JP5324487B2 - Calibration apparatus for fluorescence detection, calibration method for fluorescence detection, and fluorescence detection apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To calibrate a fluorescence detector constituted so as to perform the accurate detection of fluorescence when the fluorescence emitted from a measuring target is detected by receiving the irradiation of a laser beam. <P>SOLUTION: When the fluorescence detector is calibrated, first, the transmission of the electric signal which is generated from the measuring laser beam modulated in intensity using a modulation signal by photoelectric conversion is delayed. A light detection element detects the calibrating laser beam outputted on the basis of the electric signal delayed in transmission to output a calibrating light detection signal. Furthermore, the calibrating light detection signal is mixed with the modulation signal to generate calibrating light detection data containing data of a phase and intensity. Thereafter, a calibrating phase delay to the modulation signal of the calibrating light detection signal is calculated and a phase delay caused by a transmitting delay time of the electric signal is subtracted from the calculated calibrating phase delay to calculate a system phase delay. When fluorescence is detected, the system phase delay is used to correct the measured phase delay. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a fluorescence detection calibration apparatus used in a fluorescence detection apparatus that detects fluorescence emitted from a measurement object by being irradiated with a laser beam, a fluorescence detection calibration method, and a fluorescence detection apparatus that implements this calibration method. About.

  In the medical and biological fields, flow cytometers are widely used. This flow cytometer uses a photoelectric converter such as a photomultiplier tube or an avalanche photodiode to receive fluorescence emitted from a measurement object by irradiating a laser beam, and the types of biological substances such as cells and genes Analyze the frequency and also the characteristics of the measurement object.

  Specifically, a flow cytometer is used to measure a measurement object made by labeling a biological substance such as a cell, DNA, RNA, enzyme, protein or the like with a fluorescent reagent within about 10 m per second by applying pressure. The laminar sheath flow is formed by flowing into the sheath liquid flowing through the pipe line at a speed of 2 mm. When the flow cytometer irradiates the measurement object in the flow with laser light, the flow cytometer receives the fluorescence emitted by the fluorescent dye attached to the analysis object and identifies this fluorescence as the label of the analysis object. By doing so, the analysis object is specified.

This flow cytometer can measure, for example, the relative amounts of intracellular DNA, RNA, enzymes, proteins, and the like, and can analyze their functions in a short time. Further, the flow cytometer discriminates a predetermined type of cell or chromosome by fluorescence, and selects and collects only the identified cell or chromosome in a living state in a short time using a cell sorter or the like.
In the use of such a flow cytometer, it is required to specify more measurement objects accurately from fluorescent information in a short time.

For example, when signal processing is performed by receiving fluorescence from a sample by irradiating a sample that is a measurement object, signal processing can identify many types of fluorescence, especially in a short time. A fluorescence detection apparatus for efficiently identifying fluorescence is known (Patent Document 1).
For example, a fluorescence detection apparatus includes a laser light source unit that emits laser light that irradiates a sample to be measured, a light source that receives fluorescence emitted from the sample irradiated with the laser light, and outputs a fluorescence signal thereof, and a laser light source. A control / processing unit that generates a modulation signal of a predetermined frequency to time-modulate the intensity of the laser light emitted from the unit, and the fluorescence signal output from the light receiving unit, using the modulation signal, the fluorescence of the measurement object And an analyzer for calculating the fluorescence relaxation time.

JP 2006-226698 A

The fluorescence detection apparatus obtains a phase delay of the fluorescence signal with respect to the modulation of the laser light, and calculates a fluorescence relaxation time based on the phase delay. However, since the phase delay that is the basis for calculating the fluorescence relaxation time includes the phase delay of the fluorescence signal caused by the structure of the fluorescence detection device itself, it is necessary to correct the calculated phase delay.
The phase delay due to the structure of the fluorescence detection device itself is, for example, a phase shift caused by the transmission time for transmitting the optical path of the laser beam, the transmission time of the fluorescence signal transmitting through the transmission line, or the modulation signal used for emitting the laser beam. And a phase shift between the modulated signal used for detection of the fluorescence signal and the like. For this reason, the fluorescence detection apparatus performs correction to remove these phase shifts.

  For the correction, for example, when a fluorescence detection device is used to measure the fluorescence relaxation time with a known fluorescence dye, the calibration relaxation time is set so that the fluorescence relaxation time obtained by the measurement matches the known fluorescence relaxation time. For this purpose, a method for obtaining the reference phase delay can be considered. Specifically, the reference phase lag is subtracted from the fluorescence data obtained in the fluorescence detection apparatus, and the reference phase lag is calculated so that the fluorescence relaxation time calculated using the phase lag of the subtraction result becomes a known fluorescence relaxation time. Ask for.

