JP2007127415A - Method and device for calculating fluorescence intensity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To increase the kind of distinguishable fluorescence compared with hitherto, when determining each fluorescence intensity by using a detection value of fluorescence emitted by irradiating simultaneously a labeled sample including a plurality of fluorescent pigments with laser light. <P>SOLUTION: The labeled sample is irradiated with performing time modulation of the laser light intensity by a prescribed frequency, and fluorescence of the labeled sample at this point is received by a plurality of detection sensors having each different light-receiving wavelength band, to thereby collect each detection value including phase information from each detection sensor. A correction transformation matrix is generated by using a parameter of a transfer function when the fluorescence of the labeled sample when being irradiated with the laser light is relaxation response of a primary delay system. A set of the detection values including the phase information collected from each detection sensor is used as a vector, and the intensity of the fluorescence emitted from the labeled sample is determined by applying an inverse matrix generated from the generated correction transformation matrix to the vector. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fluorescence intensity calculation method for obtaining each fluorescence intensity using detection values of fluorescence of a plurality of labeled samples emitted by simultaneously irradiating a plurality of labeled samples with laser light using a flow cytometer, and the like. Relates to the device.

  In the medical and biological fields, the fluorescence emitted from the measurement object when irradiated with laser light is received using a photoelectric converter such as a photomultiplier or avalanche photodiode, and the type of measurement object such as a cell or gene Flow cytometers that analyze frequency and characteristics are widely used.

Specifically, a flow cytometer uses a binding such as an antigen-antibody reaction to make a sample solution by suspending a sample such as a cell or a microbead labeled with a fluorescent dye in physiological saline or the like. A laminar sheath flow in which each labeled sample flows is formed by flowing the sample solution so as to wrap the sample solution in another solution called, and this sheath flow is irradiated with laser light, and fluorescence and scattered light for each sample. It is a device that measures. For example, when measuring fluorescence intensity and identifying which sample has a fluorescent characteristic among many types of samples to be analyzed, different types of fluorescent dyes are attached to the various types of samples. Labeled samples are used. This labeled sample is irradiated with laser light, and the fluorescence emitted at that time is measured. At this time, it is necessary to simultaneously measure and identify each fluorescence emitted from various types of fluorescent dyes and autofluorescence emitted from a sample such as a cell or microbead. For this reason, the flow cytometer is provided with a plurality of photoelectric converters having different light reception wavelength bands, and fluorescent dyes matching the light reception wavelength bands are selected and used. At that time, the measured values obtained by the plurality of photoelectric converters represent the fluorescence intensity in each fluorescent dye. However, when a plurality of fluorescent lights are simultaneously received in the light receiving wavelength range of the photoelectric converter, the measurement result of the fluorescent intensity is deviated from the actual fluorescent intensity. In order to correct this deviation, the detection value is generally corrected.
Examples of such correction include a fluorescence value correction method disclosed in Patent Document 1 below.

JP 2003-83894 A

In Patent Document 1, the measurement values obtained by a plurality of photoelectric converters are represented as vectors, while an inverse matrix of a preset correction matrix is created, and this inverse matrix is applied to the vector to It is supposed that the fluorescence intensity can be calculated. In this case, the correction matrix is a matrix of geometric transformation that corrects the positional relationship in the two-dimensional correlation diagram (scattergram), as shown in FIGS. For this reason, in order to create an inverse matrix from a correction matrix and to apply this inverse matrix to a vector having measured values as elements, the correction matrix needs to be a square matrix. The matrix size of this correction matrix is determined by the number of photoelectric converters that output measurement values and the total number of types of autofluorescence emitted by samples such as microbeads and cells and the types of fluorescence emitted by fluorescent dyes attached to the samples. Therefore, in order for the correction matrix to be a square matrix, the number of photoelectric converters and the number of received fluorescence must be equal. That is, if there are four types of fluorescent dyes to be attached to a sample such as microbeads or cells, it is necessary to perform measurement using a number corresponding to one type of autofluorescence, that is, five photoelectric converters. Measurement using a large number of photoelectric converters increases the number of photoelectric converters arranged, and also increases the number of processing circuits that process the measurement values, thereby increasing the cost of the flow cytometer and its processing device.
For this reason, there arises a problem that the types of distinguishable fluorescence that are simultaneously measured are limited according to the number of arranged photoelectric converters.

  Therefore, in order to solve the above problems, the present invention measures simultaneously when obtaining each fluorescence intensity using the detected value of fluorescence emitted by irradiating a labeled sample labeled with a plurality of fluorescent dyes with laser light. It is an object of the present invention to provide a fluorescence intensity calculation method and a fluorescence intensity calculation apparatus that can increase the types of fluorescent dyes that can be identified as compared with conventional ones.

In order to achieve the above object, the present invention is a fluorescence intensity calculation method for obtaining each fluorescence intensity from a detection value of fluorescence emitted by irradiating a labeled sample labeled with a plurality of fluorescent dyes with laser light. The intensity of the light is time-modulated at a predetermined frequency and irradiated to the labeled sample. The fluorescence of the labeled sample at this time is received by a plurality of detection sensors having different light receiving wavelength bands, thereby detecting each detection value including phase information. Collecting from the sensor, and setting the matrix elements of the correction transformation matrix using the parameters of the transfer function when each fluorescence of the labeled sample irradiated with the laser beam is a relaxation response of the first-order lag system, the correction A step of creating a conversion matrix, and a set of detection values including the phase information collected from each detection sensor is set as a vector, and the vector is used as the vector from the correction conversion matrix. Providing fluorescence intensity calculating method characterized by comprising the steps of: determining the fluorescence intensity of the fluorescence emitted by each beacon samples by applying an inverse matrix made.
The labeled sample is, for example, a cell or microbead labeled with a fluorescent dye.

The labeled sample has a plurality of different types by attaching different types of fluorescent dyes to the sample that emits autofluorescence upon irradiation with laser light, and by irradiating the labeled sample with laser light, The fluorescence emitted from at least one of the fluorescent dyes and the autofluorescence emitted from the sample may partially overlap each other in the wavelength region. In this case, although it is difficult to set the detected value to each fluorescence intensity emitted from the labeled sample, each fluorescence intensity can be obtained using the fluorescence intensity calculation method. The fluorescent dye is attached to the sample by chemical or physical bonding. The fluorescent dye may be selected so as to emit fluorescence having a wavelength (color) depending on the type of the fluorescent dye, but if the fluorescence relaxation characteristics (fluorescence relaxation time constant) of the fluorescent dye are different, the same wavelength ( It is also possible to select a fluorescent dye that emits light in (color).
Further, it is preferable that 2 · m ≧ n + 1 is satisfied when the number of the detection sensors is m and the number of the fluorescent dyes is n.

