WO2010092752A1 - 蛍光検出装置及び蛍光検出方法 - Google Patents
蛍光検出装置及び蛍光検出方法 Download PDFInfo
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- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims description 13
- 238000005259 measurement Methods 0.000 claims abstract description 58
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- 238000012935 Averaging Methods 0.000 abstract 1
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- 238000004458 analytical method Methods 0.000 description 6
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
- G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1429—Signal processing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N2015/0038—Investigating nanoparticles
Definitions
- the present invention relates to a fluorescence detection apparatus that receives fluorescence emitted from a measurement object by irradiating the measurement object with laser light and performs signal processing of a fluorescence signal obtained at this time.
- the present invention also relates to a fluorescence detection method for receiving fluorescence emitted from a measurement object by irradiating the measurement object with laser light and performing signal processing of a fluorescence signal obtained at this time.
- an analyzer for analyzing a measurement object such as a flow cytometer used in the medical and biological fields, using a fluorescence emitted by a fluorescent dye to identify a measurement object such as a cell or DNA or RNA.
- the present invention relates to an applied fluorescence detection device.
- a flow cytometer used in the medical and biological fields incorporates a fluorescence detection device that receives fluorescence from a fluorescent dye of a measurement object by irradiating laser light and identifies the type of the measurement object. .
- a flow cytometer labels biological substances such as cells, DNA, RNA, enzymes, proteins, etc. in a turbid solution with a fluorescent reagent, and applies pressure to flow through the pipeline at a speed of about 10 m / sec.
- the measurement object is poured into the sheath liquid. Thereby, a laminar sheath flow is formed.
- the fluorescent light emitted from the fluorescent dye attached to the measurement object is received, and the measurement object is identified by identifying this fluorescence as a label.
- This flow cytometer can measure, for example, the relative amounts of intracellular DNA, RNA, enzymes, proteins, and the like, and can analyze these functions in a short time.
- a cell sorter or the like that identifies a predetermined type of cell or chromosome by fluorescence and selects and collects only the identified cell or chromosome in a live state is used.
- a fluorescent dye is previously attached to the biological material with a fluorescent reagent.
- This biological material includes microbeads having a diameter of 5 to 20 ⁇ m, which are labeled with a fluorescent dye different from the fluorescent dye attached to the microbeads described later, and have a specific structure such as a carboxyl group on the surface. Mixed in liquid. The structure such as the carboxyl group acts on (couples) a biological material having a certain known structure. Therefore, when the fluorescence from the microbead and the fluorescence of the biological material are detected at the same time, it can be seen that the biological material is bound to the structure of the microbead. Thereby, the characteristic of a biological substance can be analyzed. In order to prepare a variety of microbeads having a variety of coupling structures and analyze the characteristics of biological materials in a short time, a very wide variety of fluorescent dyes are required.
- Patent Document 1 describes that microbeads or the like are used as an object to be measured, and this object to be measured is irradiated with laser light whose intensity is modulated at a predetermined frequency, and the fluorescence relaxation time of the fluorescence emitted at that time is obtained. Since this fluorescence relaxation time varies depending on the type of fluorescent dye, this fluorescence relaxation time can be used to identify the type of fluorescence and further the type of measurement object.
- Patent Document 1 although fluorescence can be efficiently identified in a short time based on the fluorescence relaxation time, the measurement accuracy of the fluorescence relaxation time is not necessarily high. For example, when the measurement object includes microbeads having a relatively long fluorescence relaxation time such that the fluorescence relaxation time exceeds 20 ns, the measurement accuracy has been lowered.
- An object of the present invention is to provide a fluorescence detection apparatus and a fluorescence detection method that improve the measurement accuracy of the fluorescence relaxation time.
- the fluorescence detection apparatus of the present invention is a fluorescence detection apparatus that receives fluorescence emitted from the measurement object by irradiating the measurement object with laser light, and performs signal processing of the fluorescence signal obtained at this time,
- a laser light source unit that irradiates the measurement target with laser light
- a light receiving unit that outputs a fluorescence signal of fluorescence emitted from the measurement target irradiated with the laser light
- a laser beam emitted from the laser light source unit is a fluorescence detection apparatus that receives fluorescence emitted from the measurement object by irradiating the measurement object with laser light, and performs signal processing of the fluorescence signal obtained at this time.
