the Y axis side. In the same equation, the sixth term on the right side; is the independence ρ element of the dye phosphor U (a component of the amount of variation proportional to the number of photons or photoelectrons that has no correlation across axes such as X and Y), and means the variance value of the influence of the independence ρ element on the X-axis side on the Y-axis side. In the same equation, the seventh term on the right side; means the correlation ρ element of the dyed phosphor V (an element that has correlation across axes such as X and Y among the amount of variation proportional to the number of photons or photoelectrons), and the first term in the curly brackets of that term corresponds to the variance value of the correlation system ρ element on the Y-axis side, and the second term in the curly brackets of that term corresponds to the variance value of the influence that the correlation system ρ element on the X-axis side has on the Y-axis side.

[0042] The calculation unit 202 applies various parameters stored in a memory such as the RAM 102 to the theoretical model expressed by the above formulas (1) to (4), thereby determining whether the expected value of the number of virtual photons (or the number of effective photoelectrons) of each channel is less than S Ｕ >, <S Ｖ > and the dispersion (standard deviation) of the expected value. Alternatively, the target expected value <S Ｕ > and the expected value of the target <S Ｖ> is defined, the calculation unit 202 can obtain in advance the above formulas (1) and (3), which are theoretical formulas for the average signal strength in the space where compensation has been performed, and the above formulas (2) and (4), which are theoretical formulas for the degree of variation.

[0043] The calculation unit 202 also has a parameter determination function that determines in advance the parameters in the above formulas (1) to (4) and the parameters required for compensation, prior to calculation of an analytical evaluation value through measurement of an analyte to which two dye fluorophores U and V have been given. The parameter determination function will be described below.

[0044] Among the parameters included in the above formulas (1) to (4), G Ｘ , G Ｙ , Q Ｘ , Q Ｙ , C are input into the data processing device 12 by the user and are set as known values ​​in advance.

[0045] When the measurement is performed in a state where the analyte is dyed only with the dyeing fluorescent substance U, the average value A of the intensity signal X is Ｘ , and the standard deviation σ of the intensity signal X Ｘ is expressed by the following theoretical formula: Ｕ ' denotes the degree of variability in measurements involving dye fluorophore U before separation into independent and correlated components.

[0046] In addition, when the measurement is performed in a state where the object to be analyzed is stained only with the dyeing fluorescent substance V, the average value A of the intensity signal Y is Ｙ , and the standard deviation σ of the intensity signal Y Ｙ is expressed by the following theoretical formula: Ｖ ' denotes the degree of variability in measurements involving dye fluorophore V before separation into independent and correlated components.

[0047] The calculation unit 202 starts detection by the flow cytometer memory for a test object that is not stained with the dyeing fluorescent materials U and V, acquires intensity signals X and Y from the photomultiplier tubes 11c and 11d for a plurality of events, and calculates the average value of these signals as A. Ｘ | Ｕ＝０，Ｖ＝０ , A Ｙ | Ｕ＝０，Ｖ＝０ Here, the symbol "|" attached to these values ​​indicates the value when the condition shown after the symbol is imposed (the same applies in the following description). Furthermore, "U=0" indicates the condition in which the test object is not dyed with the dyeing fluorescent substance U, and "V=0" indicates the condition in which the test object is not dyed with the dyeing fluorescent substance V (the same applies in the following description). In addition, the calculation unit 202 calculates the variance σ of the intensity signals X and Y for multiple events based on these signals. Ｘ ２ | Ｕ＝０，Ｖ＝０ , σ Ｙ ２ | Ｕ＝０，Ｖ＝０ Get.