Further, in the step of creating the correction conversion matrix, when the fluorescent dye is not attached and the sample consisting of the sample that emits the autofluorescence is referred to as an unlabeled sample, the autofluorescence is delayed by the first order for the unlabeled sample. A first calibration for obtaining a fluorescence relaxation time constant and a gain constant when the relaxation response of the system is assumed is performed. In the first calibration, the unlabeled sample is used as a measurement object at the predetermined frequency. By irradiating with time-modulated laser light, detection values including phase information are collected from each detection sensor, and the fluorescence relaxation time constant and gain constant of the autofluorescence emitted from the label-free sample are obtained from these detection values, The correction conversion matrix is generated using the fluorescence relaxation time constant and the gain constant obtained by the first calibration. It is preferable.
At that time, in the creation of the correction conversion matrix, when using the gain constant obtained by the first calibration to create the correction conversion matrix, the gain constant obtained from the detection value of each detection sensor, It is preferable to standardize and use the maximum gain constant among these gain constants.

  In the first calibration, the detection values used when obtaining the fluorescence relaxation time constant and the gain constant are the amplitude values of the cos component and the sin component of the signal waveform detected by the detection sensor. It is preferable that detection values including phase information are collected for each of the labeled samples, and representative values are extracted from the plurality of detection values and used for the first calibration.

  Further, in the step of creating the correction conversion matrix, for all types of the labeled sample, the fluorescence relaxation time constant and the gain constant when the fluorescence emitted by the fluorescent dye is a relaxation response of a first-order lag system, A second calibration is performed for each type of labeled sample, and in this second calibration, a labeled sample attached to a sample in which one of the fluorescent dyes emits autofluorescence is used as a measurement object. By irradiating the laser beam time-modulated at the predetermined frequency, detection values including phase information are collected from each detection sensor, and the fluorescence relaxation time constant of the fluorescence emitted from the fluorescent dye of the labeled sample is detected from these detection values. And a gain constant, and the fluorescent dye that attaches the fluorescence relaxation time constant and gain constant to the sample that emits autofluorescence While changing the type, the fluorescence relaxation time constant and gain constant of the fluorescence emitted by all the fluorescent dyes contained in the labeled sample are obtained, and the fluorescence relaxation time constant and gain constant in the labeled sample obtained by the second calibration are obtained. It is preferable to create the correction transformation matrix using

  At that time, when obtaining the fluorescence relaxation time constant and gain constant for each fluorescence emitted by the fluorescent dye of the labeled sample, when the fluorescent dye is not attached and the sample made of the autofluorescent sample is referred to as an unlabeled sample The first calibration for obtaining the fluorescence relaxation time constant and the gain constant when the autofluorescence is a first order lag relaxation response is performed on the unlabeled sample. In this first calibration, By irradiating a laser beam time-modulated at the predetermined frequency with the label-free sample as a measurement object, detection values including phase information are collected from each detection sensor, and the label-free sample is emitted from these detection values. A fluorescence relaxation time constant and a gain constant of the fluorescence are obtained, and the fluorescence relaxation time constant and the gain obtained by the first calibration are obtained. With constant, in the second calibration, it is preferable to determine the fluorescence relaxation time constant and the gain constant per fluorescence fluorescent dye of the labeled sample is emitted.

Further, in the creation of the correction conversion matrix, when using the gain constant obtained by the second calibration to create the correction conversion matrix, each detection sensor is used for each fluorescence emitted by the fluorescent dye of each labeled sample. It is preferable to use the gain constant obtained from the detected value normalized by the maximum gain constant among these gain constants.
In the second calibration, the detection values used when obtaining the relaxation time constant and the gain constant are the amplitude values of the cos component and the sin component of the signal waveform detected by the detection sensor, and this phase Preferably, detection values including information are collected for each of the labeled samples, and representative values are extracted from the detection values of the plurality of labeled samples and used for the second calibration.

  The present invention relates to a fluorescence intensity calculation device for obtaining each fluorescence intensity from the fluorescence detection values of a plurality of labeled samples emitted by irradiating a labeled sample labeled with a plurality of fluorescent dyes with laser light, the intensity of the laser light The detected value including phase information is collected from each detection sensor by irradiating the labeled sample with time modulation at a predetermined frequency, and receiving the fluorescence of the labeled sample at a plurality of detection sensors with different receiving wavelength bands. The correction conversion matrix by setting a matrix element of the correction conversion matrix using a transfer function parameter when each of the fluorescence of the labeled sample irradiated with the laser light is a relaxation response of a first-order lag system A set of matrix generation means for generating the detection value including the phase information collected from each detection sensor is set as a vector, and the vector is generated from the correction conversion matrix. Reacted with inverse matrix is to provide a strength calculating means for determining the fluorescence intensity of each emitted fluorescence of the labeled sample, the fluorescence intensity calculating apparatus, comprising a.

  In this case, the labeled sample has a plurality of different types by attaching different types of fluorescent dyes to the sample that emits autofluorescence upon irradiation with laser light, and the labeled sample is irradiated with laser light. The fluorescence emitted from at least one of the fluorescent dyes and the autofluorescence emitted from the sample may partially overlap each other in the wavelength region. In this case, although it is difficult to set the detected value to each fluorescence intensity emitted from the labeled sample, each fluorescence intensity can be obtained using the fluorescence intensity calculation method. The fluorescent dye is attached to the sample by chemical or physical bonding.

  At this time, when the fluorescent dye is not attached and the sample made of the sample emitting the autofluorescence is referred to as an unlabeled sample, the autofluorescence is assumed to be a first order delayed relaxation response for the unlabeled sample. First calibration means for obtaining the fluorescence relaxation time constant and gain constant of the laser beam, and the first calibration means irradiates a laser beam time-modulated at the predetermined frequency with the unlabeled sample as a measurement object. Thus, the detection values including phase information are collected from each detection sensor, and the fluorescence relaxation time constant and gain constant of the autofluorescence emitted from the label-free sample are obtained from these detection values. It is preferable to create the correction conversion matrix using the fluorescence relaxation time constant and gain constant obtained by the first calibration means.