- the fluorescence detection method of the present invention is a fluorescence detection method for receiving fluorescence emitted from the measurement object by irradiating the measurement object with laser light, and performing signal processing of the fluorescence signal obtained at this time, A step of irradiating the measurement object with laser light, a step of outputting a fluorescence signal of fluorescence emitted from the measurement object irradiated with the laser light, and time-modulating the intensity of the laser light with at least two frequency components Generating a modulation signal, and using the fluorescence signal and the modulation signal, obtaining a fluorescence relaxation time of fluorescence of the measurement object, and obtaining the fluorescence relaxation time, Obtaining a phase delay of the fluorescence signal with respect to the modulation signal in at least two frequency components; and obtaining a fluorescence relaxation time at each frequency using the phase delay; Characterized in that it and a step of determining the average fluorescence relaxation time by performing a weighted average of the fluorescence relaxation time.
- the measurement accuracy of the fluorescence relaxation time can be improved.
- FIG. 1 is a schematic configuration diagram showing an example of a flow cytometer 10 using a fluorescence detection apparatus using intensity-modulated laser light according to the present invention.
- the flow cytometer 10 includes a signal processing device 20 and an analysis device (computer) 80.
- the signal processing device 20 detects a fluorescent fluorescence signal emitted from a fluorescent dye provided in the sample 12 when the sample 12 such as microbeads or cells, which are measurement objects, is irradiated with laser light, and performs signal processing. I do.
- the analysis device (computer) 80 analyzes the measurement object in the sample 12 from the processing result obtained by the signal processing device 20.
- the signal processing device 20 includes a laser light source unit 22, light receiving units 25 and 26, a control / processing unit 28, and a conduit 30.
- 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 modulates the intensity of the laser light from the laser light source unit 22.
- the signal processing unit 42 identifies the fluorescence signal from the sample 12.
- the controller 44 manages all operations of the flow cytometer 10.
- the pipe line 30 flows by including the sample 12 in a sheath liquid that forms a high-speed flow, thereby forming a laminar sheath flow. In this flow, for example, the channel diameter is 100 ⁇ m, and the flow velocity is 1 to 10 m / sec.
- the sphere diameter of the microbeads is several ⁇ m to 30 ⁇ m.
- a recovery container 32 is provided at the outlet of the conduit 30.
- a cell sorter for separating a biological substance such as a specific cell in the sample 12 can be arranged within a short time by irradiation with laser light, and separated into separate collection containers.
- the laser light source unit 22 emits laser light whose intensity is modulated at a predetermined frequency.
- the laser light source unit 22 is provided with a lens system so as to be focused at a predetermined position in the pipe line 30.
- the sample 12 is measured at a position (measurement point) where the laser beam is focused.
- FIG. 2 is a schematic configuration diagram showing an example of the configuration of the laser light source unit 22.
- the laser light source unit 22 includes a light source 23, a lens system 24a, and a laser driver 34.
- the light source 23 emits CW (continuous wave) laser light having a constant intensity, and modulates the intensity of the CW laser light to emit laser light.
- the lens system 24 a focuses the laser light emitted from the light source 23 on a measurement point in the pipe 30.
- the laser driver 34 drives the light source 23.
- the light source 23 that emits laser light is, for example, a semiconductor laser.
- the output of the laser beam is, for example, about 5 to 100 mW.
- the wavelength of the laser light is, for example, a visible light band of 350 nm to 800 nm.
- the laser driver 34 is connected to the control / processing unit 28. In addition, the laser driver 34 generates a drive signal for intensity-modulating the laser light using a modulation signal composed of at least two frequencies, and supplies the drive signal to the light source 23.
- Fluorescent dye excited by laser light is attached to a sample 12 (measurement object) such as a biological material or microbeads.
- a sample 12 such as a biological material or microbeads.
- the sample 12 emits fluorescence upon receiving laser light irradiation at the measurement point.
- the laser beam is emitted after being intensity-modulated at two frequencies.
- the light receiving unit 25 is disposed so as to face the laser light source unit 22 with the duct 30 interposed therebetween.
