Patent Application
20110313725
The present invention relates to a device for detecting fluorescence by receiving fluorescence emitted by a measurement object irradiated with laser light and processing a fluorescent signal obtained at this time. The present invention also relates to a method for detecting fluorescence by receiving fluorescence emitted by a measurement object irradiated with laser light and processing a fluorescent signal obtained at this time. Particularly, the present invention relates to a fluorescence detecting device to be used in an analyzing device, such as a flow cytometer for use in medical and biological fields, which analyzes a measurement object, such as cells, DNA, or RNA, by identifying the measurement object based on fluorescence emitted by a fluorochrome.
A flow cytometer used in medical and biological fields includes a fluorescence detecting device that identifies the type of a measurement object by receiving fluorescence emitted by a fluorochrome in the measurement object irradiated with laser light.
More specifically, in the flow cytometer, a suspension liquid containing a measurement object such as a biological material (e.g., cells, DNA, RNA, enzymes, or proteins), labeled with a fluorescent reagent is allowed to flow through a tube together with a sheath liquid flowing under pressure at a speed of about 10 m/sec or less to form a laminar sheath flow. The flow cytometer receives fluorescence emitted by a fluorochrome attached to the measurement object by irradiating the measurement object in the laminar sheath flow with laser light and identifies the measurement object by using the fluorescence as a label.
The flow cytometer can measure, for example, the relative amounts of DNA, RNA, enzymes, proteins, etc. contained in a cell and can quickly analyze their functions. Further, a cell sorter or the like is used to identify a predetermined type of cell or chromosome based on fluorescence and selectively and quickly collect only the identified cells or chromosomes alive.
For example, when a biological material such as DNA is analyzed by a flow cytometer, a fluorochrome is previously attached to the biological material with a fluorescent reagent. The biological material is labeled with the fluorochrome different from a fluorochrome attached to a microbead (which will be described later), and is mixed with a liquid containing microbeads having a diameter of 5 to 20 μm. Each of the microbeads has a specific structure, such as a carboxyl group, provided on the surface thereof. The specific structure, such as a carboxyl group, acts on and is coupled to a biological material having a certain known structure. Therefore, simultaneous detection of fluorescence derived from the microbead and fluorescence derived from the biological material indicates that the biological material has been coupled to the specific structure of the microbead. This makes it possible to analyze the characteristics of the biological material. In order to prepare various microbeads having different structures for coupling to quickly analyze the characteristics of the biological material, a very wide variety of fluorochromes are required.
Patent Document 1 discloses a fluorescence detecting device that determines the fluorescence relaxation time of fluorescence emitted by a measurement object, such as a microbead, by irradiating the measurement object with laser light whose intensity is modulated at a predetermined frequency. The fluorescence relaxation time varies depending on the type of fluorochrome used, and therefore the type of fluorescence can be identified based on the fluorescence relaxation time, which makes it possible to identify the type of the measurement object.
The fluorescence detecting device disclosed in Patent Document 1 can quickly and efficiently identify fluorescence based on a fluorescence relaxation time, but the accuracy of fluorescence relaxation time measurement is not always high. For example, when a measurement object includes a microbead or the like that emits fluorescence having a relatively long fluorescence relaxation time exceeding 20 nsec, the accuracy of fluorescence relaxation time measurement is lowered.
It is therefore an object of the present invention to provide a fluorescence detecting device and a fluorescence detecting method which make it possible to achieve higher accuracy of fluorescence relaxation time measurement.
The inventor of the present invention found that a range of fluorescence relaxation time of a measurement object with higher accuracy varies depending on the modulation frequency of the laser light. In other words, when the fluorescence relaxation time of a measurement object extends over a wide range, the measurement accuracy is to be improved by modulating a intensity of the laser light with multiple frequency components, not with single frequency component.