  Further, for all types of the labeled sample, a second time for obtaining a fluorescence relaxation time constant and a gain constant when the fluorescence emitted from the fluorescent dye is a relaxation response of a first-order lag system is obtained for each type of the labeled sample. The second calibration unit time-modulates the labeled sample attached to the sample that emits autofluorescence at the predetermined frequency as a measurement object. By irradiating laser light, detection values including phase information are collected from each detection sensor, and from these detection values, the fluorescence relaxation time constant and gain constant of the fluorescence emitted by the fluorescent dye of this labeled sample are obtained, and this fluorescence While changing the kind of fluorescent dye to be attached to the sample that emits autofluorescence, the relaxation time constant and the gain constant are changed. Fluorescence relaxation time constants and gain constants of fluorescence emitted from all the fluorescent dyes are obtained, and the matrix creation means uses the fluorescence relaxation time constants and gain constants in the labeled sample obtained by the second calibration means. It is preferable to create the correction conversion matrix.

  In the present invention, a gain constant corresponding to the fluorescence intensity can be obtained even when a labeled sample labeled with a plurality of fluorescent dyes is irradiated with laser light. At this time, since the laser light is time-modulated at a predetermined frequency, the phase difference information can be detected from one photoelectric converter. Therefore, since the fluorescence intensity is obtained using these values, the number of labeled samples that can be obtained by measurement as in the conventional case is the same as the type of fluorescent light that can be identified when the number of arranged photoelectric converters is the same. It can be more than that.

  In addition, before the fluorescence intensity for a plurality of types of labeled samples, the first calibration and the second calibration for calculating the fluorescence relaxation time constant and the gain constant using the unlabeled sample and the labeled sample for each type are performed. As a result, the fluorescence intensity for a plurality of types of labeled samples can be obtained with high accuracy.

Hereinafter, the fluorescence intensity calculation apparatus of the present invention that implements the fluorescence intensity calculation method of the present invention will be described in detail based on a flow cytometer.
FIG. 1 is a schematic configuration diagram of a flow cytometer 10 using a fluorescence intensity detection apparatus using intensity-modulated laser light according to the present invention.

  The flow cytometer 10 irradiates a labeled sample 12 with a fluorescent dye attached to a receptor sample (referred to as a sample) such as a microbead or a specific cell by chemical binding or physical binding, and irradiates the labeled sample 12 with laser light. A signal processing device 20 that detects and processes a fluorescence signal of fluorescence emitted from the labeled sample 12 and an analysis device (computer) 80 that analyzes the labeled sample 12 from the processing result obtained by the signal processing device 20 are provided. The sample is autofluorescent in response to laser light irradiation.

The signal processing device 20 processes the fluorescence signal from the laser light source unit 22, the light receiving units 24 and 26, a control unit that modulates the intensity of the laser light from the laser light source unit 22 at a predetermined frequency, and the label sample 12. A control / processing section 28 having a section, and a conduit 30 for forming a flow cell by flowing the labeled sample 12 together with a sheath liquid forming a high-speed flow.
A recovery container 32 is provided at the outlet of the conduit 30. The flow cytometer 10 includes a cell sorter for separating biological substances such as specific cells in the labeled sample 12 within a short period of time by laser light irradiation, and is separated into separate collection containers. You can also

The laser light source unit 22 is a portion that emits a continuous wave laser beam in a visible light band of 350 nm to 800 nm, for example, a laser beam having a wavelength of 405 nm, with intensity modulation at a predetermined frequency.
For example, a semiconductor laser is used as a light source for emitting laser light, and the laser light is emitted with an output of, for example, about 5 to 100 mW. On the other hand, the frequency (modulation frequency) for modulating the intensity of the laser light has a period slightly longer than the fluorescence relaxation time, for example, 10 to 100 MHz.
In the present invention, in addition to one laser beam, three laser beams having different wavelengths such as λ ₁ = 405 nm, λ ₂ = 533 nm, and λ ₃ = 650 nm are emitted at the same time. May be. In this case, it is preferable to use a dichroic mirror to irradiate three laser beams into one light beam. In addition, each laser beam includes encoded signal information orthogonal to each other so that it can be identified which of the R, G, and B fluorescent light is reacted by the laser beam irradiation. It is good to make it. Details are described in Japanese Patent Application No. 2005-37399, which is an application of the present applicant.

  The laser light source unit 22 oscillates in a predetermined wavelength band so that the laser light excites the fluorescent dye to emit fluorescence in a specific wavelength band. The fluorescence emitted by the laser light is the self-emission fluorescence of the labeled sample 12 to be measured and the fluorescence emitted by the fluorescent dye in the labeled sample 12. When the labeled sample 12 passes through the conduit 30, the laser beam is measured at the measurement point. It emits fluorescence at a specific wavelength upon irradiation.

  The light receiving unit 24 is disposed so as to face the laser light source unit 22 with the pipe line 30 interposed therebetween, and the labeled sample 12 passes the measurement point to cause the labeled sample 12 to move the measurement point. A photoelectric converter is provided that outputs a detection signal indicating that it passes. The signal output from the light receiving unit 24 is supplied to the control / processing unit 28, and is used as a trigger signal informing the timing at which the marker sample 12 passes through the measurement point in the pipe 30.

On the other hand, the light receiving unit 26 is arranged in a direction orthogonal to the emission direction of the laser light emitted from the laser light source unit 22 and in a direction orthogonal to the moving direction of the marker sample 12 in the conduit 30. And a photoelectric converter such as a photomultiplier (photomultiplier tube) or an avalanche photodiode that receives fluorescence emitted from the labeled sample 12 irradiated at the measurement point.
FIG. 2 is a schematic configuration diagram illustrating a schematic configuration of an example of the light receiving unit 26.

The light receiving unit 26 shown in FIG. 2 includes a lens system 26a for focusing the fluorescence signal of the fluorescence from the labeled sample 12, dichroic mirrors 26b ₁ and 26b ₂ , bandpass filters 26c _{1 to} 26c ₃ , a photomultiplier tube and an avalanche. And photoelectric converters 27a to 27c such as photodiodes.
The lens system 26a is configured to focus the fluorescence incident on the light receiving unit 26 on the light receiving surfaces of the photoelectric converters 27a to 27c.

The dichroic mirrors 26b ₁ and 26b ₂ are mirrors that reflect fluorescence in a wavelength band within a predetermined range and transmit the other fluorescence. The reflection wavelength band and the transmission wavelength band of the dichroic mirrors 26b ₁ and 26b ₂ are set so as to be filtered by the band-pass filters 26c _{1 to} 26c ₃ and to capture fluorescence of a predetermined wavelength band by the photoelectric converters 27a to 27c. .

The band-pass filters 26c _{1 to} 26c ₃ are filters that are provided in front of the light receiving surfaces of the photoelectric converters 27a to 27c and transmit only fluorescence in a predetermined wavelength band. The wavelength band of the transmitted fluorescence is set corresponding to the wavelength band of the fluorescence, and is a different wavelength band.