- the light receiving unit 25 includes a photoelectric converter that outputs a detection signal indicating that the sample 12 passes through the measurement point when the laser beam is scattered forward by the sample 12 passing through the measurement point.
- a signal output from the light receiving unit 25 is supplied to the control / processing unit 28.
- the signal output from the light receiving unit 25 is used as a trigger signal informing the timing at which the sample 12 passes the measurement point in the pipe 30 in the control / processing unit 28.
- 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. Yes.
- the light receiving unit 26 includes a photoelectric converter that receives fluorescence emitted from the sample 12 irradiated with laser light at a measurement point.
- FIG. 3 is a schematic configuration diagram illustrating an example of the light receiving unit 26.
- the light receiving unit 26 includes a lens system 24 b and a photoelectric converter 27.
- the lens system 24b focuses the fluorescence signal from the sample 12.
- the lens system 24 b is configured to focus the fluorescence incident on the light receiving unit 26 on the light receiving surface of the photoelectric converter 27.
- the photoelectric converter 27 includes, for example, a photomultiplier tube, and converts light received by the photocathode into an electrical signal.
- the electrical signal (fluorescence signal) converted by the photoelectric converter 27 is supplied to the control / processing unit 28.
- FIG. 4 is a schematic configuration diagram illustrating an example of the control / processing unit 28.
- the control / processing unit 28 includes a signal generation unit 40, a signal processing unit 42, and a controller 44.
- a light source control unit that generates a modulation signal for modulating the intensity of the laser light is formed by the signal generation unit 40 and the controller 44.
- the signal generator 40 includes oscillators 46a and 46b, power splitters 48a and 48b, a power divider 48c, and amplifiers 50a, 50b, and 50c.
- the signal generation unit 40 generates a modulation signal, supplies the modulation signal to the laser driver 34 of the laser light source unit 22, and supplies the modulation signal to the signal processing unit 42.
- the reason why the modulation signal is supplied to the signal processing unit 42 is to use it as a reference signal for detecting the fluorescence signal output from the photoelectric converter 27, as will be described later.
- the oscillators 46a and 46b output sine wave signals having different frequencies.
- the frequency of the sine wave signal is set in the range of 1 to 50 MHz, for example.
- the sine wave signal having the frequency f 1 (angular frequency ⁇ 1 ) output from the oscillator 46a is distributed to the power divider 48c and the amplifier 50a by the power splitter 48a.
- the sine wave signal of frequency f 2 (angular frequency ⁇ 2 ) output from the oscillator 46b is distributed to the power divider 48c and the amplifier 50b by the power splitter 48b.
- the sine wave signals distributed from the power splitters 48a and 48b to the power divider 48c are combined by the power divider 48c to generate a modulation signal.
- the modulation signal generated by the power divider 48c is amplified by the amplifier 50c and then supplied to the laser driver 34.
- the signal processing unit 42 uses the fluorescence signal output from the photoelectric converter 27 to extract information on the phase delay of the fluorescence emitted from the measurement object such as microbeads when irradiated with the laser beam.
- the signal processing unit 42 includes a power splitter 48d, amplifiers 54 and 55, and IQ mixers 58 and 59.
- the power splitter 48d distributes the fluorescent signal output from the photoelectric converter 27 to the amplifiers 54 and 55.
- the amplifiers 54 and 55 amplify the fluorescence signal distributed from the power splitter 48d and supply the amplified signals to the IQ mixers 58 and 59, respectively.
- the IQ mixer 58 is supplied with a sine wave signal of frequency f 1 supplied from the amplifier 50a as a reference signal. Further, the IQ mixer 59, a sine wave signal supplied frequency f 2 is supplied as a reference signal from the amplifier 50b.
- the IQ mixers 58 and 59 are devices that synthesize the fluorescence signal supplied from the photoelectric converter 27 using the sine wave signals of frequencies f 1 and f 2 supplied from the signal generation unit 40 as reference signals. Specifically, each of the IQ mixers 58 and 59 multiplies the reference signal by the fluorescence signal (RF signal) to calculate a processing signal including the cos component and the high frequency component of the fluorescence signal. Each of the IQ mixers 58 and 59 multiplies the fluorescence signal by a signal obtained by shifting the phase of the reference signal by 90 degrees, and calculates a processing signal including a sin component 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 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 and manages all operations of the flow cytometer 10. Further, the system controller 60 controls the oscillators 46a and 46b of the signal generator 40 to generate a sine wave signal having a predetermined frequency.