However, the fluorescence relaxation time inherent to the fluorescent dye changes over time, and the fluorescence relaxation time changes depending on the storage conditions of the fluorescent dye. Furthermore, the fluorescence relaxation time also changes depending on the temperature condition when measuring the fluorescence relaxation time. For this reason, it is not always preferable from the viewpoint of reliability to obtain the reference phase delay using a fluorescent dye. Further, when a plurality of light receiving channels are used for detecting a plurality of fluorescence in the fluorescence detection apparatus, it is necessary to obtain a reference phase delay by changing the fluorescent dye for each light receiving channel, and the correction process becomes complicated. Further, since the value of the fluorescence relaxation time of the fluorescent dye varies slightly depending on the measurement method, it may not always be appropriate to use a known fluorescence relaxation time value.
For the above reasons, obtaining the reference phase delay obtained using a known fluorescent dye and using it for correction may not be preferable in calculating an accurate fluorescence relaxation time.

  Therefore, the present invention provides a fluorescence detection calibration method for calibrating a fluorescence detection apparatus so that accurate fluorescence detection can be performed when detecting fluorescence emitted from a measurement object by receiving laser light irradiation, An object of the present invention is to provide a fluorescence detection calibration apparatus and a fluorescence detection apparatus using the calibration apparatus.

One embodiment of the present invention is a fluorescence detection device that processes a fluorescence signal of fluorescence emitted from a measurement object when irradiated with laser light.
The fluorescence detection device is
A light emitting element for emitting intensity-modulated laser light for measurement with a modulation signal of a predetermined frequency;
A light receiving element that receives the fluorescence emitted by the measurement object located at the measurement position by being irradiated with the measurement laser light, and outputs a fluorescence signal;
A first processing unit that generates fluorescence data including phase and intensity information by mixing the fluorescence signal with the modulation signal of the frequency;
By calibrating the fluorescence data using a reference phase delay acquired in advance, a phase delay of the fluorescence with respect to the modulation signal is calculated, and a fluorescence relaxation time of the fluorescence is calculated using the calculated phase delay. A second processing unit;
A calibration unit arranged at the measurement position in place of a measurement object to obtain the reference phase delay, the transmission of the measurement laser light, or the electrical signal photoelectrically converted from the measurement laser light A fluorescence adjustment calibration unit that includes a delay adjustment unit that delays transmission and guides the measurement laser light or the calibration laser light output based on the electrical signal to the light receiving element.

  In the fluorescence detection device, the light receiving element receives the calibration laser light and outputs a calibration light reception signal, and the first processing unit mixes the calibration light reception signal with the frequency modulation signal. Thus, the calibration light reception data including phase information is generated, and the second processing unit calculates a calibration phase delay of the calibration light reception signal with respect to the modulation signal from the calibration light reception data, The reference phase delay is calculated by subtracting the phase delay derived from the fluorescence detection calibration unit including the phase delay derived from the transmission delay time set by the delay adjustment unit from the calculated calibration phase delay. It is preferable to do.

  The delay adjustment unit of the calibration unit for fluorescence detection includes a delay circuit that delays transmission of the electrical signal, and the calibration unit for fluorescence detection receives and photoelectrically converts the measurement laser beam, It is preferable to include a calibration light-receiving element that generates the electrical signal and a calibration light-emitting element that emits the calibration laser light based on the electrical signal delayed by the delay adjustment unit.

  Alternatively, it is also preferable that the delay adjustment unit of the fluorescence detection calibration unit includes a line stretcher that variably adjusts the transmission line length of the electrical signal.

Another embodiment of the present invention is a fluorescence detection calibration apparatus used for a fluorescence detection apparatus that detects fluorescence emitted from a measurement object by receiving a measurement laser beam by a light receiving element.
The calibration apparatus for fluorescence detection includes a delay adjustment unit that takes in the measurement laser light and delays transmission of the measurement laser light or electrical signal photoelectrically converted from the measurement laser light, The measurement laser beam or the calibration laser beam output based on the electrical signal is guided to a light receiving element that receives the fluorescence of the fluorescence detection device.

  In the fluorescence detection calibration apparatus, the delay adjustment unit includes a delay circuit that delays transmission of the electrical signal, and further, a calibration that generates the electrical signal by receiving and photoelectrically converting the measurement laser beam. It is preferable to include a light receiving element for calibration and a light emitting element for calibration that emits a calibration laser beam based on the electrical signal delayed by the delay adjustment unit.

  Alternatively, it is also preferable that the delay adjustment unit includes a line stretcher that variably adjusts the transmission line length of the electrical signal.