[0048] Then, the calculation unit 202 uses the above-mentioned theoretical formula to calculate the parameter X ０ , Y ０ , (σ ｃＸ ) ２ , (σ ｃＹ ) ２ These parameters are stored in a memory such as the RAM 102. ０ = A Ｘ | Ｕ＝０，Ｖ＝０ , (σ ｃＸ ) ２ = σ Ｘ ２ | Ｕ＝０，Ｖ＝０ , Y ０ = A Ｙ | Ｕ＝０，Ｖ＝０ , (σ ｃＹ ) ２ = σ Ｙ ２ | Ｕ＝０，Ｖ＝０

[0049] Furthermore, the calculation unit 202 starts detection by the flow cytometer memory on a test object stained only with one dyeing fluorescent substance U when the intensity of the signal light is sufficiently high, acquires intensity signals X from the photomultiplier tube 11c for a plurality of events, and calculates an average value A of the intensity signals X. Ｘ | Ｕ＞＞Ｈ，Ｖ＝０ and the variance σ of the intensity signal X Ｘ ２ | Ｕ＞＞Ｈ，Ｖ＝０ Furthermore, the calculation unit 202 starts detection by the flow cytometer memory for a test object stained only with one dyeing fluorescent substance V when the intensity of the signal light is sufficiently high, acquires intensity signals Y from the photomultiplier tube 11d for a plurality of events, and calculates an average value A of the intensity signals Y. Ｙ | Ｕ＝０，Ｖ＞＞Ｈ and the variance σ of the intensity signal Y Ｙ ２ | Ｕ＝０，Ｖ＞＞Ｈ Then, using the theoretical formula described above, the parameter ρ is obtained using the following formula: Ｕ ', ρ Ｖ ' and store these parameters in a memory such as the RAM 102.

[0050] Furthermore, the calculation unit 202 executes detection using a flow cytometer memory for a test object stained only with one of the dyeing fluorescent substances U while varying the intensity of the signal light between two levels, high and low, and acquires intensity signals X from the photomultiplier tube 11c for multiple events, and calculates an average value A of the intensity signals X when the intensity of the signal light is high. Ｘ | Ｕ＝Ｈ，Ｖ＝０ and the variance σ of the intensity signal X in that case Ｘ ２ | Ｕ＝Ｈ，Ｖ＝０ and the average value A of the intensity signal X when the intensity of the signal light is low. Ｘ | Ｕ＝Ｌ，Ｖ＝０ and the variance σ of the intensity signal X in that case Ｘ ２ | Ｕ＝Ｌ，Ｖ＝０The calculation unit 202 also executes detection using a flow cytometer memory on a test object stained only with one of the dyeing fluorescent substances V, while varying the intensity of the signal light between two levels, high and low, and acquires intensity signals Y from the photomultiplier tube 11d for multiple events, and calculates an average value A of the intensity signals Y when the intensity of the signal light is high. Ｙ | Ｕ＝０，Ｖ＝Ｈ and the variance σ of the intensity signal X in that case Ｙ ２ | Ｕ＝０，Ｖ＝Ｈ and the average value A of the intensity signal Y when the intensity of the signal light is low. Ｙ | Ｕ＝０，Ｖ＝Ｌ and the variance σ of the intensity signal Y in that case Ｙ ２ | Ｕ＝０，Ｖ＝Ｌ Then, using the above-mentioned theoretical formula, the parameter F is obtained using the following formula: Ｘ , F Ｙ These parameters are stored in a memory such as the RAM 102.

[0051] Furthermore, the calculation unit 202 executes detection using a flow cytometer memory for a test object stained only with one dyeing fluorescent substance V, with the intensity of the signal light set to a normal value, acquires intensity signals X and Y from the photomultiplier tubes 11c and 11d for a plurality of events, and calculates an average value A of the intensity signals X. Ｘ | Ｕ＝０ and the average value A of the intensity signal Y Ｙ | Ｕ＝０ Furthermore, with the intensity of the signal light set to a normal value, detection is performed using a flow cytometer memory on a test object stained only with one of the staining fluorescent substances U, and intensity signals X and Y are acquired from the photomultiplier tubes 11c and 11d for a plurality of events, and the average value A of the intensity signals X is obtained. Ｘ | Ｖ＝０ and the average value A of the intensity signal Y Ｙ | Ｖ＝０ Here, when the measurement is performed in a state where the object to be analyzed is dyed with both dyeing fluorescent materials U and V, the average value A of the intensity signal X is obtained. Ｘ , and the average value A of the intensity signal Y Ｙ is expressed by the following theoretical formula: The calculation unit 202 uses the above-mentioned theoretical formula to calculate the parameter R Ｖ→Ｘ , R Ｕ→Ｙ and stores these parameters in a memory such as the RAM 102. Here, the calculation unit 202 calculates the virtual photon number expectation value<S using the above theoretical formula. Ｕ >, <S Ｖ > and store them in a memory such as RAM 102.