- the low-pass filter 62 removes the high frequency component from the processing signal obtained by adding the high frequency component to the cos component and sin component calculated by the signal processing unit 42, and the processing signal of the cos component and sin component of the two frequencies f 1 and f 2. Get.
- the amplifier 64 amplifies the cos component and sin component processing signals.
- the A / D converter 66 samples the amplified processing signal.
- FIG. 5 is a schematic configuration diagram illustrating an example of the analysis apparatus (computer) 80.
- the analyzer 80 is configured by starting a predetermined program on a computer.
- the analyzer 80 includes a phase delay acquisition unit 86, a fluorescence relaxation time acquisition unit 88, a weighting factor acquisition unit 90, an average fluorescence relaxation time formed by starting up software.
- a display 100 is connected to the analyzer 80.
- the CPU 82 is an arithmetic processor provided in the computer.
- the CPU 82 substantially executes various calculations of the phase delay acquisition unit 86, the fluorescence relaxation time acquisition unit 88, the weighting factor acquisition unit 90, and the average fluorescence relaxation time acquisition unit 92.
- the memory 84 is a hard disk or ROM that stores a program that forms a phase delay acquisition unit 86, a fluorescence relaxation time acquisition unit 88, a weighting factor acquisition unit 90, and an average fluorescence relaxation time acquisition unit 92 by being executed on a computer, And a RAM for storing the processing results calculated by these units and the data supplied from the input / output port 94.
- the input / output port 94 receives input of detection values of cos component and sin component corresponding to each of at least two frequency components f 1 and f 2 supplied from the controller 44. Further, the input / output port 94 outputs information on the processing result created by each unit to the display 100.
- the display 100 displays values of processing results such as information on the phase delay of fluorescence, phase relaxation time, weighting factor, average fluorescence relaxation time, and the like obtained by each unit.
- the phase delay acquisition unit 86 determines the frequency component f 1 (angular frequency ⁇ 1 ) from the detected values of the cos component and the sin component corresponding to each of the at least two frequency components f 1 and f 2 supplied from the controller 44. Phase lag ⁇ ⁇ 1 , and phase lag ⁇ ⁇ 2 for the frequency component f 2 (angular frequency ⁇ 2 ) are obtained.
- the fluorescence relaxation time acquisition unit 88 calculates fluorescence relaxation times ⁇ ( ⁇ ⁇ 1 ) and ⁇ ( ⁇ ⁇ 2 ) based on the phase delays ⁇ ⁇ 1 and ⁇ ⁇ 2 determined by the phase delay acquisition unit 86.
- the weighting factor acquisition unit 90 weights each of the fluorescence relaxation times ⁇ ( ⁇ ⁇ 1 ) and ⁇ ( ⁇ ⁇ 2 ) obtained by the fluorescence relaxation time acquisition unit 88 and weights m ( ⁇ ⁇ 1 ) and m ( ⁇ ⁇ 2 ). )
- the weight coefficient is a value between 0 and 1.
- the average fluorescence relaxation time acquisition unit 92 includes the fluorescence relaxation times ⁇ ( ⁇ ⁇ 1 ) and ⁇ ( ⁇ ⁇ 2 ) determined by the fluorescence relaxation time acquisition unit 88, and the weighting factors m ( ⁇ ⁇ 1 ) and m determined by the weighting factor acquisition unit 90. Based on ( ⁇ ⁇ 2 ), an average fluorescence relaxation time ⁇ ave is obtained.
- the fluorescence relaxation time (the above-described average fluorescence relaxation time ⁇ ave ) with high measurement accuracy can be obtained by using the detection values of the fluorescence signals corresponding to each of at least two frequency components f 1 and f 2. it can. Then, the type of the sample 12 is specified by identifying the fluorescent dye using the average fluorescence relaxation time ⁇ ave .
- the improvement in measurement accuracy according to the present invention will be described in more detail.
- phase delay ⁇ of the fluorescence signal with respect to the modulation signal that modulates the intensity of the laser light depends on the fluorescence relaxation time of the fluorescence emitted by the fluorescent dye.