Furthermore, another aspect of the present invention is a calibration method used in a fluorescence detection apparatus that processes a fluorescence signal of fluorescence emitted from a measurement object when irradiated with laser light. In that case, the fluorescence detection device,
A light emitting element for emitting intensity-modulated laser light for measurement with a modulation signal of a predetermined frequency;
A light receiving element that receives fluorescence emitted by the measurement object positioned at the measurement position by being irradiated with the measurement laser light, and outputs a fluorescence signal;
A first processing unit that generates fluorescence data including phase and intensity information by mixing the fluorescence signal with the modulation signal of the frequency;
By calibrating the fluorescence data using a system phase delay acquired in advance, a phase delay of the fluorescence with respect to the modulation signal is calculated, and a fluorescence relaxation time of the fluorescence is calculated using the calculated phase delay. And a second processing unit.
When calibrating fluorescence detection,
A first step of emitting measurement laser light from the light emitting element;
A second step of delaying transmission of the measurement laser beam or transmission of an electrical signal photoelectrically converted from the measurement laser beam;
A third step in which the light-receiving element receives the calibration laser beam output based on the measurement laser beam delayed in transmission or the electrical signal and outputs a calibration light-receiving signal;
A fourth step in which the first processing unit generates calibration light reception data including phase and intensity information by mixing the calibration light reception signal with the modulation signal of the frequency;
The second processing unit calculates a calibration phase delay of the calibration light reception signal with respect to the modulation signal from the calibration light reception data, and is set by the delay adjustment unit from the calculated calibration phase delay. A fifth step of calculating the system phase lag by subtracting the phase lag in the second step and the third step including the phase lag derived from the transmission delay time, and the second processing unit Calibrating the fluorescence data using the system phase lag.

  In the fluorescence detection calibration method and the fluorescence detection calibration apparatus of the above aspect, an accurate reference phase delay can be calculated. Therefore, the fluorescence detection apparatus of the above aspect can perform accurate fluorescence detection.

It is a schematic block diagram of the flow cytometer using the fluorescence detection apparatus of this embodiment. It is a figure which shows an example of a structure of the laser light source part used for the flow cytometer shown in FIG. It is a schematic block diagram which shows the schematic structure of an example of the light-receiving part used for the flow cytometer shown in FIG. It is a figure which shows the structure of the outline of the control and the process part used for the flow cytometer shown in FIG. It is a figure which shows the schematic structure of the analyzer used for the flow cytometer shown in FIG. It is a figure explaining the correction | amendment performed with the analyzer shown in FIG. (A), (b) is a figure which shows the schematic structure of the calibration apparatus for fluorescence detection of this embodiment. It is a figure explaining an example of calculation of the data for calibration used with the analyzer shown in FIG. It is a figure explaining the coaxial line stretcher used instead of the delay circuit shown in FIG.7 (b).

  Hereinafter, a fluorescence detection calibration apparatus, a fluorescence detection calibration method, and a fluorescence detection apparatus according to the present invention will be described in detail.

(Fluorescence detector)
FIG. 1 is a schematic configuration diagram of a flow cytometer 10 used for calibration of fluorescence detection according to the present embodiment.
In the embodiment described below, a biological substance M having a fluorescent dye suspended in a buffer solution will be described as an object to be measured. However, in addition to the biological substance M, a predetermined fluorescent dye is provided, and the biological substance M and the biological binding are provided. It is also possible to include the microbeads to be included in the buffer solution together with the biological material M, and use the biological material M and the microbeads as the measurement object.

  The flow cytometer 10 includes a signal processing device 20 and an analysis device (computer) 80. The signal processing device 20 irradiates a sample 12 configured by suspending a biological material M that emits fluorescence upon receiving laser light irradiation in a buffer solution, and the fluorescent dye included in the biological material M in the sample 12. Signal processing is performed by detecting the fluorescence signal emitted by The analysis device (computer) 80 calculates the fluorescence intensity and fluorescence relaxation time of the fluorescence detected from the processing result obtained by the signal processing device 20, and analyzes the measurement object in the sample 12 using this calculation result. .

The signal processing device 20 includes a laser light source unit 22, light receiving units 24 and 26, a control / processing unit 28, and a conduit 30.
The control / processing unit 28 includes a control unit that controls to generate a modulation signal that modulates the intensity of the laser light from the laser light source unit 22 at a predetermined modulation frequency, and a signal processing unit that identifies the fluorescence signal from the sample 12. Including. The pipe line 30 forms a flow cell by flowing the sample 12 made of a buffer solution containing the biological material M into a sheath solution that forms a high-speed flow. A recovery container 32 is provided at the outlet of the conduit 30. The flow cytometer 10 may be configured such that a cell sorter for identifying and separating the biological material M in the sample 12 is arranged within a short time by laser light irradiation and separated into separate collection containers. it can.

  The laser light source unit 22 emits laser light having a predetermined wavelength, for example, laser light having a wavelength of λ = 405 nm. A lens system 23 is provided so that the laser beam is focused at a predetermined position in the pipe 30. This focusing position forms the measurement position of the sample 12.

