JP2908742B2 - Photometric device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a photometric device for counting the number of photons in a light beam. For example, in the field of biochemistry, etc., the fluorescence generated from a sample excited by a pulsed light source is counted by photon counting to obtain the sample. It is used to quantitatively measure the number of fluorescent molecules in the sample.

[0002]

2. Description of the Related Art Conventionally, in order to quantitatively measure a fluorescent substance (or a fluorescent molecule) in a sample, 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 value of the photomultiplier tube.

[0003] However, when the amount of the fluorescent substance in the sample is very small and the fluorescence intensity is below the detection limit, the fluorometer cannot be used. Therefore, in such a case, a laser light source is used as the excitation light source. 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 output value of the photomultiplier.

A method for exciting a fluorescent substance in a sample with a pulsed laser beam output from a laser light source and quantifying the fluorescent substance in the sample from the result of fluorescence intensity measurement, that is, the output value from a photomultiplier tube, is a photomultiplier. There is a so-called photon counting method using the count value of the output pulse from the tube, and a method using the peak value of the output pulse from the photomultiplier tube. Furthermore, in the quantitative method by the photon counting method, there is a method of estimating that there is a fluorescent substance in proportion to the counted value, and a method of estimating a photoelectron emitted from a photoelectric conversion surface of a photomultiplier tube per sample irradiation of one pulse of laser light. There is a method of estimating the amount of the fluorescent substance from the probability p (0) that the number is 0.

[0005]

However, when it is estimated that there is a fluorescent substance in an amount proportional to the count value in the conventional quantification method based on the photon counting method, the following problem occurs. The number of photoelectrons emitted from the photoelectric conversion surface of the photomultiplier tube per sample irradiation of one pulse of laser light follows a photoelectron number distribution according to the amount of incident light. The probability that the number of photoelectrons emitted according to this photoelectron number distribution is 1 is p (1), and the probability that the number is 2 or more is p (x ≧ 2). It is assumed that the photoelectron number distribution on the photoelectric conversion surface follows the Poisson distribution. At this time, for example, if the average value λ of the number of photoelectrons emitted from the photoelectric conversion surface when the photomultiplier tube receives the fluorescence generated from the fluorescent material excited by the pulse laser light of one pulse exceeds 0.1, p (x ≧ 2) / p (1) is 5
% Or more. The larger the ratio, the greater the amount of fluorescence incident on the photomultiplier and the count of output pulses from the photomultiplier deviate from the proportional relationship. The error increases when the substance is determined.

[0006] The quantitative method using the zero probability has the following problems. In this quantitative method, assuming that the photoelectron number distribution on the photoelectric conversion surface follows the Poisson distribution, the average value λ of the photoelectron number is estimated by the following equation: λ = −log (p (0)) (1) and is proportional to this. Quantitatively determine the fluorescent substance. Here, the number of times of excitation by the pulse laser light is N
Assuming that the count value of the output pulse from the photomultiplier tube is n, p (0) = 1−p (x ≧ 1) = 1−n / N (2) , Λ = −log (1-n / N) (3) Even when the λ value is estimated by the above equation, when the λ value increases (for example, λ> 1.5), that is, when n is N
, The estimation error of the λ value increases.

In addition, the conventional method using the peak value of the output pulse from the photomultiplier tube has the following problems. In this method, the fluorescent substance is quantified on the assumption that the average number of photoelectrons emitted from the photoelectric conversion surface per pulse excitation is proportional to the average value <h> of the peak value of the output pulse.
Where <h> is

[0008]

(Equation 4)

Where N is the number of excitations,
n (h) is the count value of the output pulse of the photomultiplier tube whose peak value is h, and h _max is the maximum peak value determined by the number of bits of the AD converter that converts the peak value into a digital value; h
_min is the minimum peak value used for calculating the average peak value. h _min = 0 is desirable, but due to the noise of the amplifier system superimposed on the output pulse from the photomultiplier tube,
h _min = 0 cannot be set. Therefore, the average peak value calculated by equation (4) is different from the true value. Further, when the average number of photoelectrons increases, the frequency of outputting a pulse having a peak value larger than h _max increases. However, since the pulse having such a peak value is not counted, the average peak value is calculated by Expression (4). Calculating and quantifying the fluorescent substance will underestimate.