- the cos component and the sin component are expressed by the following formulas (1) and (2).
- ⁇ is the modulation angular frequency of the laser beam
- ⁇ is the fluorescence relaxation time.
- the fluorescence relaxation time tau when the initial fluorescence intensity and I 0, the fluorescence intensity from the point (the e base of natural logarithms, e ⁇ 2.71828) I 0 / e refers to the time up to the point of the.
- phase delay ⁇ is obtained from the ratio tan ( ⁇ ) between the cos component and the sin component of the fluorescence signal, and the fluorescence relaxation time ⁇ can be obtained from the above equations (1) and (2) using this phase delay ⁇ . .
- tan ⁇ is represented by the following formula (3).
- the flow cytometer 10 efficiently uses a modulation frequency in which the fluctuation of the fluorescence relaxation time with respect to the fluctuation of the phase delay is small, that is, ⁇ / ⁇ becomes large, so that the fluorescence relaxation time (average fluorescence relaxation time) is high. ⁇ ave ) can be obtained.
- FIG. 6 is a graph showing the amount of change ( ⁇ / ⁇ ) in the phase delay ⁇ with respect to the fluorescence relaxation time ⁇ .
- the amount of change in phase delay ⁇ ( ⁇ / ⁇ ) when the modulation frequency f is 7.5 MHz, 15 MHz, and 30 MHz is shown.
- the curve with a modulation frequency of 30 MHz and the curve with 15 MHz intersect at a point where the fluorescence relaxation time ⁇ is 7.5 nsec.
- the curve with the modulation frequency of 15 MHz and the curve with 7.5 MHz intersect at a point where the fluorescence relaxation time ⁇ is 15 nsec.
- the amount of change ( ⁇ / ⁇ ) is the largest when the modulation frequency f is 30 MHz.
- the amount of change ( ⁇ / ⁇ ) is the largest when the modulation frequency f is 15 MHz.
- the fluorescence relaxation time ⁇ is 15 nsec or longer, the amount of change ( ⁇ / ⁇ ) is the largest when the modulation frequency f is 7.5 MHz.
- the measurement accuracy improves. Therefore, when the fluorescence relaxation time of the measurement object covers a wide range, for example, if the intensity of the laser light is modulated only at 30 MHz, the S / N ratio becomes low in a range where the fluorescence relaxation time exceeds 20 nsec. It becomes difficult to obtain a high measurement accuracy.
- the average value of the fluorescence relaxation times is obtained by weighting the fluorescence relaxation times for the respective frequencies. That is, assuming that the frequency f 1 oscillated by the oscillator 46a is 30 MHz and the frequency f 2 oscillated by the oscillator 46b is 15 MHz, the phase delays ⁇ ⁇ 1 and ⁇ ⁇ 2 for the frequencies f 1 and f 2 are obtained.
- the average fluorescence relaxation time acquisition unit 92 multiplies each of ⁇ ( ⁇ ⁇ 1 ) and ⁇ ( ⁇ ⁇ 2 ) by a weighting factor m ( ⁇ ⁇ i ) as shown in the following formula (6).
- the weighting factor acquisition unit 90 sets the weighting factor m ( ⁇ i ) so that the weighting factor m ( ⁇ i ) increases as the change amount ( ⁇ / ⁇ ) of the phase delay ⁇ increases during a certain fluorescence relaxation time ⁇ . Determine.
- the fluorescence relaxation time ⁇ (7.5 nsec) at the point where the curve with the modulation frequency of 30 MHz and the curve with 15 MHz intersect is 0.9555 [rad] for the phase delay ⁇ ⁇ 1 and 0. 0 for the phase delay ⁇ ⁇ 2 .
- the weighting factor acquisition unit 90 determines the weighting factor.
- the weighting factor acquisition unit 90 determines the weighting factor.
- the value of the phase delay at which the magnitude relationship of the weight coefficients is reversed is determined according to the modulation frequencies f 1 and f 2 to be used. For this reason, the weighting coefficient is stored in advance in the memory 84 so as to satisfy the magnitude relationship of the weighting coefficient as described above.
- the type of the sample 12 can be specified with higher accuracy.