  The photoelectric converters 27a to 27c are sensors that include, for example, a sensor including a photomultiplier tube, and convert light received by the photoelectric surface into an electrical signal. Here, since the received fluorescence is received as an optical signal having signal information, the output electric signal is a fluorescence signal having phase difference signal information. This fluorescence signal is supplied to the control / processing unit 28 and amplified by an amplifier.

As shown in FIG. 3, the control / processing unit 28 includes a signal generation unit 40, a signal processing unit 42, and a controller 44. The signal generation unit 40 and the controller 44 form a light source control unit that generates a modulation signal having a predetermined frequency.
The signal generator 40 is a part that generates a modulation signal for modulating (amplitude modulation) the intensity of the laser light at a predetermined frequency.
Specifically, the signal generation unit 40 includes an oscillator 46, a power splitter 48, and amplifiers 50 and 52, and supplies a generated modulation signal to the laser light source unit 22 and also to the signal processing unit 42. It is. The reason why the modulation signal is supplied to the signal processing unit 42 is that it is used as a reference signal for detecting the phase difference of the fluorescence signals output from the photoelectric converters 27a to 27c, as will be described later. The modulation signal is a sine wave signal having a predetermined frequency and is set to a frequency in the range of 10 to 50 MHz.

  The signal processing unit 42 is a part that extracts information (phase difference) related to the phase delay of the fluorescence emitted from the labeled sample 12 by the irradiation of the laser light, using the fluorescence signals output from the photoelectric converters 27a to 27c. The signal processing unit 42 amplifies the fluorescence signals output from the photoelectric converters 27a to 27c, and the modulated signals that are sine wave signals supplied from the signal generation unit 40 to the amplified fluorescence signals. And a phase difference detector 56 having an IQ mixer (not shown) for combining the amplified fluorescence signal with the modulated signal.

  An IQ mixer (not shown) provided in the phase difference detector 56 includes a photoelectric converter for synthesizing the fluorescence signals supplied from the photoelectric converters 27a to 27c using the modulation signal supplied from the signal generation unit 40 as a reference signal. 27a to 27c are provided separately. Specifically, each of the IQ mixers multiplies the reference signal by the fluorescence signal (RF signal) to calculate a processing signal including a cos component (real part) and a high frequency component of the fluorescence signal, and the phase of the reference signal. Is multiplied by the fluorescence signal to calculate a processing signal including a sin component (imaginary part) and a high frequency component of the fluorescence signal. The processing signal including the cos component and the processing signal including the sin component are supplied to the controller 44.

  The controller 44 controls the signal generation unit 40 to generate a sine wave signal having a predetermined frequency, and from the processing signal including the cos component and the sin component of the fluorescence signal obtained by the signal processing unit 42, the high frequency component. This is a part for obtaining the cos component and the sin component of the fluorescence signal by removing.

  Specifically, the controller 44 gives an instruction for controlling the operation of each part, and also manages a system controller 60 that manages all operations of the flow cytometer 10, a cos component calculated by the signal processing unit 42, sin A low-pass filter 62 that removes the high-frequency component from the processing signal in which the high-frequency component is added to the component, an amplifier 64 that amplifies the processing signal of the cos component and sin component from which the high-frequency component has been removed, and A that samples the amplified processing signal / D converter 66. In the A / D converter 66, the processing signals of the cos component and the sin component from which the high frequency component has been removed are sampled and supplied to the analyzer 80.

The analysis device 80 uses the cos component (real part) and sin component (imaginary part) of the fluorescence signal as a vector component of the processed signal value (detected value), and an inverse matrix created from a predetermined correction conversion matrix. On the other hand, it is a device that calculates a fluorescence intensity by applying a vector.
The analyzer 80 corresponds to the fluorescence intensity calculation apparatus of the present invention, and implements the fluorescence intensity calculation method described later.

FIG. 4 is a schematic configuration diagram of the analyzer 80.
The analysis device 80 is a device configured by starting a predetermined program on a computer. In addition to the CPU 82, the memory 84, and the input / output port 86, the first calibration unit formed by starting software. 88, a second calibration unit 90, and an intensity calculation unit 92. A display 94 is connected to the analyzer 80.

  The processing performed by the analyzer 80 is the processing shown in FIGS. The intensity calculating unit 92 executes the main part of the processing flow shown in FIG. 5, the first calibration unit 88 executes the main part of the processing flow shown in FIG. 6, and the second calibration unit 90 is shown in FIG. Execute the main part of the processing flow.

The CPU 82 is an arithmetic processor provided in the computer, and substantially executes various calculations of the first calibration unit 88, the second calibration unit 90, and the intensity calculation unit 92.
The memory 84, a ROM that stores programs that form the first calibration unit 88, the second calibration unit 90, and the intensity calculation unit 92, and the first calibration unit 88, the second calibration, and the like are executed on the computer. A RAM for storing the processing results calculated by the operation unit 90 and the intensity calculating unit 92 and the data supplied from the input / output port 86.
The input / output port 86 accepts input of detected values of the cos component (real part) and sin component (imaginary part) of the fluorescence signal supplied from the controller 44, and the first calibration unit 88 and the second calibration unit 90. In addition, it is used to output information such as the value of the processing result created by the intensity calculation unit 92 and the scattergram to the display 94.
The display 94 displays graphs such as values of processing results such as fluorescence relaxation time constants and gain constants obtained by the first calibration unit 88, the second calibration unit 90, and the intensity calculation unit 92, and scattergrams.

The intensity calculation unit 92 is a part that executes the main part of the processing flow shown in FIG. 5, and obtains each fluorescence intensity from the detected values of the cos component and the sin component supplied from the controller 44.
That is, the intensity calculation unit 92 corrects using the parameters (gain constant, fluorescence relaxation time constant) of the transfer function when the fluorescence of the labeled sample irradiated with the laser light is assumed to be a first order lag relaxation response. A correction conversion matrix is created by setting the matrix elements of the conversion matrix and obtaining the matrix elements of the correction conversion matrix. Next, a set of detection values (detection values including phase information) of the cos component and the sin component collected from each detection sensor supplied from the controller 44 is set as a vector, and this vector is used as a correction conversion matrix previously created. The fluorescence intensity of the fluorescence emitted from the labeled sample is calculated by applying the created inverse matrix. Details of the processing of the intensity calculation unit will be described later.
The labeled sample 12 is a labeled sample that is labeled by attaching different types of fluorescent dyes to a sample such as a cell or a microbead. The number of labeled samples at this time is n, and a photoelectric detector M and n are set so as to satisfy 2 · m ≧ n + 1.