FIG. 2 is a diagram illustrating an example of the configuration of the laser light source unit 22.
The laser light source unit 22 emits intensity-modulated laser light. The laser light source unit 22 includes, for example, a light source 22a that emits blue laser light B as CW (continuous wave) laser light and modulates the intensity of the CW laser light at a predetermined frequency.
Further, the laser light source unit 22 includes a lens system 23 and a laser driver 34. The lens system 23 focuses the laser beam on the measurement position in the pipe 30. The laser driver 34 drives the light source 22a.

  For example, a semiconductor laser is used as a light source for emitting these laser beams. The laser beam has an output of about 5 to 100 mW, for example. On the other hand, the frequency (modulation frequency) for modulating the intensity of the laser beam is slightly longer than the fluorescence relaxation time constant, for example, 10 to 50 MHz.

  The light source 22a oscillates in a predetermined wavelength band so that the laser light excites the fluorescent dye to emit fluorescence in a specific wavelength band. The fluorescent dye excited by the laser beam is attached to the biological material M to be measured, and when the biological material M passes through the conduit 30 as a measurement object, the fluorescent pigment is irradiated with the laser beam at the measurement position and specified. Fluoresce at wavelength.

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 biological material M moves the measurement position by the forward scattering of the laser light by the biological material M passing through the measurement position. 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 for notifying the timing at which the biological material M passes through the measurement position in the conduit 30 in the control / processing unit 28.
In addition, when the time when the biological material M passes the measurement position is T, the average diameter of the biological material M is D, the velocity of the biological material M flowing through the flow cell is V, and the spot width at the measurement position of the laser beam is W The time T is T = (D + W) / V. This time T is a known value. The signal processing device 20 starts detection of fluorescence using the trigger signal generated by the light receiving unit 24 as a measurement start timing, and continues measurement for a time 2 · T that is twice the time T. The measurement means that during the period of time T, fluorescence emitted from the sample 12 is received, signal processing is performed by the control / processing unit 28 described later, and phase difference information is supplied to the analyzer 80. By this measurement, when the biological material M passes through the measurement position, the fluorescence signal of the fluorescence received by the light receiving unit 26 is collected.

  On the other hand, the light receiving unit 26 is arranged in a direction perpendicular to the emitting direction of the laser light emitted from the laser light source unit 22 and perpendicular to the moving direction of the sample 12 in the pipe 30. . The light receiving unit 26 includes a photoelectric converter that receives fluorescence emitted from the sample 12 irradiated at the measurement position. FIG. 3 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. 3 includes a lens system 26a that focuses a fluorescent signal from the biological material 12 in the sample 12, dichroic mirrors 26b ₁ and 26b ₂ , bandpass filters 26c _{1 to} 26c _3, and photomultiplier. And photoelectric converters 27a to 27c such as tubes.
The lens system 26a focuses 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 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 emitted by the fluorescent dye. The reflection wavelength band and the transmission wavelength band of the dichroic mirrors 26b ₁ and 26b ₂ are set so that the photoelectric converters 27a to 27c can capture fluorescence in a predetermined wavelength band by filtering by the band pass filters 26c _{1 to} 26c _3. ing.

  The photoelectric converters 27a to 27c are sensors that include a sensor including, for example, 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 phase difference information with respect to the modulation signal of the laser light, the output electric signal is a fluorescence signal having phase difference information. The fluorescence signal is amplified by an amplifier and supplied to the control / processing unit 28.

As illustrated in FIG. 4, 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 generation unit 40 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 the generated modulation signal to the laser driver 34 of the laser light source unit 22, and the signal processing unit 42. To supply. 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 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 processes a signal including phase difference information of fluorescence emitted from the sample 12 by irradiation with laser light, using the fluorescence signals output from the photoelectric converters 27a to 27c. The signal processing unit 42 includes amplifiers 54a to 54c, a power splitter 56, and IQ mixers 58a to 58c. The amplifiers 54a to 54c amplify the fluorescence signals output from the photoelectric converters 27a to 27c. The power splitter 56 distributes a modulated signal that is a sine wave signal supplied from the signal generation unit 40 to each of the amplified fluorescence signals.

  The IQ mixers 58a to 58c mix (synthesize) the fluorescent signals supplied from the photoelectric converters 27a to 27c using the modulation signal supplied from the signal generator 40 as a reference signal. Specifically, each of the IQ mixers 58a to 58c multiplies the reference signal by the fluorescence signal (RF signal), and a processing signal including a cos component (signal component having the same phase as the reference signal) and a high-frequency component of the fluorescence signal. And a signal obtained by shifting the phase of the reference signal by 90 degrees is multiplied by the fluorescence signal to include a sin component of the fluorescence signal (a signal component having a phase difference of 90 degrees with respect to the reference signal) and a high-frequency component. A processing signal is calculated. 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 modulation signal (sine wave signal) having a predetermined frequency, and further, the value of the cos component and the sin component of the fluorescence signal obtained by the signal processing unit 42. This is a part for obtaining the value of the cos component and the value of the sin component of the fluorescence signal by removing the high frequency component from the processing signal including the value.