- ⁇ Second Embodiment> In the first embodiment, two oscillators are provided and the respective frequencies are different from each other. However, three or more oscillators may be provided and the respective frequencies may be different. In the present embodiment, three oscillators are provided, and the frequencies are set so that f 1 is 30 MHz, f 2 is 15 MHz, and f 3 is 7.5 MHz.
- the phase lag acquisition unit 86 obtains phase lags ⁇ ⁇ 1 , ⁇ ⁇ 2 , and ⁇ ⁇ 3 for the frequencies f 1 , f 2 , and f 3 , respectively.
- the average fluorescence relaxation time acquisition unit 92 assigns a weight coefficient m ( ⁇ ⁇ i ) to each of ⁇ ( ⁇ ⁇ 1 ), ⁇ ( ⁇ ⁇ 2 ), and ⁇ ( ⁇ ⁇ 3 ) as shown in the above equation (6).
- the average fluorescence relaxation time ⁇ ave is obtained by multiplication.
- N 3.
- the weighting factor acquisition unit 90 sets the weighting factor m ( ⁇ i ) so that the weighting factor m ( ⁇ i ) increases as the change amount ( ⁇ / ⁇ ) of the phase delay ⁇ increases during a certain fluorescence relaxation time ⁇ . Determine.
- the fluorescence relaxation time ⁇ (7.5 nsec) at the point where the curve with the modulation frequency of 30 MHz and the curve with 15 MHz intersect is 0.9555 [rad] for the phase delay ⁇ ⁇ 1 and 0. 0 for the phase delay ⁇ ⁇ 2 . This corresponds to 6153 [rad].
- the fluorescence relaxation time ⁇ (15 nsec) at the point where the curve with the modulation frequency of 15 MHz and the curve with 7.5 MHz intersects is 0.9555 [rad] for the phase delay ⁇ ⁇ 2 and 0.6153 for the phase delay ⁇ ⁇ 3 . Corresponds to [rad].
- the fluorescence relaxation time ⁇ is shorter than 7.5 nsec (phase delay ⁇ ⁇ 1 is 0 ⁇ ⁇ ⁇ 1 ⁇ 0.9555 [rad]), m ( ⁇ ⁇ 1 )> m ( ⁇ ⁇ 2 ), m ( ⁇
- the weighting factor acquisition unit 90 determines the weighting factor so that ⁇ 3 ). Further, fluorescence relaxation time ⁇ is shorter than 15n seconds at least 7.5n seconds (phase delay theta .omega.2 is 0.6153 [rad] ⁇ ⁇ ⁇ 2 ⁇ in 0.9555 [rad]), m ( ⁇ ⁇ 2)>
- the weight coefficient acquisition unit 90 determines the weight coefficient so that m ( ⁇ ⁇ 1 ) and m ( ⁇ ⁇ 3 ).
- the weighting factor acquisition unit 90 determines the weighting factor so as to be ( ⁇ ⁇ 2 ).
- the type of the sample 12 can be specified with higher accuracy.
- the frequencies of the plurality of oscillators are a plurality of frequencies having a large difference in frequency, rather than a plurality of relatively close frequencies.
- the value of one frequency of at least two frequencies is twice or more the value of other frequencies.
- the magnitude relationship between the weighting factors m ( ⁇ ⁇ 1 ), m ( ⁇ ⁇ 2 ), and m ( ⁇ ⁇ 3 ) is defined according to the fluorescence relaxation time ⁇ , but in this embodiment, the weighting factor m
- the difference from the second embodiment is that only 0 or 1 is used as the values of ( ⁇ ⁇ 1 ), m ( ⁇ ⁇ 2 ), and m ( ⁇ ⁇ 3 ).
- the weighting factor m is defined according to the fluorescence relaxation time ⁇ , but in this embodiment, the weighting factor m
- the type of the sample 12 can be specified with higher accuracy.