In order to solve such a problem, a photodetector using an avalanche photodiode (hereinafter, APD) (reference: Shawn J. Fagen, "Vacuum avalanc")
he photodiodes can count single photons ", Laser Fo
cus World, Nov. (1993) pp.125-132). A photodetector using this APD has lower noise and higher sensitivity than a photomultiplier tube, and can count even a single photon. Furthermore, in a peak distribution of a current signal obtained by measuring a light beam of a constant light amount many times, a k-photoelectron event, that is, an event in which the number of photoelectrons emitted by the light beam incident on the photoelectric conversion surface is k (k = 1, 2, 3,...) Has an excellent feature that peaks corresponding to each of them can be identified. Because of these features, it is expected that the number of photoelectrons emitted from the photoelectric conversion surface, that is, the quantity of the measured light beam can be accurately measured.

However, even in the photodetector using the APD, when the number of photoelectrons generated on the photoelectric conversion surface is about several (about 5 to 8 or less), the number of photoelectrons can be measured accurately. Yes, but if the number of photoelectrons is large,
The number of photoelectrons cannot be measured accurately.

For example, regarding spectrum measurement by a spectroscope, an intensity peak may appear at many wavelengths around a center wavelength. At this time, the amount of light in the entire wavelength band of the measured light beam is adjusted so that measurement by the photon counting method can be ideally performed for the largest peak intensity. However, when the difference between the peak intensities at the respective wavelengths is large, even if the maximum peak intensity can be measured with high accuracy, the quantum noise increases when the small peak intensity is measured, resulting in poor measurement accuracy. On the other hand, in order to measure a small peak intensity with high accuracy, it is necessary to increase the light amount of the whole light beam to be measured, and this time, the measurement accuracy of the large peak intensity deteriorates.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problem, and is intended to solve the above problem even when the light beam to be measured is incident on the photoelectric conversion surface of the photodetector and the number of photoelectrons emitted is large.
Alternatively, it is an object of the present invention to provide a photometric device capable of accurately measuring the light amount or spectrum of a measured light beam even when the peak intensity difference is large when measuring the spectrum of the measured light beam.

[0014]

According to the present invention, there is provided a photometric device comprising: (1) a photoelectric conversion surface which emits a number of photoelectrons according to a distribution corresponding to a light amount of a light beam to be measured; A predetermined number of multiplying units for injecting and multiplying the number of photoelectrons and outputting a current signal, and an entrance window for transmitting a light beam to be measured having a photoelectric conversion surface and a predetermined number of multiplying units therein. A vacuum vessel including a photodetector, (2) integrating means for integrating each of the current signals output from each of the predetermined number of multipliers for a predetermined time and converting them into a voltage signal, and (3) a voltage signal. AD conversion means for converting into a corresponding digital value;
(4) a pulse height distribution generating means for cumulatively adding a predetermined value to each of the predetermined number of multipliers and the address corresponding to the digital value, and generating a voltage signal peak distribution for each of the predetermined number of multipliers; 5) a single photoelectron event wave height distribution generating means for acquiring a wave height distribution generated by the wave height distribution generating means as a single photoelectron event wave height distribution when the number of photoelectrons incident on each of the predetermined number of multipliers is one; 6) The k-photoelectron event height distribution in each case where the number k of photoelectrons incident on each of the predetermined number of multipliers is 2 or more and a certain number or less is recursively calculated based on the single photoelectron event height distribution. K-photoelectron event wave height distribution generating means calculated by a volume calculation; and (7) a wave height to be measured is incident on the photoelectric conversion surface, and the emitted photoelectrons are incident on any of a predetermined number of multipliers to generate a wave height distribution. Wave height generated by means Based on the single photoelectron event wave height distribution and the k-photoelectron event wave height distribution in each case of the number k of photoelectrons, a predetermined number of multiplication units when the measured light flux enters the photoelectric conversion surface A photoelectron number distribution estimating means for estimating the photoelectron number distribution to obtain the light amount of the measured light beam.