Specifically, the controller 44 includes a system controller 60, a low-pass filter 62, an amplifier 64, and an A / D converter 66.
The system controller 60 gives instructions for operation control of each part of the signal processing device 20 and manages all operations of the flow cytometer 10. The low pass filter 62 removes the high frequency component from the processed signal obtained by adding the high frequency component to the cos component (Re component) and the sin component (Im component) calculated by the signal processing unit 42. The amplifier 64 amplifies the processing signal of the cos component (Re component) and the sin component (Im component) from which the high frequency component has been removed. The A / D converter 66 samples the amplified processing signal.
The system controller 60 determines the oscillation frequency of the oscillator 46 for intensity modulation of the laser light.
The controller 44 supplies the digitized cos component (Re component) and sin component (Im component) processing signals to the analyzer 80.

  The analyzer 80 obtains the fluorescence intensity and fluorescence relaxation time of the fluorescence emitted by the biological material M, and identifies the type of fluorescence of the biological material M in the sample 12 using the fluorescence intensity and fluorescence relaxation time. It is also possible to examine the characteristics of the biological material M, such as whether or not the biological material M is biologically bound, by including microbeads with a predetermined fluorescent dye attached to the sample 12.

FIG. 5 is a diagram illustrating a configuration of the analysis device 80.
The analysis device 80 is configured by a computer having a CPU 82 and a memory 84. By starting the software stored in the memory 84, each part of the analyzer 80 is formed as a software module. Specifically, the analysis device 80 includes, as software modules, a fluorescence intensity calculation unit 86, a phase delay calculation unit 88, a fluorescence relaxation time calculation unit 90, and a calibration data calculation unit 92. Of course, these parts can also be constituted by dedicated circuits.

The fluorescence intensity calculation unit 86 obtains the absolute value of a complex number (= (Re component: value of cos component) + j (Im component: sin component)) for the fluorescence data supplied from the A / D converter 66. The fluorescence intensity is calculated.
The phase delay calculation unit 88 calculates the complex argument (tan ⁻¹ (Im component of fluorescence data / Re component of fluorescence data)) of the fluorescence data supplied from the A / D converter 66 as the phase delay θ _meas. To do. Further, as shown in FIG. 6, the phase lag calculation unit 88 subtracts the system phase lag θ _sys (calibration data) obtained in advance by a method described later from the calculated phase lag θ _meas to obtain a fluorescent dye. The phase lag θ _fl when the _light is fluorescent is calculated. The system phase delay θ _sys is supplied from the calibration data calculation unit 92.
The fluorescence relaxation time calculation unit 90 calculates the fluorescence relaxation time τ according to the equation of τ = 1 / (2πf) · tan (θ _fl ) using the phase delay θ _fl calculated by the phase delay calculation unit 88. Here, f is a modulation frequency used for intensity modulation of laser light. The reason why the fluorescence relaxation time τ can be calculated according to the equation of τ = 1 / (2πf) · tan (θ _fl ) is that the fluorescence phenomenon shows a change along the first-order relaxation process.
The fluorescence intensity calculated by the fluorescence intensity calculator 86 and the fluorescence relaxation time calculated by the fluorescence relaxation time calculator 90 are output to an output device such as a printer or a display (not shown). Further, it is used for analysis of an analysis object.

The system phase delay θ _sys used in the above-described phase delay calculation unit 88 is data that is calculated and acquired by the calibration data calculation unit 92 by fluorescence detection calibration described later, and is stored in the memory 84.
The acquisition of the system phase delay θ _sys is performed by setting the pipe line 30 to a measurement position of the pipe line 30 instead of a fluorescent detection calibration apparatus 100 described later and executing a fluorescent detection calibration method described later.

(Calibration device for fluorescence detection)
FIG. 7A is a diagram showing a mode in which a fluorescence detection calibration device 100 is arranged instead of the pipe line 30 of the flow cytometer 10, and FIG. 7B is a configuration of the fluorescence detection calibration device 100. FIG.
The fluorescence detection calibration apparatus 100 takes in intensity-modulated laser light emitted from the laser light source unit 22 of the flow cytometer 10. Further, the calibration apparatus 100 for fluorescence detection emits laser light whose phase is delayed with respect to the intensity modulation of the captured laser light toward the light receiving unit 26.

  Specifically, the fluorescence detection calibration apparatus 100 includes a mirror 102, a light receiving element 104, a delay circuit 106, an amplifier 108, and a laser light source unit 110. Hereinafter, the laser light emitted from the laser light source unit 22 is referred to as measurement laser light, and the laser light emitted from the laser light source unit 110 is referred to as calibration laser light.