A fluorescence detecting device of the present invention for receiving fluorescence emitted by a measurement object by irradiating the measurement object with laser light and processing a fluorescent signal obtained at this time, the device comprising: a laser light source unit that irradiates the measurement object with laser light; a light-receiving unit that outputs a fluorescent signal of fluorescence emitted by the measurement object irradiated with the laser light; a light source control unit that generates a modulation signal for time-modulating an intensity of the laser light emitted from the laser light source unit by at least two frequency components; and a processing unit that determines a fluorescence relaxation time of the fluorescence emitted by the measurement object by using the fluorescent signal outputted by the light-receiving unit and the modulation signal, wherein the processing unit determines phase delays of the fluorescent signal with respect to the modulation signal at the two frequency components, and determines a fluorescence relaxation time at each of the frequency components by using the phase delay, and determines an average fluorescence relaxation time by weighted averaging of the fluorescence relaxation times.
A fluorescence detecting method of the present invention by receiving fluorescence emitted by a measurement object by irradiating the measurement object with laser light and processing a fluorescent signal obtained at this time, the method comprising the steps of: irradiating the measurement object with laser light; outputting a fluorescent signal of fluorescence emitted by the measurement object irradiated with the laser light; generating a modulation signal for time-modulating an intensity of the laser light by at least two frequency components; and determining a fluorescence relaxation time of the fluorescence emitted by the measurement object by using the fluorescent signal and the modulation signal, wherein the step of determining a fluorescence relaxation time includes the step of determining phase delays of the fluorescent signal with respect to the modulation signal at the two frequency components, the step of determining a fluorescence relaxation times at each of the frequency components by using the phase delay, and the step of determining an average fluorescence relaxation time by weighted averaging of the fluorescence relaxation times.
According to the fluorescence detecting device and the fluorescence detecting method of the present invention, it is possible to achieve higher accuracy of fluorescence relaxation time measurement.
Hereinbelow, a flow cytometer that appropriately uses a fluorescence detecting device according to the present invention using intensity-modulated laser light will be described based on the following embodiments.
First, the overall structure of a flow cytometer according to a first embodiment will be described with reference to
The flow cytometer 10 includes a signal processing unit 20 and an analyzing device (computer) 80. The signal processing device 20 detects and processes a fluorescent signal of fluorescence emitted by a fluorochrome introduced into a sample 12, which is a measurement object such as a microbead or a cell, by irradiation with laser light. The analyzing device (computer) 80 analyzes the measurement object in the sample 12 from processed results obtained by the signal processing device 20.
As will be described later, 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 tube 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 laser light emitted from the laser light source unit 22. The signal processing unit 42 identifies a fluorescent signal from the sample 12. The controller 44 manages all the operations of the flow cytometer 10.
The tube 30 allows a sheath liquid forming a high speed flow to flow therethrough together with the samples 12 to form a laminar sheath flow. The laminar sheath flow has a diameter of, for example, 100 μm and a flow rate of 1 to 10 m/sec. When a microbead is used as the sample 12, the diameter of the microbead is several micrometers to 30 μm. A collection vessel 32 is provided at the outlet of the tube 30.
The flow cytometer 10 may include a cell sorter to quickly separate a biological material such as specific cells in the sample 12 after irradiation with laser light into different collection vessels.
The laser light source unit 22 emits laser light whose intensity is modulated at a predetermined frequency. The laser light source unit 22 has a lens system so that the laser light is focused on a predetermined position in the tube 30. The sample 12 is measured at a position (measurement point) on which the laser light is focused.
(Laser Light Source Unit)
The laser light source unit 22 will be described with reference to
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 of constant intensity while modulating the intensity of the CW laser light. The lens system 24a focuses laser light emitted from the light source 23 on the measurement point in the tube 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 light is, for example, about 5 to 100 mW. The laser light has a wavelength within, for example, a visible light band of 350 nm to 800 nm.
The laser driver 34 is connected to the control/processing unit 28. The laser driver 34 generates a driving signal for modulating the intensity of laser light by using a modulation signal including at least two frequencies, and supplies the driving signal to the light source 23.
The fluorochrome to be excited by laser light is attached to the sample 12 (measurement object) such as a biological material or a microbead. When passing through the tube 30 in several microseconds to several tens of microseconds, the sample 12 is irradiated with laser light at the measurement point and emits fluorescence. At this time, the laser light is emitted while being intensity-modulated at two frequencies.
