JPH09159527A - Fluorescent material determining device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately perform the measurement of a quantity of the fluorescent light, that is, the determination of a fluorescent material even if a quantity of the fluorescent light generated from a trace quantity of a fluorescent material in a sample is extremely feeble. SOLUTION: The exciting pulse light outputted from a pulse laser light source 80 excites a fluorescent material contained in a sample 90. The generated fluorescent light is received by a light detector 10 through a barrier filter 92. A trigger circuit 82 controls inverse bias voltage outputted from an inverse bias power source 18 on the basis of a timing signal to generate the exciting pulse light, and a multiplication rate of the light detector 10 becomes large when falling within a constant time range in which a quantity of the fluorescent light among the measuring object light made incident on the light detector 10 on the basis of the inverse bias voltage is dominant, and becomes small when it is not so. An electric current signal outputted from the light detector 10 is integrated for constant time by an integrator 20, and a result appears on a point P as a voltage signal. In this voltage signal, the wave height distribution is generated in a histomemory 60 through an amplifier 30, a sample hold circuit 40 and an A/D converter 50, and a quantity of the fluorescent light is measured on the basis of this.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent substance quantification apparatus for measuring the amount of fluorescence emitted from a sample excited by a pulsed light source and quantitatively measuring the number of fluorescent molecules in the sample in fields such as biochemistry. It is about.

[0002]

2. Description of the Related Art Conventionally, fluorescent substances (or
In order to quantitatively measure (fluorescent molecule), the sample is irradiated with excitation light and the intensity of fluorescence generated from the fluorescent substance is measured. As a device used for such a measurement, there is a fluorometer. In this fluorometer, for example, a xenon lamp is used as a light source, and a photomultiplier tube is used as a fluorescence detector. Generally, a xenon lamp is lit continuously instead of pulsed, and a photomultiplier tube receives fluorescence generated by a fluorescent substance and continuously outputs an output current according to the intensity of the fluorescent light. In this way, the fluorescence intensity is measured, and the fluorescent substance is quantified based on the output current value of the photomultiplier tube.

However, when the amount of the fluorescent substance in the sample is very small and the amount of fluorescent light is below the detection limit of the fluorometer, the quantitative method based on the output current value of the photomultiplier cannot be used. Therefore, in such a case, the fluorescent substance is quantified by the so-called photon counting method based on the count value of the output pulse of the photomultiplier tube. A laser light source is used as the excitation light source to increase the intensity of the excitation light. Furthermore,
A pulsed laser light source is used to obtain a larger excitation light intensity and to eliminate the dark noise of the photomultiplier tube. In this case, similarly, the intensity of the fluorescence generated from the fluorescent substance in the sample excited by the laser light is measured by the photomultiplier, and the fluorescent substance is quantified based on the count value of the output pulse of the photomultiplier. .

[0004]

However, there are the following problems in the quantification of the fluorescent substance based on the count value of the output pulse from the photomultiplier tube using the above-mentioned conventional laser light source.

When the amount of the fluorescent substance in the sample becomes extremely small, regardless of whether the excitation light is continuous light or pulsed light, the amount of scattered light that is scattered by the sample and enters the fluorescence detector, The amount of background light that enters the fluorescence detector as stray light from the background becomes relatively larger than the amount of fluorescence that enters the fluorescence detector, making it difficult to measure the amount of fluorescence, that is, quantify the fluorescent substance.

In order to prevent such scattered light or background light from entering the fluorescence detector, the fluorescence wavelength band is transmitted through an optical system between the light receiving surface of the fluorescence detector and the sample. A barrier filter that blocks the wavelength band of background light (particularly scattered light) is provided. In this way, only the fluorescent component is received by the fluorescence detector, and the amount of fluorescent light that is not affected by the scattered light or the disturbance of the background light is measured.

However, even though this barrier filter blocks the scattered light and the background light, there is no real thing that can completely block the scattered light and the background light. Incident on the fluorescence detector. Therefore, when the amount of fluorescent light to be measured is extremely weak and the amount of scattered light partially transmitted through the barrier filter is greater than or equal to the amount of fluorescent light,
Unable to measure the amount of fluorescent light.

The present invention has been made in order to solve the above-mentioned problems, and it is possible to measure the fluorescent light amount, that is, to quantify the fluorescent substance even if the amount of fluorescence emitted from the extremely small amount of fluorescent substance is extremely weak. It is an object of the present invention to provide a fluorescent substance quantification device that can be accurately performed.