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Abstract
Description
(フローサイトメータの全体構成)
まず、図1を参照して、本実施形態のフローサイトメータの全体構成について説明する。図1は、本発明の強度変調したレーザ光による蛍光検出装置を用いたフローサイトメータ10の一例を示す概略構成図である。
制御・処理部28は、信号生成部40と、信号処理部42と、コントローラ44と、を含む。信号生成部40は、レーザ光源部22からのレーザ光を強度変調させる。また、信号処理部42は、試料12からの蛍光信号を識別する。また、コントローラ44は、フローサイトメータ10の全動作を管理する。
管路30は、高速流を形成するシース液に試料12を含ませて流し、ラミナーシースフローを形成する。このフローは、例えば、流路径が100μmであり、流速が1~10m/秒である。また、試料12としてマイクロビーズを用いる場合、マイクロビーズの球径は数μm~30μmである。管路30の出口には、回収容器32が設けられている。
フローサイトメータ10には、レーザ光の照射により短時間内に試料12中の特定の細胞等の生体物質を分離するためのセル・ソータを配置して、別々の回収容器に分離することもできる。
レーザ光源部22は、所定の周波数で強度変調したレーザ光を出射する。レーザ光源部22には、管路30中の所定の位置に集束するようにレンズ系が設けられる。レーザ光が集束する位置(測定点)において、試料12が測定される。
次に、図2を参照して、レーザ光源部22について説明する。図2は、レーザ光源部22の構成の一例を示す概略構成図である。
図1に戻り、受光部25は、管路30を挟んでレーザ光源部22と対向するように配置されている。受光部25は、測定点を通過する試料12によってレーザ光が前方散乱されることにより、試料12が測定点を通過する旨の検出信号を出力する光電変換器を備えている。受光部25から出力される信号は、制御・処理部28に供給される。受光部25から出力される信号は、制御・処理部28において、試料12が管路30中の測定点を通過するタイミングを知らせるトリガ信号として用いられる。
次に、図4を参照して、制御・処理部28の概略構成について説明する。図4は、制御・処理部28の一例を示す概略構成図である。制御・処理部28は、信号生成部40と、信号処理部42と、コントローラ44と、を備える。レーザ光の強度を変調させる変調信号を生成する光源制御部は、信号生成部40及びコントローラ44によって形成される。
コントローラ44は、システム制御器60と、ローパスフィルタ62と、アンプ64と、A/D変換器66を備えている。
次に、図5を参照して、分析装置(コンピュータ)80の概略構成について説明する。図5は、分析装置(コンピュータ)80の一例を示す概略構成図である。分析装置80は、コンピュータ上で所定のプログラムを起動させることにより構成される。分析装置80は、CPU82、メモリ84、入出力ポート94の他に、ソフトウェアを起動することによって形成される位相遅れ取得ユニット86、蛍光緩和時間取得ユニット88、重み係数取得ユニット90、平均蛍光緩和時間取得ユニット92、を備える。また、分析装置80には、ディスプレイ100が接続されている。
上記式(1)、(2)より、tanθは下記式(3)で表される。
第1の実施形態においては、発振器を2つ設け、それぞれの周波数が異なる構成としたが、発振器を3つ以上設け、それぞれの周波数が異なるような構成としてもよい。本実施形態では発振器を3つ設け、周波数はf1を30MHz、f2を15MHz、f3を7.5MHzとする。位相遅れ取得ユニット86は、周波数f1、f2、f3のそれぞれに対する位相遅れθω1、θω2、θω3を求める。
上記式(5)から明らかなように、蛍光緩和時間τが小さい領域では、変調角周波数ωが大きいほど位相遅れθの変化量(δθ/δτ)が大きくなる。一方、蛍光緩和時間τが大きい領域では、変調角周波数ωが小さいほど位相遅れθの変化量(δθ/δτ)が大きくなる。
第2の実施形態においては、蛍光緩和時間τに応じて、重み係数m(θω1)、m(θω2)、m(θω3)の大小関係を規定したが、本実施形態では重み係数m(θω1)、m(θω2)、m(θω3)の値として0又は1のみを用いた点が第2の実施形態と異なる。その他の構成については、第2の実施形態と同様である。
12 試料
20 信号処理装置
22 レーザ光源部
23 光源
24a,24b レンズ系
25,26 受光部
27 光電変換器
28 制御・処理部
30 管路
32 回収容器
34 レーザドライバ
40 信号生成部
42 信号処理部
44 コントローラ