(Light-Receiving Unit)
Referring to
On the other hand, the light-receiving unit 26 is arranged in a direction perpendicular to a direction in which laser light emitted from the laser light source unit 22 travels and to a direction in which the sample 12 moves in the tube 30. The light-receiving unit 26 is equipped with a photoelectric converter that receives fluorescence emitted by the sample 12 irradiated with laser light at the measurement point.
The general structure of the light-receiving unit 26 will be described with reference to
The photoelectric converter 27 is equipped with, for example, a photomultiplier to convert light received by its photoelectric surface to an electrical signal. The electrical signal (fluorescent signal) converted by the photoelectric converter 27 is supplied to the control/processing unit 28.
(Control/Processing Unit)
The general structure of the control/processing unit 28 will be described with reference to
The signal generation unit 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 and supplies the modulation signal to the laser driver 34 of the laser light source unit 22 and the signal processing unit 42. As will be described later, the modulation signal supplied to the signal processing unit 42 is used as a reference signal for detecting a fluorescent signal outputted from the photoelectric converter 27.
Each of the oscillators 46a and 46b outputs a sinusoidal signal, but a sinusoidal signal outputted from the oscillator 46a and a sinusoidal signal outputted from the oscillator 46b are different in frequency. The frequency of each of the sinusoidal signals is set to a value in the range of, for example, 1 to 50 MHz. The sinusoidal signal outputted from the oscillator 46a has a frequency of f1 (angular frequency of ω1), and is split by the power splitter 48a and sent to the power divider 48c and the amplifier 50a. The sinusoidal signal outputted from the oscillator 46b has a frequency of f2 (angular frequency of ω2), and is split by the power splitter 48b and sent to the power divider 48c and the amplifier 50b. The sinusoidal signal sent from the power splitter 48a to the power divider 48c is combined with the sinusoidal signal sent from the power splitter 48b to the power divider 48c 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 supplied to the laser driver 34.
The signal processing unit 42 uses the fluorescent signal outputted from the photoelectric converter 27 to extract information about the phase delay of fluorescence emitted by the measurement object such as a microbead by irradiation with laser light. The signal processing unit 42 includes a power splitter 48d, amplifiers 54 and 55, and IQ mixers 58 and 59.
The power splitter 48d splits the fluorescent signal outputted from the photoelectric converter 27 and sends the split fluorescent signals to the amplifiers 54 and 55. The amplifier 54 amplifies the split fluorescent signal sent from the power splitter 48d and supplies the amplified fluorescent signal to the IQ mixer 58. The amplifier 55 amplifies the split fluorescent signal sent from the power splitter 48d and supplies the amplified fluorescent signal to the IQ mixer 59. To the IQ mixer 58, the sinusoidal signal having a frequency of f1 and supplied from the amplifier 50a is also supplied as a reference signal. To the IQ mixer 59, the sinusoidal signal having a frequency of f2 and supplied from the amplifier 50b is also supplied as a reference signal.
The IQ mixer 58 is a device that combines the fluorescent signal supplied from the photoelectric converter 27 with the sinusoidal signal having a frequency of f1 and supplied from the signal generation unit 40 as a reference signal. The IQ mixer 59 is a device that combines the fluorescent signal supplied from the photoelectric converter 27 with the sinusoidal signal having a frequency of f2 and supplied from the signal generation unit 40 as a reference signal. More specifically, each of the IQ mixers 58 and 59 multiplies the fluorescent signal (RF signal) by the reference signal to calculate a processing signal including a cos component and a high-frequency component of the fluorescent signal. Further, each of the IQ mixers 58 and 59 multiplies the florescent signal by a signal obtained by shifting the phase of the reference signal by 90° to calculate a processing signal including a sin component and a high-frequency component of the fluorescent 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 controlling the operations of the individual units and manages all the operations of the flow cytometer 10. Further, the system controller 60 controls the oscillators 46a and 46b of the signal generation unit 40 to generate sinusoidal signals having predetermined frequencies.