[0009]

The fluorescent substance quantification device according to the present invention is (1) an excitation light source for outputting excitation pulse light based on a pulse light generation timing signal, and (2) irradiation with excitation pulse light. A photodetector that receives the light flux to be measured containing the fluorescence generated from the fluorescent substance in the sample and outputs a current signal according to the light intensity of the light flux to be measured and the set multiplication factor, and (3) pulsed light generation timing signal Based on the above, a gate signal indicating whether or not it is within a certain time range from a predetermined time after the time when the excitation light pulse is generated and the light amount of fluorescence is dominant in the measured light flux is output. A gate signal generating means and (4) a multiplication factor changing means for setting the multiplication factor when the time is within the fixed time range to be larger than the multiplication factor when the time is outside the fixed time range based on the gate signal It is characterized by

This device operates as follows. When the sample is irradiated with the excitation pulsed light output from the excitation light source based on the pulsed light generation timing signal, the fluorescent substance contained in the sample emits fluorescence. When the measured light flux containing the fluorescence enters the photodetector, a current signal corresponding to the amount of light and the multiplication factor is output. Based on the pulsed light generation timing signal, the gate signal generation means generates a gate signal indicating whether or not the amount of fluorescence is dominant within a certain time period within the measured light flux. Based on this gate signal, the multiplication factor of the photodetector is set to a large multiplication factor within the fixed time range by the multiplication factor changing means, and set to a small multiplication factor otherwise. In this way, only fluorescence can be measured with high sensitivity,
It is possible to accurately quantify the fluorescent substance.

Further, a barrier filter may be further provided which is provided on the optical path between the sample and the photodetector, transmits the fluorescence, and blocks the scattered light scattered by the excitation pulsed light irradiated on the sample. . In this case, since the light flux other than the fluorescence is absorbed by the barrier filter, the measurement of the fluorescent light amount, that is, the quantitative determination of the fluorescent substance can be performed with higher accuracy.

Further, the photodetector emits a number of photoelectrons according to the photoelectron number distribution according to the light quantity of the light flux to be measured, multiplies the photoelectrons by a set multiplication factor, and outputs a current signal. (1) Integrating means for integrating a current signal for at least a certain period of time to convert it into a voltage signal, (2) AD converting means for converting it into a digital value corresponding to the voltage signal, and (3) predetermined at an address corresponding to the digital value. The peak height distribution generating means for cumulatively adding the values to generate the peak height distribution of the voltage signal and (4) the peak height distribution generated by the peak height distribution generating means when the number of photoelectrons emitted by the photodetector is one. A single photoelectron event wave height distribution generating means for acquiring as a photoelectron event wave height distribution, and (5) k-photoelectron event wave height distribution in each case where the number k of photoelectrons emitted by the photodetector is 2 or more and less than or equal to a predetermined number. , Based on single photoelectron event wave height distribution K-photoelectron event wave height distribution generation means calculated recursively by convolution calculation, and (6) wave height distribution and single photoelectron event wave height generated by the measured light flux incident on the photodetector. The number of photoelectrons for which the light quantity of the measured light flux is obtained by estimating the photoelectron number distribution when the measured light flux enters the photodetector based on the distribution and the k-photoelectron event wave height distribution in each case k Distribution estimation means may be further provided. In this case, from the estimated photoelectron number distribution, that is, the average value of the number of emitted photoelectrons, the amount of fluorescent light, that is, the quantification of the fluorescent substance can be accurately performed.

As the photodetector, (1) a photoelectric conversion surface that emits a number of photoelectrons according to the photoelectron number distribution according to the light quantity of the light flux to be measured, and (2) a reverse bias between the anode and the cathode. When a voltage is applied and the portion facing the photoelectric conversion surface is set to a potential higher than the potential of the photoelectric conversion surface, the electron-hole pairs generated by inputting photoelectrons are avalanche at the set multiplication factor. Avalanche photodiode that outputs a current signal according to the number of electron-hole pairs that have been multiplied and avalanche-multiplied, and (3) a photoelectric conversion surface and an avalanche photodiode that are equipped with an entrance window that transmits the light flux to be measured. It is possible to preferably use a vacuum container including an inside, and the multiplication factor changing unit is configured such that the reverse bias voltage when the gain is within the fixed time range is larger than the reverse bias voltage when the gain is outside the fixed time range. Voltage By increasing setting, changing multiplication factor, it is also possible.

Further, the photoelectron number distribution estimating means may estimate the photoelectron number distribution by the maximum likelihood method. The photoelectron number distribution may be estimated by assuming that the photoelectron number distribution is Poisson distribution.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

First, the structure of the fluorescent substance quantification device according to the present invention will be described. FIG. 1 is a configuration diagram of a fluorescent substance quantification device according to the present invention.

The fluorescent substance quantifying device according to the present embodiment is
(1) A pulsed laser light source 80 that outputs excitation pulsed light,
(2) An irradiation optical system including a reflecting mirror 83 and lenses 84, 85 and 86 for irradiating the sample 90 with excitation pulsed light, and (3) a fluorescent substance in the sample 90, which includes lenses 91, 93 and a barrier filter 92. Condensing optical system for guiding the generated fluorescence, and (4) photodetector 10, integrator 20, amplifier 3
0, a sample and hold circuit 40, an AD converter 50, a hist memory 60, a calculation unit 70, and the like, and a fluorescence detection unit that measures the fluorescence output from the condensing optical system, and (5) each unit that constitutes the fluorescence detection unit. Circuit 8 for generating a signal for controlling the operation of the optical disc based on the pulsed light generation timing
2 and (6) a counter 81 for counting the number of output pulses of the excitation pulsed light.