[0052] Furthermore, the calculation unit 202 starts detection using the flow cytometer memory for a test object stained only with one dyeing phosphor U, with the signal light intensity set to the intensity during normal measurement, and acquires intensity signals (first intensity signal) X and intensity signals (second intensity signal) Y for multiple events from the photomultiplier tubes 11c and 11d. Next, compensation is performed on the intensity signal X to obtain the correction value Comp[X]| of the intensity signal X. Ｖ＝０ and based on this, the standard deviation (first amount of variation) σ of the intensity signal X is calculated. Ｃｏｍｐ［Ｘ］ | Ｖ＝０ is obtained by calculation, and the correction value Comp[X]| Ｖ＝０ and standard deviation σ Ｃｏｍｐ［Ｘ］ | Ｖ＝０ is stored in a memory such as the RAM 102. Furthermore, compensation is also performed on the intensity signal Y, and the correction value Comp[Y]| of the intensity signal Y is Ｖ＝０ and based on this, the standard deviation (second amount of variation) σ of the intensity signal Y is calculated. Ｃｏｍｐ［Ｙ］ | Ｖ＝０ is obtained by calculation, and the correction value Comp[Y]| Ｖ＝０ and standard deviation σ Ｃｏｍｐ［Ｙ］ | Ｖ＝０ is stored in a memory such as the RAM 102.

[0053] Furthermore, the calculation unit 202 starts detection using the flow cytometer memory for a test object stained only with the other dyeing phosphor V, with the signal light intensity set to the intensity during normal measurement, and acquires intensity signals (third intensity signal) X and intensity signals (fourth intensity signal) Y for multiple events from the photomultiplier tubes 11c and 11d. Next, compensation is performed on the intensity signal X, and the correction value Comp[X]| of the intensity signal X is calculated.Ｕ＝０ Based on this, the standard deviation (third amount of variation) σ of the intensity signal X is calculated. Ｃｏｍｐ［Ｘ］ | Ｕ＝０ is obtained by calculation, and the correction value Comp[X]| Ｕ＝０ and standard deviation σ Ｃｏｍｐ［Ｘ］ | Ｕ＝０ is stored in a memory such as the RAM 102. Furthermore, compensation is also performed on the intensity signal Y, and the correction value Comp[Y]| of the intensity signal Y is Ｕ＝０ Based on this, the standard deviation (fourth amount of variation) σ of the intensity signal Y is calculated. Ｃｏｍｐ［Ｙ］ | Ｕ＝０ is obtained by calculation, and the correction value Comp[Y]| Ｕ＝０ and standard deviation σ Ｃｏｍｐ［Ｙ］ | Ｕ＝０ is stored in a memory such as the RAM 102.

[0054] Then, the calculation unit 202 calculates the various parameters stored in the memory and the two correction values ​​Comp[X]| Ｖ＝０ , Comp[Y] | Ｕ＝０ and four standard deviations σ Ｃｏｍｐ［Ｘ］ | Ｖ＝０ , σ Ｃｏｍｐ［Ｙ］ | Ｖ＝０ , σ Ｃｏｍｐ［Ｘ］ | Ｕ＝０ , σ Ｃｏｍｐ［Ｙ］ | Ｕ＝０ By solving six simultaneous equations obtained by applying the above equations (1) to (4), the four variability parameters ρ Ｕｉ , ρ Ｕｒ , ρ Ｖｉ , ρ Ｖｒ These parameters are stored in a memory such as the RAM 102. This concludes the description of the parameter determination function performed by the calculation unit 202.