46a,46b 発振器
48a,48b,48d パワースプリッタ
48c パワーデバイダ
50a,50b,50c,54,55,64 アンプ
58,59 IQミキサ
60 システム制御器
62 ローパスフィルタ
66 A/D変換器
80 分析装置
82 CPU
84 メモリ
86 位相遅れ取得ユニット
88 蛍光緩和時間取得ユニット
90 重み係数取得ユニット
92 平均蛍光緩和時間取得ユニット
94 入出力ポート
100 ディスプレイ
Claims (8)
- 測定対象物にレーザ光を照射することにより前記測定対象物が発する蛍光を受光し、このとき得られる蛍光信号の信号処理を行う蛍光検出装置であって、
前記測定対象物にレーザ光を照射するレーザ光源部と、
前記レーザ光を照射された前記測定対象物が発する蛍光の蛍光信号を出力する受光部と、
前記レーザ光源部から出射されるレーザ光の強度を、少なくとも2つの周波数成分で時間変調させる変調信号を生成する光源制御部と、
前記受光部から出力された蛍光信号と前記変調信号とを用いて、前記測定対象物の蛍光の蛍光緩和時間を求める処理部と、を有し、
前記処理部は、前記少なくとも2つの周波数成分における、前記変調信号に対する前記蛍光信号の位相遅れを求め、前記位相遅れを用いて各周波数成分における蛍光緩和時間を求め、前記蛍光緩和時間の重み付け平均を行って平均蛍光緩和時間を求めることを特徴とする蛍光検出装置。 - 前記変調信号は、少なくとも2つの周波数の信号を合成した信号である、請求項1に記載の蛍光検出装置。
- 前記処理部は、前記少なくとも2つの周波数成分における前記位相遅れの、蛍光緩和時間に対する変化量の大きさに応じた重み係数を用いて、前記平均蛍光体緩和時間を求める、請求項1又は2に記載の蛍光検出装置。
- 前記少なくとも2つの周波数のうち1つの周波数の値は、他の周波数の値の2倍以上である、請求項1乃至3のいずれかに記載の蛍光検出装置。
- 測定対象物にレーザ光を照射することにより前記測定対象物が発する蛍光を受光し、このとき得られる蛍光信号の信号処理を行う蛍光検出方法であって、
前記測定対象物にレーザ光を照射する工程と、
前記レーザ光を照射された前記測定対象物が発する蛍光の蛍光信号を出力する工程と、
前記レーザ光の強度を少なくとも2つの周波数成分で時間変調させる変調信号を生成する工程と、
前記蛍光信号と前記変調信号とを用いて、前記測定対象物の蛍光の蛍光緩和時間を求める工程と、を有し、
前記蛍光緩和時間を求める工程は、
前記少なくとも2つの周波数成分における、前記変調信号に対する前記蛍光信号の位相遅れを求める工程と、
前記位相遅れを用いて各周波数における蛍光緩和時間を求める工程と、
前記蛍光緩和時間の重み付け平均を行って平均蛍光緩和時間を求める工程と、
を有することを特徴とする蛍光検出方法。 - 前記変調信号を生成する工程は、前記少なくとも2つの周波数成分の信号を合成する、請求項5に記載の蛍光検出方法。
- 前記蛍光緩和時間を求める工程は、前記少なくとも2つの周波数成分における前記位相遅れの、蛍光緩和時間に対する変化量の大きさに応じた重み係数を用いて、前記平均蛍光緩和時間を求める、請求項5又は6に記載の蛍光検出方法。
- 前記少なくとも2つの周波数のうち1つの周波数の値は、他の周波数の値の2倍以上である、請求項5乃至7のいずれかに記載の蛍光検出方法。
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EP10741032A EP2397841A1 (en) | 2009-02-10 | 2010-01-25 | Fluorescence detection device and fluorescence detection method |
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JP2012211848A (ja) * | 2011-03-31 | 2012-11-01 | Sumitekku:Kk | 蛍光温度計及び温度計測方法 |
CN105758834A (zh) * | 2016-04-26 | 2016-07-13 | 福州大学 | 一种激光诱导与ccd采集的生物芯片检测方法 |
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US20160238506A1 (en) * | 2015-02-02 | 2016-08-18 | Derek Oberreit | Ice nucleii counter technology |
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