A pulsed laser light source 80 for outputting pulsed laser light (excitation pulsed light) with which a sample 90 containing a fluorescent substance is irradiated outputs pulsed laser light in accordance with a timing signal of the laser light output, and at the same time, its timing. It also outputs a signal. Counter 8 for inputting this timing signal
1 counts the number of pulses of the output pulsed laser light, that is, the number N of times of excitation of the fluorescent substance, based on this timing signal. A trigger circuit (gate signal generation means) 82 which also inputs this timing signal, based on the timing signal, has a reverse bias power supply (gain multiplication means) 18, a switch 21, a sample hold circuit 40 and an AD converter which will be described later. Each of the signals for controlling the operation timing of each of the 50 is generated and output to control the fluorescent light amount measurement operation.

The pulsed laser light for exciting the fluorescent substance, which is output from the pulsed laser light source 80, is reflected by the reflecting mirror 83 and the beam diameter is expanded by the lenses 84 and 85.
The light is condensed by the lens 86 and is irradiated to the sample 90, and the sample 9
The fluorescent substance in 0 is excited. The fluorescence generated from this fluorescent substance is collected by the lenses 91 and 93, passes through the barrier filter 92, and enters the entrance window 12 of the photodetector 10. The barrier filter 92 transmits the fluorescence, but blocks the pulsed laser light scattered by the sample 90 and traveling toward the incident window 12 of the photodetector 10. However, the blocking of the pulsed laser light by the barrier filter 92 is not perfect, and a small portion of the pulsed laser light is transmitted and goes to the incident window 12 of the photodetector 10.

A lens 93 is transmitted through the barrier filter 92.
The photodetector 10 that receives the light flux condensed by the above emits a number of photoelectrons according to the photoelectron number distribution according to the light quantity of the incident light flux, multiplies the photoelectrons, and outputs a current signal. is there. For example, a photodetector using a photomultiplier tube or an avalanche photodiode (hereinafter, APD) (reference: Shawn J. Fagen, "Vacuum avalanche photodi
odes can count single photons ", Laser Focus World,
Nov. (1993) pp.125-132) is used. In particular, AP
A photodetector using D is preferably used. This APD
FIG. 2 shows a cross-sectional view of a photodetector using the.

The photodetector 10 using this APD has an entrance window 1 formed in a part of a vacuum container 11 whose inside is kept vacuum.
2 is provided, and the incident light flux A passes through the incident window 12 and reaches the photoelectric conversion surface 13. Photoelectric conversion surface 13
Is, for example, −10 with respect to the anode 16 of the APD 15.
Since a high voltage of kV to −15 kV is applied by the high-voltage power supply 19, when the incident light flux A enters the photoelectric conversion surface 13, the number of photoelectrons B according to the photoelectron number distribution according to the light quantity of the incident light flux A is obtained. Is released. Then, the photoelectrons B are accelerated by the electric field between the photoelectric conversion surface 13 and the APD 15, are focused by the focusing electrode 14 having an opening at the center and set to a predetermined potential, and enter the APD 15.

In this APD 15, a reverse bias voltage is applied between the anode 16 and the cathode 17 by a reverse bias power source 18, and the potential of the anode 16 facing the photoelectric conversion surface 13 is higher than that of the photoelectric conversion surface 13. Is also set to a high potential. When photoelectrons B collide with this APD 15, a pair of electrons and holes are generated for every 3.6 eV of energy lost by photoelectrons in APD 15 due to ionization, and this electron-hole pair is avalanche multiplied in APD 15. And is output as a current signal between the anode terminal 16a and the cathode terminal 17a. However, the energy lost by the photoelectrons in the APD 15 is not a constant value but follows a certain distribution, and the multiplication factor of the APD 15 is not a constant value but also has a certain distribution, so that electrons / holes output by one photoelectron The number of pairs also has a distribution.

Therefore, when the photodetector 10 measures a light flux having a constant light quantity many times, the number distribution of photoelectrons emitted from the photoelectric conversion surface 13 (photoelectron number distribution) is around a certain average value according to the light quantity. The current signal output from the photodetector 10 has a wider distribution, and the current signal output from the photodetector 10 has a further distribution according to the number distribution of electron-hole pairs output by one photoelectron.

The photodetector 10 using this APD has low noise and high sensitivity as compared with a photomultiplier tube and can count single photons. Furthermore, in the wave height distribution of the current signal obtained by measuring the light flux of a constant light quantity many times,
k-photoelectron event, that is, an event in which the number of photoelectrons emitted when a light beam is incident on the photoelectric conversion surface 13 is k (k = 1, 2, 3,
...) It has an excellent feature that the peaks corresponding to each can be identified. Since it has such characteristics, it is suitable as a photodetector used in the fluorescent substance quantification device according to the present invention.

The avalanche multiplication factor in the APD 15 of the photodetector 10 depends on the reverse bias voltage as shown in FIG. For example, when the reverse bias voltage is 90V, the multiplication factor is 1.8. When the reverse bias voltage is 145V, the multiplication factor is 32.6. When the reverse bias voltage is changed, the multiplication factor changes greatly. To do.

That is, when the amount of fluorescent light in the light incident on the entrance window 12 of the photodetector 10 is smaller than or equal to the amount of scattered light, the reverse bias voltage of the APD 15 is reduced to increase the avalanche multiplication factor. It is possible to reduce the scattered light detection sensitivity. On the contrary, when the fluorescent light amount is larger than the scattered light amount and the scattered light amount is negligible, the reverse bias voltage of the APD 15 can be increased to increase the avalanche multiplication factor and increase the fluorescence detection sensitivity.