The present device operates as follows. When the light beam to be measured that has entered the photodetector passes through the entrance window of the vacuum vessel and enters the photoelectric conversion surface, the number of photoelectrons corresponding to the amount of light is emitted from the photoelectric conversion surface. When a number of photoelectrons according to the photoelectron number distribution enter each of a predetermined number of multipliers in the photodetector, the photomultipliers are multiplied and a current signal is output. Each of the current signals output from each of the predetermined number of multipliers is integrated for a predetermined time by the integration means and converted into a voltage signal.
The conversion unit converts the voltage value into a digital value corresponding to the voltage signal. This digital value is input to a peak distribution generating means, and a peak distribution is generated for each of a predetermined number of multiplication units. Then, the single photoelectron event height distribution obtained by the single photoelectron event height distribution generating means and the k-photoelectron event height distribution calculated by the k-photoelectron event height distribution generating means (k = 2,3,
.., K) and the wave height distribution of each of the predetermined number of multipliers generated by the wave height distribution generating means when the light beam to be measured is incident on the photodetector. The distribution of the number of photoelectrons incident on each of the multipliers is estimated, and the light quantity of the measured light flux is obtained.

Each of a predetermined number of photomultipliers of the photodetector used in the present apparatus has a reverse bias voltage applied between an anode and a cathode, and a portion facing the photoelectric conversion surface has a photoelectric conversion surface. Is set higher than the potential of the avalanche, and avalanche multiplies the electron-hole pairs generated by inputting photoelectrons, and outputs a current signal corresponding to the number of avalanche-multiplied electron-hole pairs. Preferably, the photodiode is an avalanche photodiode. In this case, excellent quantitative performance is maintained, and the measurement range of the light amount of the measured light beam is expanded.

Further, the photoelectron number distribution estimating means may estimate the photoelectron number distribution by the maximum likelihood method, or calculate the photoelectron number distribution by assuming that the photoelectron number distribution is a predetermined distribution such as a Poisson distribution. It may be estimated.

Further, there is further provided a lens system provided on the front surface of the entrance window of the photodetector for enlarging or reducing the diameter of the incident light beam to be measured and for causing the enlarged or reduced light beam to be incident to the incident window. If it is provided, it is effective when measuring a measured light beam having a large light amount.

[0019]

Embodiments of the present invention will be described below in detail 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 configuration of the photometric device according to the present invention will be described. FIG. 1 is a configuration diagram of a photometric device according to the present invention.

The photodetector 10, which receives the incident light beam A, emits a number of photoelectrons in accordance with a distribution corresponding to the amount of light when the incident light beam A enters the photoelectric conversion surface 13, and the photoelectric conversion surface 13 emits the photoelectrons. This is to multiply the photoelectrons incident on each of a predetermined number of multipliers arranged in an array so as to face the conversion surface 13 and output a current signal. For example, an avalanche photodiode (hereinafter, APD) is suitably used as the multiplier.

In this photodetector 10, an entrance window 12 is provided in a part of a vacuum vessel 11 in which the inside is kept in a vacuum, and an incident light beam A is transmitted through the entrance window 12 and photoelectrically converted. The plane 13 is reached. Opposing the photoelectric conversion surface 13,
The APDs 15a to 15d are arranged in a one-dimensional or two-dimensional array, and the array is substantially parallel to the photoelectric conversion surface 13. In this figure, four APDs are arranged in a one-dimensional array. The photoelectric conversion surface 13 has, for example, -1 with respect to the APDs 15a to 15d arranged in an array.
Since a high voltage of 0 kV to −15 kV is applied by the high voltage power supply 19, the incident light beam A
, A number of photoelectrons B are emitted in accordance with the distribution according to the amount of the incident light flux A, and the photoelectrons B
Are accelerated by an electric field between the photoelectric conversion surface 13 and the APDs 15a to 15d, and are incident on any of the APDs 15a to 15d.

The APD 15a, one of which, will be described. Between the anode 16a and the cathode 17a,
A reverse bias voltage (for example,
+2.5 kV) is applied, and the potential of the anode 16 a facing the photoelectric conversion surface 13 is set to be higher than the potential of the photoelectric conversion surface 13. When the photoelectrons B are emitted from the region of the photoelectric conversion surface 13 facing the APD 15a, the photoelectrons B collide with the anode 15a, and the energy of the photoelectrons lost in the APD 15a by the ionization action is 3.6.
a pair of electrons and holes is generated per eV, and
This electron-hole pair is subjected to avalanche multiplication in the APD 15a, and is output as a current signal between the anode terminal and the cathode terminal. However, since the energy lost by the photoelectrons in the APD 15a is not a constant value but follows a certain distribution, and the multiplication factor of the APD 15a is not a constant value but follows a certain multiplication factor distribution. The magnitude of the signal also has a certain distribution.