  When the first calibration unit 88 is composed of a sample that does not have a fluorescent dye attached and emits autofluorescence such as microbeads, it is referred to as an unlabeled sample. This is a part for obtaining a fluorescence relaxation time constant and a gain constant when it is assumed to be a response. Specifically, the first calibration unit 88 collects detection values including phase information from each photoelectric converter by irradiating laser light that is time-modulated at a predetermined frequency with an unlabeled sample as a measurement object. The fluorescence relaxation time constant and gain constant of autofluorescence emitted from the unlabeled sample are calculated from these detection values, and stored in the memory 84. Details will be described later.

The second calibration unit 90 is a part that calculates a fluorescence relaxation time constant and a gain constant for each of the types of labeled samples having a fluorescent dye separately. Also in this case, it is assumed that the fluorescence emitted from the fluorescent dye is a first order lag relaxation response.
That is, the second calibration unit 90 prepares a labeled sample (a sample such as a microbead that emits autofluorescence to which one type of fluorescent dye is attached) labeled using only one type of fluorescent dye, and this labeled sample. Is used as a measurement object, and a detection value including phase information is collected from each detection sensor by irradiating a laser beam time-modulated at a predetermined frequency, and a fluorescent dye of a selected labeled sample is emitted from these detection values. The fluorescence relaxation time constant and gain constant of fluorescence are calculated. Then, while sequentially changing the type of fluorescent dye in the labeled sample, the relaxation time constant and gain constant of all the fluorescence emitted by the fluorescent dye of the labeled sample are calculated, and the calculated relaxation time constant and gain constant are stored in the memory 84. . Details will be described later.

As described above, the fluorescence calculation time constant and the gain constant of the autofluorescence and the fluorescence emitted by the fluorescent dye calculated by the first calibration unit and the second calibration unit and stored in the memory 84 are corrected and converted by the intensity calculation unit 92. When creating a matrix, it is used as a parameter of a transfer function, and is used to calculate a matrix element of a correction transformation matrix. The reason for using the fluorescence relaxation time constant and gain constant of the autofluorescence calculated by the first calibration unit 88 is that the accuracy of the calculation result of the n fluorescence intensities emitted by the fluorescent dye is degraded by the autofluorescence emitted from the labeled sample. Is to prevent.
In addition, when calculating the value of the transfer function, the fluorescence relaxation time constant and gain constant calculated by the second calibration unit 90 are used when the fluorescence emitted from the n fluorescent dyes is measured simultaneously. This is for obtaining the above with high accuracy.
That is, the intensity calculation unit 92 stores the fluorescence relaxation time constant and gain constant of autofluorescence that are stored in the memory 84 and become known, and the fluorescence relaxation time constant and gain of the fluorescent dye that is stored in the memory 84 and becomes known. A correction conversion matrix is created using the constants, and the fluorescence intensity is obtained using this. Detailed description is omitted.
The above is the configuration of the analyzer 80.

In such a flow cytometer 10, the process as shown in FIG. 5 is performed, and the fluorescence intensity of each fluorescence is obtained.
First, n types of labeled samples are prepared (step S10). The n types of labeled samples refer to those in which n types of fluorescent dyes are attached to a sample such as microbeads and are labeled, and are those that are turbid in the measurement solution. The labeled sample solution forms a flow cell in the conduit 30 using sheath liquid. The flow cell is irradiated with laser light intensity-modulated at a predetermined frequency to measure fluorescence (step S20).

The fluorescence measurement is performed using m photoelectric converters having different wavelength bands (shown in FIG. 2) according to a trigger signal generated by the light receiving unit 24 that informs the timing when the labeled sample 12 passes through the measurement point in the pipe 30. In the embodiment, measurement according to m = 3) is started.
From the fluorescence signal obtained by the measurement, the processing signal including the cos component and the sin component of the fluorescence signal is extracted by the phase difference detector 56 of the signal processing unit 42. This processing signal is obtained as a detection value in the controller 44 by removing the high frequency signal by the low-pass filter 62 and AD converting the cos component and the sin component of the fluorescence signal.

The obtained cos component and sin component are supplied to the analyzer 80, and a scattergram (two-dimensional correlation diagram) is displayed on the display 94 using information on the cos component and sin component obtained within a predetermined measurement time. (Step S30).
A scattergram is displayed on the display 94, and the detected values of the cos component and the sin component detected for each photoelectric converter are combined into a vector as a vector component, and this vector is an inverse matrix created from a correction conversion matrix described later. (Step S50). By applying an inverse matrix to a vector, a gain constant in fluorescence emitted from each labeled sample is obtained, and this gain constant is obtained as a vector of fluorescence intensity ratios.
The ratio of the fluorescence intensity thus obtained and the fluorescence relaxation time constant are displayed on the display 94 (step S50).

Here, the matrix elements of the correction transformation matrix are created as follows.
Assuming that the fluorescence emitted from the labeled sample is a first-order lagging relaxation process, the detection values (cos component, sin component) output from each photoelectric converter are the fluorescence from n types of fluorescent dyes emitted from the labeled sample. And the transfer functions of each first-order lag system of one type of autofluorescence are added and expressed as the following formulas (1) and (2).

Here, τ _i (i = 1 to n) is a fluorescence relaxation time constant of the fluorescence emitted by the i-th type fluorescent dye. Κ _ij (i = 1 to n, j = 1 to m) is a gain constant when the fluorescence emitted from the i-th type of fluorescent dye is detected by the j-th photoelectric converter, and m The gain constant standardized by the maximum gain constant among the gain constants detected by one photoelectric converter.
Further, τ ₀ is a fluorescence relaxation time constant when a sample emitting autofluorescence emits autofluorescence. Κ _0j (j = 1 to m) is a gain constant when autofluorescence when the sample emits autofluorescence is detected in the jth photoelectric converter, and m photoelectric converters The gain constant that is standardized by the maximum gain constant among the gain constants detected in (1).
These values are calculated and stored in the memory 84 by a first calibration (the process shown in FIG. 6) and a second calibration (the process shown in FIG. 7) described later. Note that ω _M is an angular frequency obtained by multiplying the modulation frequency of the laser light by 2π.