With respect to the change in the reverse bias voltage applied to the APD 15, the trigger circuit 82 controls the output voltage of the reverse bias power source 18 based on the timing signal of the pulse laser light generation output from the pulse laser light source 80. By. That is, after the pulsed laser light is output from the pulsed laser light source 80, the fluorescence enters from the predetermined time when the light amount of the scattered light entering the entrance window 12 of the photodetector 10 becomes sufficiently smaller than the fluorescence light amount. During a certain period of time of being incident on the window 12 (for example, about 4 to 5 times the fluorescence lifetime), the trigger circuit 82 instructs the reverse bias power source 18 to have a relatively large reverse bias voltage (for example, 145).
V) is output. The trigger circuit 82 instructs the reverse bias power source 18 to output a relatively small reverse bias voltage (for example, 90 V) during a time other than the fixed time.

By doing so, the scattered light is measured at a low multiplication factor, and the fluorescence is measured at a high multiplication factor to eliminate or reduce the influence of the scattered light and accurately measure the fluorescent light amount. A current signal that can be measured and is proportional to the number of electron-hole pairs generated and multiplied by the APD 15 is detected by the photodetector 10.
As the output of the anode terminal 16a and the cathode terminal 17a
Is output between and.

The integrator 20, which receives the current signal output from the photodetector 10, integrates the current signal for a certain period of time and converts it into a voltage signal. In the integrator 20, a switch 21 and a capacitor 22 are provided in parallel between the anode terminal 16a of the photodetector 10 and the ground terminal.
The switch 21 opens and closes according to a gate signal output from the trigger circuit 82 based on a timing signal for generating pulsed laser light. The capacitor 22 is a switch 21
The current signal output from the photodetector 10 is integrated only during the period when is open, and the potential as the integration result appears at one terminal (point P in the figure). When the switch 21 is closed, the electric charge accumulated in the capacitor 22 is discharged and the potential at the point P becomes the ground potential.

This gate signal instructs the APD 15 to open the switch 21 at the same time as the large reverse bias voltage is applied or during the period when the large reverse bias voltage is applied, and the excitation pulse generation time and the next excitation. It is instructed to close the switch 21 at least once before the pulse generation time. This gate signal may have the same timing as the signal for controlling the reverse bias power source 18 described above.

The potential at the point P, which is the result of the integration of the current signal by the integrator 20, is amplified by the amplifier 30 so as to have an appropriate amplitude when the sample-hold circuit 40 and the AD converter 50 in the subsequent stage operate. The amplified voltage signal is sampled and held by the sample hold circuit 40, and is further converted by the AD converter 50 into a digital value corresponding to the voltage signal. The sample hold circuit 40 and the AD converter 50 also operate in synchronization with the pulse laser light generation timing. That is, the trigger circuit 8 is based on the timing signal for generating the pulsed laser light.
Controlled by 2, the digital value corresponding to the potential of the point P at a predetermined time immediately before the time when the switch 21 changes from open to closed is output.

A hist memory (wave height distribution generating means) 60 for inputting this digital value accumulates a predetermined value (for example, 1) at an address corresponding to the digital value every time integration is performed in the integrator 20 for a fixed time. to add. Integrator 20
By cumulatively adding the integration result of a large number of times to the hist memory 60, the peak value distribution of the voltage signal output from the AD converter 50 can be obtained.

On the basis of the pulse height distribution generated by the hist memory 60 and the pulse number N of the pulsed laser light counted by the counter 81, the calculation unit 70 calculates the photoelectrons emitted from the photoelectric conversion surface 13 of the photodetector 10. The number distribution (photoelectron number distribution) is estimated, and the light quantity of the incident light flux A is obtained based on the estimated photoelectron number distribution. For example, a computer is used as the arithmetic unit 70.

Next, a method of estimating the distribution of the number of photoelectrons emitted by the incident light flux on the photoelectric conversion surface 13 of the photodetector 10 will be described in detail. This estimation is performed in the calculation unit 70.

Prior to the estimation, first, the arithmetic unit 70 acquires the single photoelectron event wave height distribution generated in the hist memory 60 in the case of a single photoelectron event. Here, a single photoelectron event refers to an event in which a light beam is incident on the photoelectric conversion surface 13 of the photodetector 10 and one photoelectron is emitted, and a single photoelectron event wave height distribution refers to the event in which the event occurs. This is a wave height distribution generated by the history memory 60 in the case of the above. That is, the single photoelectron event wave height distribution is 1
Since one photoelectron is incident on APD15, APD1
5 shows the distribution of the number of electron-hole pairs output from No. 5. This single photoelectron event peak height distribution is represented by p ₁ (h) as a function of the peak value h. h is an output value from the AD converter 50. It should be noted that this single photoelectron event wave height distribution is obtained by making a very weak light beam incident on the photodetector 10 such that the probability of occurrence of a single photoelectron event is overwhelmingly dominant.