[0055] The analysis unit 203 calculates the number of virtual photons for each channel calculated by the calculation unit 202 as follows: Ｕ >,<S Ｖ > and standard deviation σ for each channel Ｃｏｍｐ［Ｘ］ , σ Ｃｏｍｐ［Ｙ］ Specifically, the number of virtual photons in multiple channels is less than S. Ｕ >,<S ＶThe analysis unit 203 generates histograms and dot plots based on the data and outputs them to an input / output device. The analysis unit 203 also performs gating on the generated dot plots to define boundaries between different groups of analytes. Furthermore, the analysis unit 203 can control the fluid system 2 to execute a sorting process that classifies and separates groups based on the boundaries of the groups of analytes.

[0056] 4 and 5 are graphs showing examples of dot plots generated and output in data analysis by the analysis unit 203. FIG. 4 shows a dot plot plotting the relationship between the virtual photon number of the fluorescence channel corresponding to antibody A and the virtual photon number of the fluorescence channel corresponding to antibody B, with the boundaries defined by the gating process indicated by solid lines. Thus, the gating process calculates and outputs the proportions of populations within the entire analyte to be detected, with the proportion of populations negative for both antibody A and antibody B being "36.6%," the proportion of populations positive for only antibody B being "34.7%, the proportion of populations positive for only antibody A being "27.7%, and the proportion of populations positive for both antibodies A and B being "1.05%." FIG. 5 shows a dot plot plotting the relationship between the virtual photon number of the fluorescence channel using the fluorescent dye Cy5 and the virtual photon number of the fluorescence channel using the fluorescent dye TR. Thus, when defining the boundaries of populations by the gating process, the average value of the virtual photon number of the population <S Ｕ >, and its average value <S Ｕ >The standard deviation σ corresponding to Ｃｏｍｐ［Ｘ］ The boundary of the range W of the gate section determined by the standard deviation σ can be automatically defined. Ｃｏｍｐ［Ｘ］ The range W determined by, for example, the number of virtual photons is <S Ｕ >±3×σ Ｃｏｍｐ［Ｘ］ / CG Ｘ Q Ｘ is set to the range.

[0057] Next, with reference to Figures 6 and 7, we will explain the procedure for processing the output signal of the photomultiplier tube using the flow cytometer system 1. Figure 6 shows the preparatory processing of parameters for each channel by the flow cytometer system 1, and Figure 7 shows the analytical processing by flow cytometry of a sample fluid.

[0058] First, referring to FIG. 6, the user inputs the conversion coefficient C and gain G, which are known parameters for each channel. Ｘ , G Ｙ , and the quantum efficiency Q of the signal light Ｘ , Q Ｙ is input to the data processing device 12 (step S101). Thereafter, in the flow cytometer system 1, detection by the flow cytometer memory is started for a test object that is not stained with the dyeing fluorescent materials U and V, and the data processing device 12 acquires data of the digital value DN of each channel for a plurality of events in a state where no signal light is incident on each channel (step S102). Then, in the data processing device 12, the parameter X of each channel is ０ , Y ０ , (σ ｃＸ ) ２ , (σ ｃＹ ) ２ is acquired and saved (step S103).

[0059] Next, in the flow cytometer system 1, the laser light is changed to a predetermined intensity, beads or dyed fluorescent materials that emit an appropriate amount of light are used, or a calibration light source is installed on the flow cytometer side, and the data processing device 12 repeatedly detects the signal light from the sample fluid dyed with either one of the dyed fluorescent materials U or V under a condition where the intensity of the signal light is sufficiently high, thereby acquiring data of the digital value DN of each channel for multiple events (step S104). Ｕ ', ρ Ｖ ' is acquired and saved (step S105).

[0060] Furthermore, in the flow cytometer system 1, the intensity of the signal light is changed to two levels, high and low, by changing the intensity of the laser light, using beads or fluorescent dyes that emit an appropriate amount of light, using a light source for calibration of the changed light intensity, or using an ND filter, and in this state, the data processing device 12 repeatedly detects the signal light from the sample fluid dyed with either one of the dyeing fluorescent materials U and V, thereby acquiring data of the digital value DN of each channel for multiple events (step S106). Ｘ , F Ｙ In addition, in the data processing device 12, with the intensity of the signal light set to a normal value, data of the digital value DN of each channel for a plurality of events is acquired for the sample fluid dyed only with the dyeing phosphor U, and data of the digital value DN of each channel for a plurality of events is acquired for the sample fluid dyed only with the dyeing phosphor V, and based on these data, the parameter R Ｖ→Ｘ , R Ｕ→Ｙ may be calculated and stored in a memory such as RAM 102.