On the other hand, α _i (i = 1 to n) is a gain constant of fluorescence emitted by the fluorescent dye when n kinds of fluorescent dyes and one kind of autofluorescent sample emit fluorescence simultaneously. Represents the fluorescence intensity when the laser beam is irradiated simultaneously. α ₀ is a gain constant of autofluorescence emitted by the sample when n types of fluorescent dyes and one type of sample emit fluorescence simultaneously. These gain constants α _i and α ₀ are unknown numbers representing the fluorescence intensity when a labeled sample labeled with n kinds of fluorescent dyes is simultaneously irradiated with laser light, and are to be obtained.
Therefore, an equation is expressed from the equations (1) and (2) using the correction conversion matrix M as in the following equation (3). Here, the matrix element of the correction transformation matrix M is, for example, the matrix element of 1 row and 1 column is κ ₁₁ / (1+ (τ ₁ · ω _M ) ² ).
In this way, a correction conversion matrix M in which the value of the transfer function is a matrix element is created.

The matrix size of the correction transformation matrix M is 2 · m × (n + 1) (vertical direction × horizontal direction). Here, m and n are set to satisfy 2 · m ≧ n + 1 as described above. For this reason, the vertical size of the correction conversion matrix is reduced, and for example, rows with small values of κ _ij are removed to obtain a matrix size of (n + 1) × (n + 1). Thereby, the matrix of the correction transformation matrix M is reduced in size, and an inverse matrix M ′ ^{−1 of the} reduced square matrix M ′ is obtained, and this inverse matrix M ′ ⁻¹ is obtained as the left-side vector A of Expression (3). In operation, the right side vector X in the equation (3) is calculated (step S40).

In addition to the method of reducing the matrix size as described above, both sides of the equation (3) are multiplied by the transposed matrix M ^t of the correction conversion matrix M to obtain the correction conversion matrix of (n + 1) × (n + 1). The matrix matrix size is changed to a square matrix M ^t · M, and the inverse matrix (M ^t · M) ^-1 of this square matrix is applied to M ^t A obtained by multiplying the vector A on the left side in Equation (3) by the transpose matrix. Thus, the vector X (= (M ^t · M) ⁻¹ · M ^t A) on the right side of the equation (3) can also be calculated.
The vector X on the right side in the equation (3) thus obtained includes known numbers τ _i (i = 1 to n) and τ ₀ , so these values are substituted and the calculated vector Α _i (i = 1 to n) and α ₀ are calculated from X.
Finally, the fluorescence relaxation time constants τ _i , τ ₀ and gain constants α _i , α _{0 of} each fluorescence are displayed on the display 94. The gain constants α _i and α ₀ represent the fluorescence intensity of each fluorescence.

As described above, when the correction conversion matrix M is created, τ ₀ and κ _0j stored in the memory 84 are used. These values are calculated by the first calibration shown in FIG.
Hereinafter, the first calibration will be described.

In the first calibration shown in FIG. 6, first, a label-free sample is prepared (step S100).
A label-free sample refers to a solution of one type of sample that autofluoresce when irradiated with laser light. Examples of such a sample include receptor samples such as microbeads and cells provided with whiskers to which antibodies and the like can be bound.

Next, measurement is performed on the unlabeled sample with a flow cytometer (step S110).
The measurement by the flow cytometer (step S110) and the display display of the scattergram (step S120) are the same processes as steps S20 and S30 shown in FIG.
Next, in order to identify the autofluorescence emitted from the unlabeled sample, a sample group in the fluorescence region of the unlabeled sample is selected in the scattergram displayed on the display 94 (step S130). This selection is performed using an input operation system such as a mouse by an operator, and the cos component and sin component of the label-free sample included in the selected sample group region, or representative values of the amplitude and phase difference of the fluorescence signal ( For example, an average value, a centroid value, and a frequency peak value) are obtained (step S140).
Next, using the obtained representative values, the fluorescence relaxation time constant and gain constant are determined using the following equations (4) to (7) that define the relationship between the representative value and the fluorescence relaxation time constant and gain constant. It is obtained (step S150).

Here, τ ₀ is a fluorescence relaxation time constant of the autofluorescence of the sample emitting autofluorescence, and ω _M is an angular frequency obtained by multiplying the modulation frequency of the laser light by 2π. α _0j is an autofluorescence gain constant obtained by the j-th (j = 1 to m) photoelectric converter. C is a proportionality constant.
The fluorescence relaxation time constant τ ₀ of autofluorescence and the gain constant α _0j of _{autofluorescence} are calculated using any two of formulas (4) to (7).
The fluorescence relaxation time constant and gain constant of the label-free sample calculated in this way are stored in the memory 84 (step S150).
The first calibration is performed as described above.

Next, the second calibration will be described.
In the second calibration shown in FIG. 7, first, a labeled sample to which one type of fluorescent dye is attached among n types of labeled samples is prepared (step S200). Here, one type of labeled sample means one type of sample such as microbeads, which is self-luminous, and one type of fluorescent dye attached thereto.
Next, fluorescence is measured by the flow cytometer 10 (step S210), the measurement result is displayed on a scattergram (step S220), and a sample group in one type of labeled sample is selected from the scattergram.

The measurement by the flow cytometer (step S210) and the display display of the scattergram (step S220) are the same processes as steps S20 and S30 shown in FIG.
The selection of the sample population is performed to identify the autofluorescence emitted by the sample in one type of labeled sample. Specifically, in the scattergram displayed on the display 94, a sample group in the fluorescent region of one type of labeled sample is selected. This selection is performed using an input operation system such as a mouse by an operator, and is representative of the cos and sin components of the fluorescence emitted by the labeled sample included in the selected sample population region, or the amplitude and phase difference of the fluorescence signal. Values (for example, average value, barycentric value, frequency peak value) are obtained (step S240).

  Next, using the obtained representative value and further using the fluorescence relaxation time constant and gain constant of the autofluorescence emitted from the sample calculated by the first calibration and stored in the memory 84, the following formula (8) to From (10), the fluorescence relaxation time constant and gain constant of the fluorescence emitted from the fluorescent dye in the labeled sample are calculated (step S250).

Here, the subscripts [1] and [2] in the equation (8) indicate the amplitudes of the fluorescence signals obtained by the plurality of photoelectric converters ((cos component) ² + (sin component) ² )) ^{(1/2 )} Is the number of the photoelectric converter having the maximum amplitude value and the number of the photoelectric converter having the second largest amplitude value. Therefore, κ _{0 [1]} and κ _{0 [2]} are ratios normalized with respect to the maximum gain of the gain constant calculated in the first calibration among the self-luminous fluorescence, and the subscript [ Since the numbers 1] and [2] are known, they are known values.
The fluorescence relaxation time constant τ _i calculated by the equation (8) is stored in the memory 84 for use in calculating the value of the matrix element of the correction transformation matrix in the equation (3) in the processing flow shown in FIG. . Further, α _0max is calculated from the equation (9) using the fluorescence relaxation time constant τ _i . α _0max is the maximum gain constant of fluorescence when the sample emitting autofluorescence in the second calibration _undergoes autofluorescence. By substituting this α _0max into the equation (10), α _{i [1]} can be calculated. α _{i [1]} is a gain constant of fluorescence received at the photoelectric converter number having the maximum amplitude value among the fluorescence emitted by the fluorescent dye.
Similarly, the gain constant α _ij of the fluorescence received by the j th photoelectric converter in the equation (11) is calculated using the following equation (11).