Since the noise of the amplifier system is superposed on the low wave height part of p ₁ (h), the actual data cannot be used. Therefore, in the low crest portion, the actual data is not used, and the extrapolated data is obtained from data of an adjacent crest region where noise is not superimposed. Also, p ₁ (h) is

[0037]

[Equation 1]

It is standardized in. h _max is the maximum peak value determined by the number of bits of the AD converter 50 that converts the peak value into a digital value.

Subsequently, the calculation unit 70 is generated in the hist memory 60 when the k-photoelectron event wave height distribution, that is, when a light beam is incident on the photoelectric conversion surface 13 of the photodetector 10 and k photoelectrons are emitted. The wave height distribution p _k (h)

[0040]

(Equation 2)

It is calculated recursively by the convolution calculation (k = 2, 3, ..., K). K is 2 or more. When an arbitrary distribution is assumed when estimating the photoelectron number distribution, the peak value that gives the peak value of p ₁ (h) is h _peak1 and K = h _max / h _peak1 . h _max = 4095,
If h _peak1 = 400, K is about 10. When the Poisson distribution is assumed as the photoelectron number distribution, K is about 2 to 3 times h _max / h _peak1 , that is, h _max = 4095 and h _peak1 = 400,
K should be about 30. Note that the evidence that it is possible to obtain the above manner height distribution p _k-1 (h) and height distribution from the convolution calculations of p ₁ and _{(h) p k (h)} , pulse height distribution p _k (h ) Is an electron avalanche-multiplied by k photoelectrons emitted from the photoelectric conversion surface 13 when entering the APD 15.
It is based on the fact that it represents the distribution of the number of hole pairs.

The photodetector 10, the integrator 20, the amplifier 30, the sample and hold circuit 40, and the AD converter 50.
If the spread of the wave height distribution due to the noise that occurs in P is not negligible, the wave height distribution p ₁ (h) can be calculated by deconvolution calculation to remove the influence of the noise from the wave height distribution p ₁ (h). Each _k (h) is calculated, and then the influence of noise is superimposed on each wave height distribution p _k (h) by convolution calculation (k =
2,3, ..., K).

As described above, a wave height distribution p _k (h) (k = 1, k = 1, considering the influence of noise peculiar to the photometric device)
2,3, ..., K) are prepared prior to the estimation of the photoelectron number distribution. FIG. 3 is a diagram showing the respective wave height distributions p _k (h) of the _k -photoelectron events thus obtained.

Then, the calculation unit 70 receives the luminous flux to be measured by the photodetector 10 to obtain the wave height distribution n (h) generated in the hist memory 60, and this wave height distribution n (h) and the above-mentioned From the wave height distribution p _k (h) in each case of a single photoelectron event and k-photoelectron event, the average value of the number of photoelectrons emitted from the photoelectric conversion surface 13 upon receiving the measured light flux, that is, the photoelectron number distribution, Estimate as required.

This wave height distribution n (h) does not need to be standardized and may be the value as it is stored in the hist memory 60.
That is, the peak height distribution n (h) represents the distribution of the events for which the peak value h was obtained. When estimating the distribution of the number of photoelectrons emitted from the photoelectric conversion surface 13 of the photodetector 10 after receiving the incident light flux (photoelectron number distribution), for example, the maximum likelihood method is used. That is, the probability of each k-photoelectron event occurring is _defined by q _k (k = 0,
1,2, ..., K)

[0046]

(Equation 3)

Q having the maximum log-likelihood represented by the following equation:
_{Find k} and use this as the estimated photoelectron number distribution. Where N
Is the number of measurements and h _min is the minimum peak value h that can be used for analysis. When the peak value h is small, the photodetector 1
Since noise due to 0, amplifier 30, etc. is superimposed, it cannot be used for analysis. Therefore, only the _peak value h _min or higher is analyzed. Also, p (h) and p _ND are

[0048]

(Equation 4)

[0049]

(Equation 5)

Is as follows. This p (h) is k-photoelectron event (k =
1,2,3, ..., K) represents the occurrence probability distribution of the peak value h in consideration of each occurrence probability (photoelectron number distribution). To obtain q _k that maximizes the log-likelihood of equation (3), a numerical calculation method such as the quasi-Newton method used for the optimization problem is applied.

When the Poisson distribution is assumed as the photoelectron number distribution, the probability q _k that each k-photoelectron event occurs
Is

[0052]

(Equation 6)

Is represented by Here, λ is the photoelectric conversion surface 1
3 is the average value of the number of photoelectrons emitted from No. 3. In this case, finding the photoelectron number distribution that maximizes the log likelihood means finding the λ value that maximizes the log likelihood, and can be found by numerical calculation such as the golden section method. In the following, a Poisson distribution is assumed as the photoelectron number distribution. FIG. 4 shows a wave height distribution (broken line) generated in the hist memory 60 by receiving the measured light flux by the photodetector 10 and a wave height distribution (solid line) calculated based on the λ value estimated by the maximum likelihood method. An example of Both have a peak value h _min (= 15
0) Good agreement is shown in the above range. The average value λ of the number of photoelectrons estimated at this time was 1.03.