Therefore, when the photodetector 10 measures a light beam of a constant light amount many times, it is emitted from the photoelectric conversion surface 13 and
The number distribution (photoelectron number distribution) of photoelectrons incident on the APD 15a becomes a distribution spread around a certain average value corresponding to the light amount, and the current signal output from the photodetector 10 is output from the APD 15 by one photoelectron. The distribution further expands in accordance with the distribution of the number of pairs of electrons and holes.

The other APDs 15b to 15d are the same as the above-described APD 15a. However, the potential difference between each of the anodes 16a to 16d and the photoelectric conversion surface 13 is as follows:
It may be a constant value or a different value. Further, the reverse bias voltages applied to the APDs 15a to 15d may be constant values or different values. When each of the APDs 15a to 15d has the same characteristics, it is convenient in the subsequent processing to apply the same potential and secure the same multiplication factor. On the other hand, when the APDs 15a to 15d have different characteristics, it is preferable to apply an appropriate potential to each of them in order to secure the same multiplication factor.

Each of the APDs 15a to 15d may be an independent element, or may be integrated on a substrate. In the latter case,
Since a large number of APDs having the same characteristics are precisely arranged at predetermined positions in an array, handling becomes easy.

The current signal output from the photodetector 10 is processed as follows. APD1 in the photodetector 10
First, the processing related to the current signal output from 5a will be described below. The same applies to the processing of the current signals output from the other APDs 15b to 15d.

The integrator 20a which receives the current signal output from the APD 15a integrates the current signal for a certain period of time and converts the current signal into a voltage signal. In the integrator 20a, a switch 21a and a capacitor 22a are provided in parallel between an anode terminal of the APD 15a of the photodetector 10 and a ground terminal. The switch 21a is opened and closed in synchronization with the timing at which the incident light beam A is incident on the photodetector 10. For example, the switch 21a is opened for a certain period from time t1 to time t2 based on the incident time of the pulsed incident light beam A. Only when the switch 21a is open, the capacitor 22a
The current signal output from the PD 15a is integrated, and the potential resulting from the integration appears at one terminal (point Pa in the figure). When the switch 21 is closed, the charge stored in the capacitor 22 is discharged, and the potential at the point Pa becomes the ground potential.

The potential at the point Pa, which is the result of the integration of the current signal by the integrator 20a, is amplified by the amplifier 30a so as to have an appropriate amplitude during the operation of the sample-hold circuit 40a and the AD converter 50 at the subsequent stage. The amplified voltage signal is sampled and held by the sample and hold circuit 40a.

Similarly, current signals output from the APDs 15i other than the APD 15a are similarly integrated for a predetermined time by the integrator 20i, and the potential resulting from the integration appears at the point Pi, and the potential at the point Pi is amplified by the amplifier 30i. Then, it is sampled and held by the sample and hold circuit 40i (i = b, c, d).

Each of the voltage signals sampled, held, and output by each of the sample-and-hold circuits 40a to 40d is sequentially input to an AD converter 50 via a multiplexer 45, and converted into a digital value corresponding to each voltage value. Is converted.

Sample hold circuits 40a through 40
d, the multiplexer 45 and the AD converter 50 also operate in synchronization with the timing at which the incident light beam A enters the photodetector 10, and for example, the point Pa at the time immediately before the time t2.
The voltage values obtained by amplifying the potentials of Pd through Pd are sampled and held by the sample and hold circuits 40a through 40d, respectively, and digital values corresponding to the respective voltage values are sequentially output from the AD converter 50. Note that an AD converter may be provided for each of the sample and hold circuits 40a to 40d without providing the multiplexer 45.

A history memory (wave height distribution generating means) 60 for inputting these digital values includes an integrator 20a to 2
Each time integration for a certain period of time is performed in each of 0d,
A predetermined value (for example, 1) is cumulatively added to an address corresponding to each of the APDs 15a to 15d and the digital value output from the AD converter 50. Integrators 20a through 20
0d, the APD 15a
The peak value distribution of the voltage signal output from the AD converter 50 is obtained for each of the signals 15 to 15d.

Based on the wave height distributions of the APDs 15a to 15d obtained in this manner, the arithmetic unit 70 calculates the number of photoelectrons emitted from the photoelectric conversion surface 13 of the photodetector 10 and incident on the APDs 15a to 15d. Is estimated (photoelectron number distribution), and the light amount of the incident light beam A is obtained based on the estimated photoelectron number distribution. For example, a computer is used as the arithmetic unit 70.