The low-pass filter 62 removes the high-frequency component from the processing signal which is calculated by the signal processing unit 42 and in which the high-frequency component is added to the cos component, and removes the high-frequency component from the processing signal which is calculated by the signal processing unit 42 and in which the high-frequency component is added to the sin component. As a result, the processing signal of the cos component with a frequency of f1, the processing signal of the cos component with a frequency of f2, the processing signal of the sin component with a frequency of f1, and the processing signal of the sin component with a frequency of f2 are obtained. The amplifier 64 amplifies the processing signals of the cos component and the processing signals of the sin component. The A/D converter 66 samples the amplified processing signals.
(Analyzing Device)
The general structure of the analyzing device (computer) 80 will be described with reference to
The CPU 82 is an arithmetic processor provided in the computer. The CPU 82 virtually performs various calculations of the phase delay acquisition unit 86, the fluorescence relaxation time acquisition unit 88, the weight coefficient acquisition unit 90, and the average fluorescence relaxation time acquisition unit 92.
The memory 84 includes a hard disk or ROM that stores a program executed on the computer to provide the phase delay acquisition unit 86, the fluorescence relaxation time acquisition unit 88, the weight coefficient acquisition unit 90, and the average fluorescence relaxation time acquisition unit 92 and a RAM that stores processed results calculated by these units and data supplied from the input/output port 94.
The input/output port 94 receives the input of detected values of the cos components corresponding to at least two frequency components f1 and f2 and of the sin components corresponding to at least two frequency components f1 and f2, which are supplied from the controller 44. The input/output port 94 outputs information about processed results obtained by the units to the display 100. The display 100 displays the values of the processed results obtained by the units such as information about the phase delay of fluorescence, a fluorescence relaxation time, a weight coefficient, and an average fluorescence relaxation time.
The phase delay acquisition unit 86 determines, from the detected values of the cos components corresponding to at least two frequency components f1 and f2 and of the sin components corresponding to at least two frequency components f1 and f2 which are supplied from the controller 44, a phase delay θω1 at the frequency component f1 (angular frequency ω1) and a phase delay θω2 at the frequency component f2 (angular frequency ω2).
The fluorescence relaxation time acquisition unit 88 determines a fluorescence relaxation time τ(θω1) and a fluorescence relaxation time τ(θω2) based on the phase delay θω1 and the phase delay θω2 determined by the phase delay acquisition unit 86, respectively.
The weight coefficient acquisition unit 90 determines weight coefficients m(θω1) and m(θω2) used to assign weights to the fluorescence relaxation times τ(θω1) and τ(θω2) determined by the fluorescence relaxation time acquisition unit 88, respectively. The weight coefficient is a value of 0 or more but 1 or less.
The average fluorescence relaxation time acquisition unit 92 determines an average fluorescence relaxation time τave based on the fluorescence relaxation times τ(θω1) and τ(θω2) determined by the fluorescence relaxation time acquisition unit 88 and the weight coefficients m(θω1) and m(θω2) determined by the weight coefficient acquisition unit 90.
As described above, by using the detected values of the fluorescent signal corresponding to at least two frequency components f1 and f2, it is possible to determine a fluorescence relaxation time (i.e., the above-described average fluorescence relaxation time τave) with high accuracy. The type of the sample 12 is identified by identifying the fluorochrome using the average fluorescence relaxation time τave. The reason why higher accuracy of fluorescence relaxation time measurement is achieved by the present invention will be described below in more detail.
The phase delay θ of the fluorescent signal with respect to the modulation signal for modulating the intensity of laser light generally depends on the fluorescence relaxation time of fluorescence emitted by the fluorochrome. When the phase delay θ is represented by, for example, a first-order relaxation process, the cos component and the sin component are expressed by the following Equations (1) and (2).
wherein ω is the modulation angular frequency of laser light and τ is the fluorescence relaxation time. When the initial fluorescence intensity is defined as I0, the fluorescence relaxation time τ is a period of time between the time point when the fluorescence intensity is I0 and the time point when the fluorescence intensity becomes I0/e (e is a base of natural logarithm, e≈2.71828).
The phase delay θ is determined from the ratio between the cos component and the sin component of the fluorescent signal, that is, tan(θ), and the fluorescence relaxation time τ can be determined by the above Equations (1) and (2) using the phase delay θ.
The tan(θ) is represented by the following Equation (3) using the above Equations (1) and (2).
Equation 3
tan θ=ωτ (3)
The phase delay θ is represented by the following Equation (4) using the above Equation (3).