Next, the operation of the fluorescent substance quantifying device according to the present invention will be described. FIG. 6 is a diagram showing changes over time in the pulse laser light (excitation light) light quantity, the fluorescence light quantity, each signal, and the like. In this figure, pulsed laser light (excitation light)
1 pulse generation time is set as time 0 (FIG. 6A).

The timing signal for pulsed laser light generation output from the pulsed laser light source 80 is input to the counter 81, and based on this timing signal, the number of pulses of the output pulsed laser light, that is, the number N of times of excitation of the fluorescent substance is N.
Is counted. The timing signal is also input to the trigger circuit 82, and the reverse bias power supply 18 and the switch 2
1. A signal for controlling the operation timing of each of the sample hold circuit 40 and the AD converter 50 is generated.

The pulsed laser light for exciting the fluorescent substance, which is output from the pulsed laser light source 80, is reflected by the reflecting mirror 83, the beam diameter is expanded by the lenses 84 and 85, and then,
The light is condensed by the lens 86 and is irradiated to the sample 90, and the sample 9
The fluorescent substance in 0 is excited. The fluorescence generated from this fluorescent substance is collected by the lenses 91 and 93, passes through the barrier filter 92, and enters the entrance window 12 of the photodetector 10. At this time, the pulsed laser light (scattered light) scattered by the sample 90 also travels in the direction of the entrance window 12, but most of it is absorbed by the barrier filter 92, and a part of it is transmitted and enters the entrance window 12. To do.

A light beam incident on the photodetector 10 (see FIG. 6)
(B)) occurs from the pulse laser light irradiation time, and then gradually decreases. This light flux has a scattered light amount larger than or about the same as the fluorescent light amount in the period of the pulse width from the irradiation time of the pulsed laser light, and thereafter, the scattered light amount becomes negligibly smaller than the fluorescent light amount. .

Then, in response to the change in the ratio of the fluorescent light amount and the scattered light amount of the incident light flux, the reverse bias power source 18
Are controlled by the trigger circuit 82 based on the timing signal of pulsed laser light generation. That is, the reverse bias power supply 18 outputs the reverse bias voltage of the voltage value V2 during the period from the time t1 to the time t2 after the generation of the pulsed laser light, and during the other period, the reverse bias power supply 18 outputs the voltage value V.
The reverse bias voltage of 1 (<V2) is output (see FIG. 6).
(C)). Each of the times t1 and t2 is a fixed time with each pulse generation time of the pulsed laser light as a reference (time 0), and at the time t1, the amount of scattered light, which was initially dominant, has a magnitude relation with the amount of fluorescent light. Is reversed, and the amount of scattered light becomes small enough to be ignored with respect to the amount of fluorescent light. At time t2, the time from time 0 becomes about 4 to 5 times the fluorescence lifetime. Therefore, in the period from time t1 to time t2, the amount of scattered light entering the photodetector 10 is sufficiently smaller than the amount of fluorescent light.

A to which such a reverse bias voltage is applied
Avalanche multiplication factor in PD15 (Fig. 6 (d))
Is G2 in the period from time t1 to time t2, and G2 in the other period according to the reverse bias voltage value.
1 (<G2). That is, in the period from time 0 to time t1 when the amount of scattered light is not negligible with respect to the amount of fluorescent light, the avalanche multiplication factor is small and the amount of scattered light is negligibly small with respect to the amount of fluorescent light or no longer occurs. During the period from t1 to time t2, the avalanche multiplication factor is large, and at time t2 when fluorescence no longer occurs.
The avalanche multiplication factor decreases again in the period from the occurrence of the next pulse.

The current signal (FIG. 6 (e)) output from the photodetector 10 in which the avalanche multiplication factor of the APD 15 changes in this way changes according to the incident light amount and this avalanche multiplication factor. That is, since the avalanche multiplication factor is small in the period from time 0 to time t1, the photodetector 1
Even if scattered light with a light amount larger than the fluorescent light amount is incident on 0,
The current signal output from the photodetector 10 is small. Time t
During the period from 1 to time t2, the avalanche multiplication factor is large, and the scattered light does not enter the photodetector 10. Therefore, the current signal output from the photodetector 10 depends on the amount of the incident fluorescence light. Will be things. After time t2, since the avalanche multiplication factor is small, even if background light enters,
The current signal output from the photodetector 10 is small.

A gate signal (FIG. 6 (f)) for controlling the integration operation in the integrator 20 for inputting this current signal, that is, the opening / closing of the switch 21 is generated based on the timing signal of the laser light output. The gate signal shown in this figure is
The instruction is to open the switch 21 only during the period from time t1 to time t2. For example, the time when the switch 21 is opened may be after the time t1 without being limited to this.

The current signal output from the photodetector 10 is
It is input to the integrator 20, and is integrated according to this gate signal only while the switch 21 is open. That is, the switch 21 opens at time t1 to start the integration operation, and the potential, which is the integrated value at each time thereafter, appears at the point P in FIG. 1 (FIG. 6 (g)). Then, at time t2, the switch 21
Is closed to complete the integration operation, and the potential at the point P becomes the ground potential. The potential at the point P is the ground potential at the time t1, then gradually increases, and eventually becomes a saturated state as the fluorescent light amount gradually decreases, and becomes a substantially constant value. The potential at the point P having the constant value corresponds to the total number of electron-hole pairs generated by the APD 15 due to the incidence of fluorescence on the photodetector 10 after time t1.