[0061] Thereafter, with the intensity of the signal light changed to normal in the flow cytometer system 1, the data processing device 12 detects the signal light from the sample fluid dyed only with the dyeing fluorophore U, thereby acquiring data for the digital values ​​DN of each channel for multiple events (step S108). Then, the data processing device 12 performs compensation on the digital values ​​DN of each channel, and the parameter Comp[X]| Ｖ＝０ , σ Ｃｏｍｐ［Ｘ］ | Ｖ＝０ , Comp[Y] | Ｖ＝０ , σ Ｃｏｍｐ［Ｙ］ | Ｖ＝０ is acquired (step S109). Then, in the data processing device 12, the parameter Comp[X]| Ｖ＝０ , σ Ｃｏｍｐ［Ｘ］ | Ｖ＝０ , σ Ｃｏｍｐ［Ｙ］ | Ｖ＝０ Based on this, the parameter ρ of the dye fluorescent material U Ｕｉ , ρＵｒ is calculated and saved (step S110).

[0062] Next, in a state in which the intensity of the signal light in the flow cytometer system 1 is changed to a normal value suitable for measurement, the data processing device 12 detects the signal light from the sample fluid dyed only with the dyeing fluorophore V, thereby acquiring data of the digital value DN of each channel for multiple events (step S111). Then, the data processing device 12 performs compensation on the digital value DN of each channel, and the parameter Comp[X]| Ｕ＝０ , σ Ｃｏｍｐ［Ｘ］ | Ｕ＝０ , Comp[Y] | Ｕ＝０ , σ Ｃｏｍｐ［Ｙ］ | Ｕ＝０ is acquired (step S112). Then, in the data processing device 12, the parameter Comp[Y]| Ｕ＝０ , σ Ｃｏｍｐ［Ｘ］ | Ｕ＝０ , σ Ｃｏｍｐ［Ｙ］ | Ｕ＝０ Based on this, the parameter ρ of the dye fluorescent substance V Ｖｉ , ρ Ｖｒ is calculated and saved (step S113). This completes the advance preparation process.

[0063] 7, in the flow cytometer system 1, the intensity of the laser light is set to a predetermined value suitable for measurement, and flow cytometry detection of the sample fluid to be measured is started. In response, the data processor 12 acquires the digital value DN of each channel (step S201). Next, the data processor 12 generates a histogram and dot plot for each channel using the digital value DN of each channel (step S202). In this case, the data processor 12 refers to the stored parameters for each channel and calculates the digital value DN of each channel so that the virtual photon number < S Ｕ >, <S Ｖ > and the virtual photon number <S Ｕ >, <S Ｖ >The standard deviation σ corresponding to Ｃｏｍｐ［Ｘ］ / CG Ｘ Q Ｘ, σ Ｃｏｍｐ［Ｙ］ / CG Ｙ Q Ｙ is calculated (step S203).

[0064] Thereafter, the data processing unit 12 calculates the calculated virtual photon number<S for the generated histogram and dot plot. Ｕ >, <S Ｖ > and standard deviation σ Ｃｏｍｐ［Ｘ］ , σ Ｃｏｍｐ［Ｙ］ Then, based on the results of the gating process, the data processing device 12 classifies the population of analytes (target population) in the data shown in the histogram and dot plot (step S205).

[0065] Next, if the data processing device 12 is set to perform sorting (step S206; Yes), the data processing device 12 is controlled to perform sorting on the classified target population (S207). On the other hand, if the data processing device 12 is set not to perform sorting (step S206; No), the data processing device 12 performs data analysis processing on the classified target population, such as calculating the proportion of the population to the whole (S208).

[0066] The effects of the flow cytometer system 1 according to the embodiment described above will now be described.