  The fluorescence detection calibration apparatus 100 converts the measurement laser beam emitted from the laser light source unit 22 into an electrical signal, delays the transmission of the electrical signal, and then emits the calibration laser beam based on the electrical signal. The calibration laser beam is received by the light receiving unit 26. Since the measurement laser light emitted from the laser light source unit 22 is intensity-modulated at a predetermined frequency, the electrical signal obtained by photoelectric conversion is an electrical signal that vibrates at the same frequency as the modulation signal. Further, the calibration laser beam emitted based on the electric signal is also intensity-modulated at the same frequency as the modulation signal. Of course, since the transmission of the electrical signal is delayed, the modulation of the calibration laser light emitted based on the electrical signal is also delayed.

The mirror 102 reflects the measurement laser light emitted from the laser light source unit 22 and directs it toward the light receiving element 104.
The light receiving element 104 converts the received measurement laser light into an electrical signal by photoelectric conversion. As the light receiving element 104, for example, a photodiode or the like is used. Since the received measurement laser beam is intensity-modulated at the same frequency as the modulation signal, the converted electric signal is a signal having the same frequency as the modulation signal.
The delay circuit 106 is a passive circuit that delays transmission of an input electric signal by a set delay time and outputs the delayed signal. The delay time can be adjusted freely. As the delay circuit 106, for example, a circuit that freely changes the line length of a transmission line such as a coaxial cable or a coaxial line stretcher can be used. Alternatively, a known circuit configured using a capacitor and an inductor can be used. Information on the delay time set by the delay circuit 106 is provided to the calibration data calculation unit 92 of the analyzer 80.
The amplifier circuit 108 amplifies the delayed electrical signal.

The laser light source unit 110 emits calibration laser light whose intensity is modulated based on the amplified electrical signal. The laser light source unit 110 emits laser light having a wavelength substantially equal to the fluorescence wavelength of the fluorescent dye used for fluorescence detection. Accordingly, the phase delay of the intensity-modulated calibration laser beam emitted from the fluorescence detection calibration device 100 can be calculated using the light receiving unit 26, the control processing unit 28, and the analysis device 80 of the flow cytometer 10. .
In the case of performing fluorescence detection using a plurality of fluorescent dyes, the laser light source unit 110 has a plurality of laser light sources so as to emit laser light having substantially the same wavelength as each of the fluorescent wavelengths of these fluorescent dyes. Also good.

Hereinafter, a fluorescence detection calibration method using the fluorescence detection calibration apparatus 100 will be specifically described.
First, the fluorescence detection calibration apparatus 100 makes the intensity-modulated measurement laser light incident from the laser light source unit 22. The light receiving element 104 receives this measurement laser beam and outputs an electric signal obtained by photoelectric conversion. Further, the delay circuit 106 delays transmission of the electric signal output from the light receiving element 104 by a set delay time. The laser light source unit 110 emits calibration laser light whose intensity is modulated based on an electrical signal whose transmission is delayed. The calibration laser beam is received by the light receiving unit 26. As a result, the control / processing unit 28 processes the calibration light reception signal of the calibration laser light received and output by the light receiving unit 26 in the same manner as the fluorescence signal, as in the case of detecting the fluorescence of the sample 12. be able to.

The signal processing unit 42 of the control / processing unit 28 mixes the calibration light reception signal together with the modulation signal provided from the signal generation unit 40. The controller 44 generates light reception data for calibration composed of digitized cos component (Re component) and sin component (Im component) as in the case of the processing of the fluorescence signal, and provides it to the analyzer 80.
The phase delay calculation unit 88 of the analyzer 80 calibrates the declination (tan ⁻¹ (Im component of calibration light reception data / Re component of calibration light reception data)) from the calibration light reception data provided from the controller 44. Calculated as phase delay θ _meas . The calibration phase delay θ _meas is provided to the calibration data calculation unit 92.
The calibration data calculation unit 92 of the analysis device 80 calculates the system phase delay θ _sys using the calibration phase delay θ _meas and the delay time information provided from the delay circuit 106 of the fluorescence detection calibration device 100. To do.