The potential of the point P amplified by the amplifier 30 is sampled and held by the sample hold circuit 40, and converted into a digital value by the AD converter 50. The timing is the integration operation end time t in the integrator 20.
Immediately before 2, that is, immediately before the gate signal is turned off. Sample signals that direct these operations (see FIG. 6).
(H) is also generated by the trigger circuit 82 based on the timing signal for generating the pulsed laser light.

The digital value is stored in the hist memory 6
When input to 0, the wave height distribution n (h) is generated. The calculation unit 70 calculates the wave height distribution n (h) and the wave height distributions p _k (h) (k) of the single photoelectron event and the k-photoelectron event separately obtained from the equations (1) and (2). = 1,2,3, ..., K),
From the value N obtained by counting the number of output pulses of the pulsed laser light by the counter 81, the λ value that maximizes the logarithmic likelihood represented by the equation (3) is obtained, and this is generated on the photoelectric conversion surface 13. It is estimated to be the average value of the number of photoelectrons. Then, the fluorescent light amount is obtained based on the average value of the photoelectron numbers thus estimated, and the fluorescent substance contained in the sample 90 is quantified.

Since it is necessary to refer to the relationship between the fluorescence lifetime and the integration time in FIG. 6, it is assumed that a large number of fluorescent photons are generated, and the amount of the fluorescent light and the current signal output from the photodetector 10 are considered. The time change is illustrated. However, when the amount of the fluorescent substance is small and the number of fluorescent photons is about several, the time change of the fluorescent light amount corresponds to the generation time of each fluorescent photon, not the gradual decrease curve shown in FIG. 6B. It will be pulsed. Even in such a case, the fluorescent substance quantification device according to the present invention can be effectively used.

In particular, in the present embodiment, since the photodetector using the APD is used, it is possible to estimate the photoelectron number distribution emitted on the photoelectric conversion surface of the photodetector with high accuracy. The amount of fluorescent light, that is, the fluorescent substance can be quantified with extremely high accuracy.

The present invention is not limited to the above embodiment, and various modifications are possible. For example, the fluorescent light amount may be immediately obtained from the current value output from the photodetector without using the integrator, the sample hold circuit, the AD converter, the hist memory, the calculation unit, and the counter. Further, it is not limited to the photodetector using the APD,
Other types of photodetectors, such as photomultiplier tubes and APDs
A single unit may be used. In these cases, the amount of fluorescent light can be obtained from the value obtained by smoothing the output from the photodetector. Further, the barrier filter provided in the condensing optical system is not essential.

In short, when the light flux entering the photodetector contains scattered light that becomes noise in addition to the fluorescent light to be measured, light detection is performed during the period in which the fluorescent light quantity is dominant compared to the scattered light quantity. The multiplication factor of the detector is increased, and when it is not, the multiplication factor of the photodetector is reduced. By doing so, only fluorescence is detected with high sensitivity, the detection sensitivity for scattered light is reduced and the influence is eliminated or reduced, the amount of fluorescence light is accurately measured, and the fluorescent substance is accurately quantified. It

The maximum likelihood method is most suitable for estimating the photoelectron number distribution, but other methods such as the least squares method can also be used.

[0070]

As described above in detail, in the fluorescent substance quantifying device according to the present invention, the excitation light source generates the excitation pulsed light to irradiate the sample containing the fluorescent substance, and the photodetector emits the fluorescent substance. The light flux to be measured containing the fluorescent light is received.
At this time, the photodetector detects the measured light flux with a large multiplication factor within a certain time range in which the amount of fluorescent light is dominant in the measured light flux, and otherwise detects the measured light flux with a small multiplication factor. To do. As a result, only the amount of fluorescent light can be detected with a high multiplication factor.

Further, in the optical path between the sample and the photodetector,
If a barrier filter that transmits fluorescence and blocks scattered light is provided, the detection sensitivity of scattered light is further reduced.

Further, by obtaining the number distribution of photoelectrons emitted by the photodetector according to the light quantity of the incident light flux from the wave height distribution, the fluorescence light quantity can be accurately measured.

As the photodetector used in this device, a photodetector using an avalanche photodiode whose gain can be changed by appropriately setting a reverse bias voltage is preferable. When estimating the number distribution of photoelectrons, the Poisson distribution is assumed and the maximum likelihood method is used.

Since this is done, even if the amount of fluorescent light generated from an extremely small amount of fluorescent substance in the sample is extremely weak, a scattered light is generated by a barrier filter provided between the sample and the photodetector. Even if the light partially penetrates and enters the photodetector together with the fluorescence, the influence of the scattered light incidence can be eliminated or reduced, and the amount of the fluorescent light, that is, the quantification of the fluorescent substance can be accurately performed. .

Therefore, for example, in the field of biochemistry and the like, it can be suitably used as an apparatus for measuring the amount of fluorescence emitted from a sample excited by a pulse laser light source and quantitatively measuring the number of fluorescent molecules in the sample. .

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a fluorescent substance quantification device according to the present invention.