Next, a method for estimating the distribution of the number of photoelectrons emitted when the incident light beam enters the photoelectric conversion surface 13 of the photodetector 10 will be described in detail. This estimation is performed in the arithmetic unit 70. Note that this estimation method uses the APD
Since this is performed for each of 15a to 15d, the APD 15a will be described below.

Prior to the estimation, first, the arithmetic unit 70 acquires a single photoelectron event wave height distribution generated in the history memory 60 in the case of a single photoelectron event. Here, the single photoelectron event refers to an event in which one photoelectron is incident on the APD 15a, and the single photoelectron event wave height distribution is a wave height distribution generated in the history memory 60 when the event occurs. That is, the single photoelectron event wave height distribution represents the distribution of the number of electron-hole pairs output from the APD 15a when one photoelectron emitted from the photoelectric conversion surface 13 enters the APD 15a. This single photoelectron event peak height distribution is denoted by p ₁ (h) as a function of the peak height h. h
Is an output value from the AD converter 50. Note that the single photoelectron event wave height distribution is obtained by causing the photodetector 10 to enter an extremely weak light beam such that the probability of occurrence of a single photoelectron event is overwhelmingly dominant.

Since the noise of the amplifier system is superimposed on the low wave height portion of p ₁ (h), 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

[0038]

(Equation 5)

Is standardized. 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 calculates the k-photoelectron event wave height distribution, that is, the wave height distribution p generated by the history memory 60 when k photoelectrons enter the APD 15a.
_k (h)

[0041]

(Equation 6)

The recursive calculation is performed by the following 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 giving the peak value of p ₁ (h) is set as h _peak1 , and K = h _max / h _peak1 . h _max = 4095,
If h _peak1 = 400, K is about 10. When a Poisson distribution is assumed as the photoelectron number distribution, K is about two to three times h _max / h _peak1 , that is, if h _max = 4095 and h _peak1 = 400,
K is set to about 30. In addition, as described above, the reason why the wave height distribution p _k (h) can be _obtained from the convolution calculation of the wave height distributions p _k-1 (h) and p ₁ (h) is as follows.
This is based on the fact that the wave height distribution p _k (h) represents the distribution of the number of avalanche-multiplied electron-hole pairs when k photoelectrons emitted from the photoelectric conversion surface 13 enter the APD 15.

If the spread of the pulse height distribution caused by noise generated in the photodetector 10, the integrator 20a, the amplifier 30a, the sample hold circuit 40a, and the AD converter 50 cannot be ignored, the pulse height distribution p ₁ (h ), The influence of the noise is removed by the deconvolution calculation, and the peak height distribution p _k (h) is calculated by the equation (5).
Each is calculated, and then the influence of noise is superimposed on each of the peak height distributions p _k (h) by convolution calculation (k = 2, 3,..., K).

As described above, the peak height distribution p _k (h) (k =
1,2,3, ..., K) are prepared before estimating the photoelectron number distribution. FIG. 2 is a diagram showing each of the peak height distributions p _k (h) of the _k -photoelectron events thus obtained.

Then, the arithmetic section 70 receives the light beam to be measured by the photodetector 10 and obtains the peak height distribution n (h) generated in the history memory 60. From the wave height distribution p _k (h) in the case of a single photoelectron event and the case of a k-photoelectron event, respectively,
The average value of the number of photoelectrons incident on the PD 15a, that is, the photoelectron number distribution is estimated in the following manner.

The peak height distribution n (h) does not need to be normalized, and may be a value stored in the history memory 60.
That is, the peak height distribution n (h) represents the distribution of the events for which the peak value h was obtained. For example, the maximum likelihood method is used for estimating the distribution (number of photoelectrons) of the number of photoelectrons emitted from the photoelectric conversion surface 13 of the photodetector 10 after receiving the incident light beam and incident on the APD 15a. That is, assuming that the probability of occurrence of each k-photoelectron event is q _k (k = 0, 1, 2,..., K),

[0047]

(Equation 7)

Where the log likelihood expressed by
_k is obtained, and this is set as an 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 APD 15
Since the noise due to a and the amplifier 30a is superimposed and cannot be used for analysis, only the _peak value h _min or more is analyzed. Also, p (h) and p _ND are

[0049]

(Equation 8)

[0050]

(Equation 9)

Is as follows. This p (h) is the 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). In order to obtain q _k that maximizes the log likelihood of the equation (7), a numerical calculation method such as a quasi-Newton method used for an optimization problem is applied.