The fluorescence relaxation time constant and gain constant calculated in this way are stored in the memory 94, and the calculated value is a parameter of fluorescence in one type of labeled sample. Therefore, it is determined whether or not each of the n types of labeled samples has been prepared and the fluorescence parameter has been obtained (step S260).
As a result of the determination, if all types of labeled samples are not prepared and the fluorescence parameter is not calculated, the process returns to step S200, and steps S200 to S250 are repeated.
In this way, the fluorescence relaxation time constant and gain constant for each fluorescent dye are calculated from the measurement of fluorescence in the labeled sample for each type and stored in the memory 84.
The fluorescence relaxation time constant and gain constant stored in the memory 84 in this way are used to create a correction conversion matrix in step S40 in the process shown in FIG.
The above is the description of the second calibration.
The gain constant α _ij (i = 1 to n, j = 1 to m) calculated in the second calibration is a gain constant that becomes a maximum value when i is fixed and j is changed among these gain constants. By normalizing with max (α _ij ), κ _ij used in the correction transformation matrix in equation (3) can be obtained. Further, the normalized κ _ij is stored in the memory 84 for use in calculating the value of the matrix element of the correction transformation matrix in Expression (3) in the processing flow shown in FIG.

As described above, in the present invention, the gain constant corresponding to the fluorescence intensity can be obtained based on the processing flow shown in FIG. 5 even when the n kinds of labeled samples are irradiated with the laser light. At that time, since the laser light is time-modulated with a predetermined frequency, the values of the cos component and the sin component can be detected from one photoelectric converter. Therefore, since the fluorescence intensity is obtained using these values, the number of labeled samples that can be obtained by measurement as in the prior art is not limited to the same number or less as the number of arranged photoelectric converters as in the past. That is, when the number of arranged photoelectric converters is the same, the number of types of fluorescence that can be identified can be increased as compared with the related art.
Further, before performing the processing shown in FIG. 5 on the n types of labeled samples, the first calibration and the first calibration for calculating the fluorescence relaxation time constant and the gain constant using the unlabeled sample and the labeled samples for each type are performed. Since the second calibration is performed, the fluorescence intensity can be obtained with high accuracy in the process shown in FIG.

  As described above, the fluorescence intensity calculation method and the fluorescence intensity calculation apparatus of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiment, and various improvements and modifications can be made without departing from the gist of the present invention. Of course it is also good.

It is a schematic block diagram of the flow cytometer using the fluorescence intensity detection apparatus of this invention. It is a schematic block diagram which shows an example of the light-receiving part of the flow cytometer shown in FIG. It is a schematic block diagram which shows an example of the control / processing part of the flow cytometer shown in FIG. It is a schematic block diagram which shows an example of the analyzer of the flow cytometer shown in FIG. It is a flowchart which shows an example of the flow of the fluorescence intensity detection method of this invention. It is a flowchart which shows an example of the flow of the 1st calibration performed with the fluorescence intensity detection method of this invention. It is a flowchart which shows an example of the flow of the 2nd calibration performed with the fluorescence intensity detection method of this invention.

Explanation of symbols

10 flow cytometer 12 labeled sample 20 signal processor 22 laser light source unit 24 receiving portion 26a lens system 26c _1, 26c _2, 26c ₃ band pass filters 27a~27c photoelectric converter 28 control and processing unit 30 conduit 32 recovered Container 40 Signal generation unit 42 Signal processing unit 44 Controller 46 Oscillator 48 Power splitter 50, 52, 54a, 54b, 54c, 64 Amplifier 56 Phase difference detector 60 System controller 62 Low-pass filter 66 A / D converter 80 Analyzer 82 CPU
84 Memory 86 Input / output port 88 First calibration unit 90 Second calibration unit 92 Intensity calculation unit

Claims (14)