FIG. 2 is a sectional view of a photodetector using an avalanche photodiode (APD).

FIG. 3 is a characteristic diagram of an avalanche multiplication factor with respect to a reverse bias voltage in APD.

FIG. 4 is a diagram showing a wave height distribution p _k (h) of a _k -photoelectron event.

FIG. 5 is a diagram showing a wave height distribution (broken line) obtained by actually measuring a light flux that is a measurement target, and a wave height distribution (solid line) calculated based on a λ value estimated by the maximum likelihood method. is there.

FIG. 6 is a diagram showing a temporal change of a pulsed laser light amount, a fluorescent light amount, each signal, and the like.

[Explanation of symbols]

10 photodetector, 11 vacuum chamber, 12 entrance window, 13
... Photoelectric conversion surface, 14 ... Focusing electrode, 15 ... Avalanche photodiode (APD), 16 ... Anode, 16a ...
Anode terminal, 17 ... Cathode, 17a ... Cathode terminal, 18 ... Reverse bias power supply, 19 ... High voltage power supply, 20 ... Integrator, 21 ... Switch, 22 ... Capacitor, 30 ... Amplifier, 40 ... Sample hold circuit, 50 ... AD conversion vessel,
60 ... Hist memory, 70 ... Calculation part, 80 ... Pulse laser light source, 81 ... Counter, 82 ... Trigger circuit, 83 ... Reflector, 84, 85, 86 ... Lens, 90 ... Sample, 91 ...
Lens, 92 ... Barrier filter, 93 ... Lens, A ... Incident light flux, B ... Photoelectron.

Claims (6)

[Claims] 1. An excitation light source that outputs an excitation pulse light based on a pulsed light generation timing signal; and a light flux to be measured including fluorescence generated from a fluorescent substance in a sample irradiated with the excitation pulse light, A photodetector that outputs a current signal according to the light amount of the measured light flux and a set multiplication factor, and based on the pulsed light generation timing signal, the measured light flux after the time when the excitation pulsed light is generated In the fixed time range, based on the gate signal, a gate signal generation unit that outputs a gate signal indicating whether or not a predetermined time range from a predetermined time after the time when the fluorescence light amount is dominant A fluorescent substance quantification device comprising: a multiplication factor changing means for setting the multiplication factor larger when the time is within the predetermined time range than the multiplication factor when the time is outside. 2. A barrier filter, which is provided on an optical path between the sample and the photodetector, transmits the fluorescence, and blocks scattered light which is scattered by the excitation pulsed light irradiated on the sample. The fluorescent substance quantifying device according to claim 1, further comprising: 3. The photodetector emits a number of photoelectrons according to a photoelectron number distribution according to the light quantity of the measured light flux,
An integrator that multiplies the photoelectrons by the multiplication factor and outputs a current signal, and that integrates the current signal for at least the fixed time to convert it into a voltage signal; and an AD that converts the current signal into a digital value according to the voltage signal. Conversion means, wave height distribution generation means for cumulatively adding a predetermined value to an address corresponding to the digital value to generate a wave height distribution of the voltage signal, and when the number of photoelectrons emitted by the photodetector is one, A single photoelectron event wave height distribution generation means for acquiring the wave height distribution generated by the wave height distribution generation means as a single photoelectron event wave height distribution; and the number k of photoelectrons emitted by the photodetector is 2 or more and a predetermined number or less. The k-photoelectron event wave height distribution generator for calculating the k-photoelectron event wave height distribution in each case by recursive convolution calculation based on the single photoelectron event wave height distribution. And the k-photoelectron event wave height for each of the wave height distribution generated by the wave height distribution generation means when the measured light flux enters the photodetector, the single photoelectron event wave height distribution, and the number k of photoelectrons. And a photoelectron number distribution estimating means for obtaining the light quantity of the measured light flux by estimating the photoelectron number distribution when the measured light flux enters the photodetector based on the distribution. The fluorescent substance quantification device according to claim 1. 4. The photodetector is applied with a reverse bias voltage between a photoelectric conversion surface that emits a number of photoelectrons according to a photoelectron number distribution according to the light intensity of the measured light flux, and an anode and a cathode. And, the portion facing the photoelectric conversion surface is set to a higher potential than the potential of the photoelectric conversion surface, the electron-hole pairs generated by inputting the photoelectrons are avalanche multiplied by the multiplication factor. An avalanche photodiode that outputs the current signal corresponding to the number of avalanche-multiplied electron-hole pairs; and an incident window that transmits the measured light flux, and the photoelectric conversion surface and the avalanche photodiode are internally provided. A vacuum container included in the above-mentioned, wherein the multiplication factor changing means has a reverse bias voltage within the fixed time range that is higher than the reverse bias voltage when outside the fixed time range. By setting the voltage increases,
The fluorescent substance quantifying device according to claim 1, wherein the multiplication factor is changed. 5. The fluorescent substance quantifying device according to claim 3, wherein the photoelectron number distribution estimating means estimates the photoelectron number distribution by a maximum likelihood method. 6. The fluorescent substance quantifying device according to claim 3, wherein the photoelectron number distribution estimating means estimates the photoelectron number distribution on the assumption that the photoelectron number distribution is a Poisson distribution.