[0067] In the flow cytometer system 1, outputs from the two photomultiplier tubes 11c and 11d that detect fluorescence from the two dye phosphors U and V are obtained for a test object to which one dye phosphor U is applied, and outputs from the two photomultiplier tubes 11c and 11d are obtained for a test object to which the other dye phosphor V is applied, and based on the amount of variation in each of these outputs, an independent variation parameter ρ Ｕｉ , ρ Ｖｉ and a correlation variation parameter ρ that represents the degree of variation in the mutual influence of the outputs of the two photomultiplier tubes 11c and 11d.Ｕｒ , ρ Ｖｒ Then, the outputs of the two photomultiplier tubes 11c and 11d for an analyte to which two dye phosphors U and V are applied are calculated in advance, and the independent variability parameter ρ Ｕｉ , ρ Ｖｉ and the correlation variability parameter ρ Ｕｒ , ρ Ｖｒ Based on this, the variation in the number of photons incident on the two photomultiplier tubes 11c and 11d or the variation in the number of photoelectrons emitted from the photoelectric conversion units of the two photomultiplier tubes 11c and 11d is evaluated. This makes it possible to accurately evaluate the variation in the output signals of the two photomultiplier tubes 11c and 11d. As a result, data analysis targeting the fluorescence from the two dye phosphors U and V can be performed with high accuracy.

[0068] Furthermore, in the flow cytometer system 1, it is preferable that the evaluation value is the variation in the number of photons. In this case, data analysis can be performed with high accuracy on the number of incident photons based on the fluorescence from the two dye fluorophores U and V.

[0069] Furthermore, in the flow cytometer system 1, it is preferable that the data analysis includes a gating process for defining the boundaries of the population to be analyzed, which allows the gating process to be performed based on quantitative data on the variation of signal light, thereby improving the accuracy of identifying the population to be analyzed.

[0070] It is also preferable that compensation processing be performed on the outputs of the two photomultiplier tubes 11c and 11d in the flow cytometer system 1. In this case, the signal component leaking from the channel of one photomultiplier tube is subtracted from the intensity signal output by the other photomultiplier tube, making it possible to perform data analysis that excludes the influence of leaking between channels, thereby further improving the accuracy of identifying the population being analyzed.

[0071] Various embodiments of the present invention have been described above, but the present invention is not limited to the above embodiments, and may be modified or applied to other things within the scope that does not change the gist of the claims.

[0072] For example, the gating process in the dot plot is not limited to being automatically set by the electronic system 4, but may be set by input by an operator, or an automatically set process may be adjusted by input by an operator. Furthermore, the photomultiplier tube in the embodiment is not limited to one equipped with a dynode or an anode which is an electron collecting electrode, but may also be an HPD (Hybrid Photo Detector) which uses a semiconductor element to multiply and detect photoelectrons emitted from a photoelectric conversion unit.

[0073] As a modification, the data processing device 12 may use the number of effective photoelectrons (number of photoelectrons) instead of the number of virtual photons as the analytical evaluation value.

[0074] 1...flow cytometer system, 2...fluid system, 3...optical system, 4...electronic system (signal processing device), 5...channel, 6...flow cell, 7...laser light source, 8...lens, 9a, 9b, 9c, 9d...filter, 10b, 10c...dichroic mirror, 11a, 11b, 11c, 11d...photomultiplier tube, 12...data processing device, 201...signal acquisition unit, 202...calculation unit, 203...analysis unit.