FIG. 8 is a diagram for explaining an example of calculation of the system phase delay θ _sys .
The calibration data calculation unit 92 subtracts the phase delay θ _ref derived from the fluorescence detection calibration device 100 from the calibration phase delay θ _meas provided from the phase delay calculation unit 88, so that the system of the flow cytometer 10 _{Find the} phase delay θ _{sys due} to. Here, the phase delay θ _ref derived from the fluorescence detection calibration apparatus 100 includes, in addition to the phase delay θ _del derived from the delay time in the delay circuit 106, a phase delay θ _opt derived from the propagation of light in the optical path, The phase delay θ _las derived from the rising operation of the laser oscillation of the laser light source unit 110, the phase delay θ _pd derived from the rising of the output of the light reception signal of the light receiving element 104, and the like are included. The phase delay θ _del can be obtained in advance by the deflection angle tan ⁻¹ (2πfτ) (f is the frequency of the modulation signal and τ is the delay time provided from the delay circuit 106). The sum of the phase delays θ _opt , θ _las , and θ _pd is a constant value that does not change even if the delay time of the delay circuit 106 changes. Therefore, the amount of change in the calibration phase delay θ _meas is obtained by changing the delay time of the delay circuit 106, and the change in the delay time is determined using the amount of change in the delay time and the amount of change in the calibration phase delay θ _meas. Thus, the total value of the phase delays θ _opt , θ _las , θ _pd whose values do not change can be obtained. Therefore, the phase delay θ _ref caused by the fluorescence detection calibration apparatus 100 is known.
Therefore, the calibration data calculation unit 92 calculates the phase delay so that the calibration phase delay θ _meas calculated using the fluorescence detection calibration device 100 is equal to the phase delay θ _ref caused by the fluorescence detection calibration device 100. The system phase delay θ _sys used in the unit 88 is calculated. That is, the calibration data calculation unit 92 calculates θ _sys = θ _meas −θ _ref . The calculated system phase delay θ _sys is stored in the memory 84.
At the time of fluorescence detection, the calibration data calculation unit 92 calls the system phase delay θ _sys stored in the memory 84 from the memory 64 and supplies it to the phase delay calculation unit 88 as described above. As shown in FIG. 6, the phase delay calculation unit 88 uses the system phase delay θ _sys when calculating the phase delay θ _fl of the fluorescence derived from the fluorescent dye itself from the calculated phase delay θ _meas . That is, the system phase delay θ _sys is used to calculate θ _fl = θ _meas −θ _sys .

As described above, the analysis device 80 calculates the system phase delay θ _sys using the calibration device 100 for fluorescence detection, and further calibrates the fluorescence data using the system phase delay θ _sys , so that the accurate fluorescence can be obtained. The fluorescence relaxation time can be determined, and therefore accurate fluorescence detection can be performed.

In this embodiment, a delay circuit that delays an electric signal is used. However, a coaxial line stretcher 107 that mechanically changes the coaxial tube length as shown in FIG. 9 can be used. In this case, by adjusting the coaxial tube length of the simultaneous line stretcher 107 with an accuracy of μm, the transmission delay time can be adjusted with an accuracy of picoseconds.
In addition, unlike fluorescent dyes having a known fluorescence relaxation time, the fluorescence detection calibration apparatus 100 operates based on a certain delay time without being affected by the surrounding environment such as storage conditions and temperature conditions. The calibration apparatus 100 can obtain an accurate system phase delay θ _sys .
Since the calibration laser light emitted from the laser light source unit 110 has a wavelength substantially equal to the fluorescence wavelength of the fluorescent dye used for fluorescence detection, the fluorescence detection calibration apparatus 100 is an apparatus that is not affected by the wavelength of the laser light. In addition, since the fluorescence detection calibration apparatus 100 can freely adjust the delay time in the delay circuit 106, an accurate system can be obtained by adjusting the delay time even if there is a large difference in the fluorescence relaxation times of a plurality of fluorescent dyes. The phase delay θ _sys can be obtained.

  Furthermore, in the fluorescence detection calibration apparatus 100 of the present embodiment, the measurement laser light is once converted into an electrical signal, the transmission of this electrical signal is delayed, and then the calibration laser light is based on the delayed electrical signal. Is emitted. However, after adjusting the optical path using an optical element including a mirror or the like so as to change the optical path length of the measuring laser beam without converting the measuring laser beam into an electrical signal, the laser beam is applied to the light receiving unit 26. You may guide. In this case, the delay circuit 106, the amplifier 108, and the laser light source unit 110 are unnecessary.

  The fluorescence detection calibration apparatus, the fluorescence detection calibration method, and the fluorescence detection apparatus according to the present invention have been described in detail above. However, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. Of course, improvements and changes may be made.

10 flow cytometer 12 sample 20 signal processing device 22,110 laser light source unit 22a source 23,26a lens system 24, 26 light receiving unit 26b _1, 26b ₂ dichroic mirror 26c _1, 26c _2, 26c ₃ band pass filters 27a, 27b, 27c Photoelectric Converter 28 Control / Processing Unit 30 Pipeline 32 Collection Container 34 Laser Driver 40 Signal Generation Unit 42 Signal Processing Unit 44 Controller 46 Oscillator 48, 56 Power Splitter 50, 52, 54a, 54b, 54c, 64 Amplifier 58a, 58b 58c IQ mixer 60 System controller 62 Low pass filter 66 A / D converter 80 Analyzer 82 CPU
84 Memory 86 Fluorescence Intensity Calculation Unit 88 Phase Delay Calculation Unit 90 Fluorescence Relaxation Time Calculation Unit 92 Calibration Data Calculation Unit 100 Fluorescence Detection Calibration Device 102 Mirror 104 Light Receiving Element 106 Delay Circuit 108 Amplifier

Claims (8)