Equation 4
θ=tan−1(ωτ) (4)
The amount of change (δθ/δτ) of the phase delay θ with respect to the fluorescence relaxation time τ is represented by the following Equation (5) using the above Equation (4).
Essentially, if the phase delay θ can be accurately determined, the fluorescence relaxation time can also be accurately determined, but if the phase delay has an error, the fluorescence relaxation time cannot be determined accurately. However, if the variance of the fluorescence relaxation time with respect to the error of the phase delay is small, the fluorescence relaxation time can be accurately determined with stability. The flow cytometer 10 can accurately determine the fluorescence relaxation time (average fluorescence relaxation time τave) by efficiently using a modulation frequency allowing the variance of the fluorescence relaxation time with respect to the variance of the phase delay to be smaller, that is, a modulation frequency allowing the ratio δθ/δτ to be larger.
As can be seen from
When the amount of change (δθ/δτ) of the phase delay θ is larger, a higher S/N ratio is obtained and therefore higher measurement accuracy is achieved. For this reason, when the fluorescence relaxation time of the measurement object extends over a wide range, modulation of the intensity of laser light only at, for example, 30 MHz decreases an S/N ratio in a range where the fluorescence relaxation time exceeds 20 nsec, which makes it difficult to achieve satisfactory measurement accuracy.
In this embodiment, as described above, weights are assigned to the fluorescence relaxation times at two frequencies to determine the average of the fluorescence relaxation times. That is, the oscillating frequency f1 of the oscillator 46a is set to 30 MHz and the oscillating frequency f2 of the oscillator 46b is set to 15 MHz, and the phase delay θω1 at the frequency f1 and the phase delay θω2 at the frequency f2 are determined.
Then, the average fluorescence relaxation time acquisition unit 92 determines the average fluorescence relaxation time τave by the following Equation (6), that is, by multiplying each of the fluorescence relaxation times τ(θω1) and τ(θω2) by the weight coefficient m(θωi). Here, N is an integer of 2 or more, and represents the number of different frequency components. In this embodiment, N is 2. The weight coefficient acquisition unit 90 determines the weight coefficient m(θωi) that increases as the amount of change (δθ/δτ) of the phase delay θ increases when the fluorescence relaxation time τ has a certain value.
Here, the fluorescence relaxation time τ(7.5 nsec) at the intersection point of the curve obtained when the modulation frequency is 30 MHz and the curve obtained when the modulation frequency is 15 MHz corresponds to a phase delay θω1 of 0.9555 [rad] and to a phase delay θω2 of 0.6153 [rad]. Therefore, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1) and m(θω2) so that m(ωω1) and m(θω2) satisfy m(θω1)>m(θω2) when the fluorescence relaxation time τ is shorter than 7.5 nsec (0≦phase delay θω1<0.9555 [rad]). Further, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1) and m(θω2) so that m(θω1) and m(θω2) satisfy m(θω1)<m(θω2) when the fluorescence relaxation time τ is 7.5 nsec or longer (0.6153 [rad]≦phase delay θω2<π/2 [rad]). The value of the phase delay allowing the magnitude relationship between the weight coefficients to be reversed depends on the modulation frequencies f1 and f2 used. Therefore, weight coefficients satisfying the above-mentioned magnitude relationship are previously stored in the memory 84.
The type of the sample 12 can be identified with higher accuracy by identifying the fluorochrome using the average fluorescence relaxation time τave.
As described above, the flow cytometer according to the first embodiment has a structure in which two oscillators different in oscillating frequency are provided. However, a flow cytometer according to a second embodiment may have a structure in which three or more oscillators different in oscillating frequency are provided. In the second embodiment, three oscillators are provided, and the frequency f1 is set to 30 MHz, the frequency f2 is set to 15 MHz, and the frequency f3 is set to 7.5 MHz. The phase delay acquisition unit 86 determines the phase delay θω1 at the frequency f1, the phase delay θω2 at the frequency f2, and the phase delay θω3 at the frequency f3.