Claims

1. A signal processing method for processing the outputs of a first photomultiplier tube and a second photomultiplier tube that detect fluorescence from two phosphors provided for a test object, the method comprising the steps of: acquiring a first intensity signal output from the first photomultiplier tube and a second intensity signal output from the second photomultiplier tube for a test object provided with one of the two phosphors; acquiring a third intensity signal output from the first photomultiplier tube and a fourth intensity signal output from the second photomultiplier tube for a test object provided with the other of the two phosphors; calculating a first variation amount, a second variation amount, a third variation amount, and a fourth variation amount as the variations of the first intensity signal, the second intensity signal, the third intensity signal, and the fourth intensity signal, respectively; calculating, based on the first variation amount, the second variation amount, the third variation amount, and the fourth variation amount, an independent variation parameter representing the degree of independent variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube do not affect each other in measuring the fluorescence from each of the two phosphors, and a correlated variation parameter representing the degree of correlated variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube affect each other in measuring the fluorescence from each of the two phosphors; calculating, based on the independent variation parameter, the correlated variation parameter, and the intensity signal output from the first photomultiplier tube and the intensity signal output from the second photomultiplier tube, an evaluation value that evaluates the variation in the number of photons of signal light incident on each of the first photomultiplier tube and the second photomultiplier tube, or the variation in the number of photoelectrons emitted from the photoelectric conversion units of each of the first photomultiplier tube and the second photomultiplier tube, and performing data analysis based on the evaluation value.

2. The signal processing method according to claim 1, wherein the evaluation value is the variation in the number of photons.

3. The signal processing method according to claim 1 or 2, wherein the data analysis includes a gating process for defining boundaries of a population to be analyzed.

4. A signal processing method according to any one of claims 1 to 3, wherein, before calculating the first and third amounts of variation, a correction process is performed on the first intensity signal and the third intensity signal to subtract a signal component that has leaked into the channel of the first photomultiplier tube from the channel of the second photomultiplier tube; and before calculating the second and fourth amounts of variation, a correction process is performed on the second intensity signal and the fourth intensity signal to subtract a signal component that has leaked into the channel of the second photomultiplier tube from the channel of the first photomultiplier tube.

5. A signal processing device comprising a processor, and processing the outputs of a first photomultiplier tube and a second photomultiplier tube that detect fluorescence from two phosphors provided for a test object, wherein the processor: acquires a first intensity signal output from the first photomultiplier tube and a second intensity signal output from the second photomultiplier tube for a test object provided with one of the two phosphors; acquires a third intensity signal output from the first photomultiplier tube and a fourth intensity signal output from the second photomultiplier tube for a test object provided with the other of the two phosphors; calculates a first variation amount, a second variation amount, a third variation amount, and a fourth variation amount as the variations of the first intensity signal, the second intensity signal, the third intensity signal, and the fourth intensity signal, respectively; calculating, based on the first variation amount, the second variation amount, the third variation amount, and the fourth variation amount, an independent variation parameter representing the degree of independent variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube do not affect each other in measuring the fluorescence from each of the two phosphors, and a correlated variation parameter representing the degree of correlated variation where the output of the first photomultiplier tube and the output of the second photomultiplier tube affect each other in measuring the fluorescence from each of the two phosphors; calculating, based on the independent variation parameter, the correlated variation parameter, and the intensity signal output from the first photomultiplier tube and the intensity signal output from the second photomultiplier tube, an evaluation value that evaluates the variation in the number of photons of signal light incident on each of the first photomultiplier tube and the second photomultiplier tube, or the variation in the number of photoelectrons emitted from the photoelectric conversion units of each of the first photomultiplier tube and the second photomultiplier tube, The signal processing device is configured to perform data analysis based on the evaluation value.

6. The signal processing device according to claim 5, wherein the evaluation value is the variation in the number of photons.

7. The signal processing device according to claim 5 or 6, wherein the data analysis includes a gating process for defining boundaries of a population to be analyzed.

8. A signal processing device according to any one of claims 5 to 7, wherein the processor performs a correction process on the first intensity signal and the third intensity signal to subtract a signal component that has leaked into the channel of the first photomultiplier tube from the channel of the second photomultiplier tube before calculating the first variation amount and the third variation amount, and performs a correction process on the second intensity signal and the fourth intensity signal to subtract a signal component that has leaked into the channel of the second photomultiplier tube from the channel of the first photomultiplier tube before calculating the second variation amount and the fourth variation amount.

9. A signal processing system comprising: a signal processing device according to any one of claims 5 to 8; the first photomultiplier tube; the second photomultiplier tube; and an optical system that guides the signal light to the first photomultiplier tube and the second photomultiplier tube.

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