A fluorescence intensity calculation method for obtaining each fluorescence intensity from a detection value of fluorescence emitted by irradiating a labeled sample labeled with a plurality of fluorescent dyes with laser light,
The intensity of the laser light is time-modulated at a predetermined frequency and irradiated to the labeled sample, and the fluorescence of the labeled sample at this time is received by a plurality of detection sensors having different light receiving wavelength bands, so that each detection value including phase information is Collecting from the detection sensor;
Creating a correction conversion matrix by setting matrix elements of a correction conversion matrix using parameters of a transfer function when each fluorescence of a labeled sample irradiated with laser light is a relaxation response of a first-order lag system; ,
A set of detection values including the phase information collected from each detection sensor is set as a vector, and an inverse matrix created from the correction conversion matrix is applied to this vector to determine the fluorescence intensity of the fluorescence emitted from the labeled sample; A method for calculating fluorescence intensity, comprising: The labeled sample has a plurality of different types by attaching different types of fluorescent dyes to a sample that emits autofluorescence by laser light irradiation,
The fluorescence emitted from at least one of the fluorescent dyes and the autofluorescence emitted from the sample are partially overlapped with each other in the wavelength region by irradiation of the labeled sample with laser light. 2. The fluorescence intensity calculation method according to 1.   3. The fluorescence intensity calculation method according to claim 2, wherein 2 · m ≧ n + 1 is satisfied, where m is the number of detection sensors and n is the type of the fluorescent dye. In the step of creating the correction conversion matrix, when the fluorescent dye is not attached and the sample composed of the sample that emits autofluorescence is referred to as a label-free sample, the autofluorescence of the label-free sample is a first order lag system. A first calibration for obtaining a fluorescence relaxation time constant and a gain constant when assuming a relaxation response is performed,
In this first calibration, a detection value including phase information is collected from each detection sensor by irradiating a laser beam time-modulated at the predetermined frequency with the unlabeled sample as a measurement object, and detecting these Obtain the fluorescence relaxation time constant and gain constant of the autofluorescence emitted from the label-free sample from the value,
The fluorescence intensity calculation method according to claim 2 or 3, wherein the correction conversion matrix is created using a fluorescence relaxation time constant and a gain constant obtained by the first calibration.   In creating the correction conversion matrix, when using the gain constant obtained by the first calibration to create the correction conversion matrix, the gain constant obtained from the detection value of each detection sensor is used as the gain constant. The fluorescence intensity calculation method according to claim 4, wherein the fluorescence intensity is normalized with a maximum gain constant.   In the first calibration, the detection values used when obtaining the fluorescence relaxation time constant and the gain constant are the amplitude values of the cos component and the sin component of the signal waveform detected by the detection sensor, and this phase information 6. The fluorescence intensity according to claim 4 or 5, wherein a detection value including a plurality of detection values is collected for each of the labeled samples, and a representative value is extracted from the plurality of detection values and used for the first calibration. Calculation method. In the step of creating the correction conversion matrix, the fluorescence relaxation time constant and the gain constant when the fluorescence emitted from the fluorescent dye is a first order lag relaxation response for all types of the labeled sample are used as the labeled sample. The second calibration required for each type of
In the second calibration, by irradiating a labeled sample attached to a sample in which one of the fluorescent dyes emits autofluorescence as a measurement object, laser light time-modulated at the predetermined frequency is used. Detection values including phase information are collected from each detection sensor, and from these detection values, the fluorescence relaxation time constant and gain constant of the fluorescence emitted by the fluorescent dye of this labeled sample are obtained, and this fluorescence relaxation time constant and gain constant are obtained. While changing the type of fluorescent dye attached to the sample that emits autofluorescence, obtain the fluorescence relaxation time constant and gain constant of the fluorescence emitted by all the fluorescent dyes contained in the labeled sample,
The fluorescence intensity calculation method according to any one of claims 2 to 5, wherein the correction conversion matrix is created using a fluorescence relaxation time constant and a gain constant in the labeled sample obtained by the second calibration. When obtaining the fluorescence relaxation time constant and gain constant for each fluorescence emitted by the fluorescent dye of the labeled sample, the sample that is not attached with the fluorescent dye and that emits the autofluorescence is referred to as an unlabeled sample. For the labeled sample, first calibration is performed to obtain a fluorescence relaxation time constant and a gain constant when the autofluorescence is a relaxation response of a first order lag system,
In this first calibration, a detection value including phase information is collected from each detection sensor by irradiating a laser beam time-modulated at the predetermined frequency with the unlabeled sample as a measurement object, and detecting these In the second calibration, the fluorescence relaxation time constant and gain constant of the fluorescence emitted from the unlabeled sample are obtained from the value, and the fluorescence relaxation time constant and gain constant obtained by the first calibration are used. The fluorescence intensity calculation method according to claim 7, wherein a fluorescence relaxation time constant and a gain constant for each fluorescence emitted by the fluorescent dye of the labeled sample are obtained.   When generating the correction conversion matrix, when using the gain constant obtained by the second calibration to generate the correction conversion matrix, detection of each detection sensor for each fluorescence emitted by the fluorescent dye of each labeled sample The fluorescence intensity calculation method according to claim 7 or 8, wherein a gain constant obtained from the value is used after being normalized by a gain constant that is maximum among these gain constants.   In the second calibration, the detection values used when obtaining the relaxation time constant and the gain constant are the amplitude values of the cos component and the sin component of the signal waveform detected by the detection sensor, and the phase information is The detection value to be included is collected for each of the labeled samples, and a representative value is extracted from the detection values of the plurality of labeled samples and used for the second calibration. The fluorescence intensity calculation method according to item. A fluorescence intensity calculation device that obtains each fluorescence intensity from detection values of fluorescence of a plurality of labeled samples emitted by irradiating a labeled sample labeled with a plurality of fluorescent dyes with laser light,
The intensity of the laser light is time-modulated at a predetermined frequency and irradiated to the labeled sample, and the fluorescence of the labeled sample at this time is received by a plurality of detection sensors having different light receiving wavelength bands, so that each detected value including phase information is detected. Input means for collecting from the detection sensor;
Matrix generation for creating the correction conversion matrix by setting the matrix elements of the correction conversion matrix using the parameters of the transfer function when each fluorescence of the labeled sample irradiated with the laser beam is a relaxation response of the first-order lag system Means,
Intensities for obtaining the fluorescence intensity of the fluorescence emitted from each of the labeled samples by using a set of detection values including the phase information collected from each detection sensor as a vector and applying an inverse matrix created from the correction transformation matrix to this vector. And a fluorescence intensity calculation device comprising: a calculation means; The labeled sample has a plurality of different types by attaching different types of fluorescent dyes to a sample that emits autofluorescence by laser light irradiation,
The fluorescence emitted from at least one of the fluorescent dyes and the autofluorescence emitted from the sample are partially overlapped with each other in the wavelength region by irradiation of the labeled sample with laser light. 11. The fluorescence intensity calculation apparatus according to 11. When the fluorescent dye is not attached and the sample made of the self-fluorescent sample is referred to as an unlabeled sample, the fluorescence relaxation when the autofluorescence is assumed to be a first-order delayed relaxation response for the unlabeled sample. A first calibration means for obtaining a time constant and a gain constant;
The first calibration means collects detection values including phase information from each detection sensor by irradiating the label-free sample with a laser beam time-modulated at the predetermined frequency as a measurement object, Obtain the fluorescence relaxation time constant and gain constant of the autofluorescence emitted from the unlabeled sample from the detection value,
13. The fluorescence intensity calculation device according to claim 12, wherein the matrix creation means creates the correction conversion matrix using the fluorescence relaxation time constant and gain constant obtained by the first calibration means. Second calibration for obtaining the fluorescence relaxation time constant and gain constant for each of the labeled sample types, assuming that the fluorescence emitted by the fluorescent dye is a first order lag relaxation response Having means,
This second calibration means uses a labeled sample attached to a sample in which one of the fluorescent dyes emits autofluorescence as a measurement object, and irradiates laser light time-modulated at the predetermined frequency. The detection values including phase information are collected from each detection sensor, and the fluorescence relaxation time constant and gain constant of the fluorescence emitted by the fluorescent dye of the labeled sample are obtained from these detection values, and the fluorescence relaxation time constant and gain constant are obtained. The fluorescence relaxation time constant and the gain constant of the fluorescence emitted by all the fluorescent dyes contained in the labeled sample are obtained while changing the type of the fluorescent dye attached to the autofluorescent sample,
The fluorescence intensity calculation device according to claim 11 or 12, wherein the matrix creation means creates the correction conversion matrix using a fluorescence relaxation time constant and a gain constant in the labeled sample obtained by the second calibration means. .

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