Then, the average fluorescence relaxation time acquisition unit 92 determines the average fluorescence relaxation time τave, by the above Equation (6), that is, by multiplying each of the fluorescence relaxation times τ(θω1), τ(θω2), and τ(θω3) by the weight coefficient m(θωi). In this embodiment, N is 3. The weight coefficient acquisition unit 90 determines the weight coefficient m(θωi) that increases as the amount of change (δθ/δτ) of the phase delay θ increases when the fluorescence relaxation time τ has a certain value.
Here, the fluorescence relaxation time τ (7.5 nsec) at the intersection point of the curve obtained when the modulation frequency is 30 MHz and the curve obtained when the modulation frequency is 15 MHz corresponds to a phase delay θω1 of 0.9555 [rad] and to a phase delay θω2 of 0.6153 [rad], and the fluorescence relaxation time τ (15 nsec) at the intersection point of the curve obtained when the modulation frequency is 15 MHz and the curve obtained when the modulation frequency is 7.5 MHz corresponds to a phase delay θω2 of 0.9555 [rad] and to a phase delay θω3 of 0.6153 [rad].
Therefore, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1), m(θω2), and m(θω3) so that m(θω1), m(θω2), and m(θω3) satisfy m(θω1)>m(θω2), m(θω3) when the fluorescence relaxation time τ is shorter than 7.5 nsec (0≦phase delay θω1<0.9555 [rad]). Further, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1), m(θω2), and m(θω3) so that m(θω1), m(θω2), and m(θω3) satisfy m(θω2)>m(θω1), m(θω3) when the fluorescence relaxation time τ is 7.5 nsec or longer but shorter than 15 nsec (0.6153 [rad]≦phase delay θω2<0.9555 [rad]). Further, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1), m(θω2), and m(θω3) so that m(θω1), m(θω2), and m(θω3) satisfy m(θω3)>m(θω1), m(θω2) when the fluorescence relaxation time τ is 15 nsec or longer (0.6153 [rad]≦phase delay θω3<π/2 [rad]).
The type of the sample 12 can be identified with higher accuracy by identifying the fluorochrome using the average fluorescence relaxation time τave.
As can be seen from the above Equation (5), in a region where the fluorescence relaxation time τ is small, the amount of change (δθ/δτ) of the phase delay θ increases as the modulation angular frequency ω increases, but on the other hand in a region where the fluorescence relaxation time τ is large, the amount of change (δθ/δτ) of the phase delay θ increases as the modulation angular frequency ω decreases.
Therefore, it is preferred that the different frequencies of two or more oscillators used are not relatively close to each other, that is, the difference between the different frequencies of two or more oscillators used is large. For example, the value of one of at least two frequencies is preferably two times or larger than that of the other frequency.
By setting one of at least two frequencies to a value two times or larger than that of the other frequency, it is possible to identify the type of the sample 12 with higher accuracy.
As described above, in the second embodiment, the magnitude relationship among the weight coefficients m(θω1), m(θω2), and m(θω3) is defined according to the fluorescence relaxation time τ. A fourth embodiment is different from the second embodiment in that the weight coefficients m(θω1), m(θω2), and m(θω3) are set to either 0 or 1, but the other structures are the same as those in the second embodiment.
More specifically, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1), m(θω2), and m(θω3) so that m(θω1)=1 and m(θω2)=m(θω3)=0 when the fluorescence relaxation time τ is shorter than 7.5 nsec (0≦phase delay θω1<0.9555 [rad]). Further, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1), m(θω2), and m(θω3) so that m(θω2)=1 and m(θω1)=m(θω3)=0 when the fluorescence relaxation time τ is 7.5 nsec or longer but shorter than 15 nsec (0.6153 [rad]≦phase delay θω2<0.9555 [rad]). Further, the weight coefficient acquisition unit 90 determines the weight coefficients m(θω1), m(θω2), and m(θω3) so that m(θω3)=1 and m(θω1)=m(θω2)=0 when the fluorescence relaxation time τ is 15 nsec or longer (0.6153 [rad]≦phase delay θω3<π/2 [rad]).
The kind of the sample 12 can be identified with higher accuracy by identifying the fluorochrome using the average fluorescence relaxation time τave.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-028325 | Feb 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/000390 | 1/25/2010 | WO | 00 | 8/8/2011 |