A fluorescence detection apparatus for processing a fluorescence signal of fluorescence emitted from a measurement object by receiving laser light irradiation,
A light emitting element for emitting intensity-modulated laser light for measurement with a modulation signal of a predetermined frequency;
A light receiving element that receives the fluorescence emitted by the measurement object located at the measurement position by being irradiated with the measurement laser light, and outputs a fluorescence signal;
A first processing unit that generates fluorescence data including phase and intensity information by mixing the fluorescence signal with the modulation signal of the frequency;
By calibrating the fluorescence data using a system phase delay acquired in advance, a phase delay of the fluorescence with respect to the modulation signal is calculated, and a fluorescence relaxation time of the fluorescence is calculated using the calculated phase delay. A second processing unit;
A calibration unit arranged at the measurement position in place of the measurement object to obtain the system phase delay, the transmission of the measurement laser light, or the electrical signal photoelectrically converted from the measurement laser light A fluorescence adjustment calibration unit that includes a delay adjustment unit that delays transmission and guides the measurement laser beam or the calibration laser beam output based on the electrical signal to the light receiving element. Fluorescence detection device. The light receiving element receives the calibration laser light and outputs a calibration light reception signal;
The first processing unit generates calibration light reception data including phase information by mixing the calibration light reception signal with the frequency modulation signal,
The second processing unit calculates a calibration phase delay of the calibration light reception signal with respect to the modulation signal from the calibration light reception data, and is set by the delay adjustment unit from the calculated calibration phase delay. The fluorescence detection device according to claim 1, wherein the system phase delay is calculated by subtracting a phase delay derived from a fluorescence detection calibration unit including a phase delay derived from a transmission delay time. The delay adjustment unit of the calibration unit for fluorescence detection includes a delay circuit that delays transmission of the electrical signal,
The fluorescence detection calibration unit receives the measurement laser light and photoelectrically converts the calibration light receiving element to generate the electrical signal, and the calibration laser based on the electrical signal delayed by the delay adjustment unit. The fluorescence detection apparatus according to claim 1, further comprising: a calibration light emitting element that emits light.   The fluorescence detection apparatus according to claim 1, wherein the delay adjustment unit of the fluorescence detection calibration unit includes a line stretcher that variably adjusts a transmission line length of the electrical signal. A fluorescence detection calibration device used in a fluorescence detection device that detects fluorescence emitted from a measurement object by receiving irradiation of a measurement laser beam with a light receiving element,
A delay adjustment unit that takes in the measurement laser light and delays transmission of the measurement laser light or electrical signal photoelectrically converted from the measurement laser light; A calibration apparatus for fluorescence detection, characterized in that a calibration laser beam output based on a signal is guided to a light receiving element that receives fluorescence of the fluorescence detection apparatus. The delay adjustment unit includes a delay circuit that delays transmission of the electrical signal,
Furthermore, a calibration light receiving element that generates the electrical signal by receiving and photoelectrically converting the measurement laser light, and a calibration light emission that emits the calibration laser light based on the electrical signal delayed by the delay adjustment unit The fluorescence detection calibration apparatus according to claim 5, further comprising an element.   The said delay adjustment part is a calibration apparatus for fluorescence detection of Claim 5 provided with the line stretcher which adjusts the transmission line length of the said electrical signal variably. A calibration method used for a fluorescence detection apparatus, which processes a fluorescence signal of fluorescence emitted from a measurement object by receiving laser light irradiation,
The fluorescence detection device comprises:
A light emitting element for emitting intensity-modulated laser light for measurement with a modulation signal of a predetermined frequency;
A light receiving element that receives fluorescence emitted by the measurement object positioned at the measurement position by being irradiated with the measurement laser light, and outputs a fluorescence signal;
A first processing unit that generates fluorescence data including phase and intensity information by mixing the fluorescence signal with the modulation signal of the frequency;
By calibrating the fluorescence data using a system phase delay acquired in advance, a phase delay of the fluorescence with respect to the modulation signal is calculated, and a fluorescence relaxation time of the fluorescence is calculated using the calculated phase delay. A second processing unit,
When calibrating fluorescence detection,
A first step of emitting measurement laser light from the light emitting element;
A second step of delaying transmission of the measurement laser beam or transmission of an electrical signal photoelectrically converted from the measurement laser beam;
A third step in which the light-receiving element receives the calibration laser beam output based on the measurement laser beam delayed in transmission or the electrical signal and outputs a calibration light-receiving signal;
A fourth step in which the first processing unit generates calibration light reception data including phase and intensity information by mixing the calibration light reception signal with the modulation signal of the frequency;
The second processing unit calculates a calibration phase delay of the calibration light reception signal with respect to the modulation signal from the calibration light reception data, and is set by the delay adjustment unit from the calculated calibration phase delay. A fifth step of calculating the system phase lag by subtracting the phase lag in the second step and the third step including a phase lag derived from the delayed transmission time,
And a sixth step of calibrating the fluorescence data using the system phase delay in the second processing unit.