When the Poisson distribution is assumed as the photoelectron number distribution, the probability q _k that each of the k-photoelectron events will occur
Is

[0053]

(Equation 10)

Is represented by Here, λ is the APD 15a
Is the average value of the number of photoelectrons incident on. 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.
Is the peak height distribution (dashed line) generated in the history memory 60 by receiving the light beam to be measured by the photodetector 10 and the peak height distribution (solid line) calculated based on the λ value estimated by the maximum likelihood method.
Here is an example. Both show good agreement in the range above the _peak value h _min (= 150). The average value λ of the number of photoelectrons estimated at this time was 1.03.

As described above, the distribution of the number of photoelectrons incident on the APD 15a (photoelectron number distribution), that is, the average value of the number of incident photoelectrons is obtained. The same applies to the APDs 15b to 15d. The average value of the number of photoelectrons incident on each of the APDs 15a to 15d is A
This is the average value of the number of photoelectrons emitted in each of the areas of the photoelectric conversion surface 13 facing the PDs 15a to 15d, and represents the light amount of the light beam to be measured incident on that area. Therefore, from the average value of the number of photoelectrons incident on each of the APDs 15a to 15d,
Is obtained, the light quantity distribution of the measured light beam incident on
From the sum of the number of photoelectrons incident on each of the PDs 15a to 15d, the sum of the light amounts of the measured light flux incident on the photoelectric conversion surface 13 is obtained.

By measuring the light quantity of the measured light beam in this manner, even if the measured light beam is incident on the photoelectric conversion surface of the photodetector and the number of photoelectrons emitted is large, a plurality of A
Since the avalanche multiplication of the electron-hole pairs is performed in each PD, the amount of light of the measured light beam can be measured with high accuracy.

When a light beam to be measured having a large light quantity is measured, the light beam to be measured is not incident on the entrance window 12 in a spot-like manner in order to effectively use the photometric device.
It is preferable that the light is incident over a wide area. Therefore, when the light beam diameter of the measured light beam is small, it is preferable to provide a lens for enlarging the light beam diameter of the measured light beam on the front surface of the entrance window. In this way, the measurement range of the light quantity of the measured light beam can be expanded while maintaining excellent quantitative performance.

On the other hand, when measuring a light beam to be measured having a small light amount, the light beam to be measured is received in the entire area of the photoelectric conversion surface 13 and photoelectrons are incident on each of the APDs 15a to 15d. The number of photoelectrons incident on the surface becomes small, and it takes time for the measurement, resulting in poor efficiency. In this case, the light beam to be measured enters only a limited area of the photoelectric conversion surface 13 so that the APD 15a
If the photoelectrons are made incident on a part of 15d through 15d and only the current signal output from the APD on which the photoelectrons are incident is processed, the measurement can be performed efficiently in a short time.

Next, a case where the photometric device according to the present invention is applied to fluorescence spectroscopy will be described. FIG. 4 is an explanatory diagram of the fluorescence spectrometry using the photometric device according to the present invention.

The pulse laser light source 80 which outputs the pulse laser light as the excitation light also outputs a timing signal of the laser light output. The control unit 91 counts the number of pulses of the output pulse laser light, that is, the number N of times of excitation of the fluorescent substance, based on the timing signal, and switches 21a to 21d, sample and hold circuits 40a to 40d, a multiplexer 45, and an AD converter. It generates and outputs signals for controlling the operation of each of the device 50 and the history memory 60, and controls these operations.

The pulse laser light for exciting the fluorescent substance output from the pulse laser light source 80 is supplied to the lenses 81 and 8.
The light is condensed at 2 and irradiates the sample 83 to excite the fluorescent substance in the sample 83. Fluorescence generated from this fluorescent substance is collected by the lenses 84 and 86, passes through the barrier filter 85, and enters the spectroscope 90. Barrier filter 85
Transmits the fluorescence, but blocks the pulsed laser light scattered by the sample 83 and traveling toward the spectroscope 90.

The spectroscope 90 to which the fluorescent light is input disperses the fluorescent light into the component wavelengths and outputs only the luminous flux of the selected wavelength, and can scan the selected wavelength.
The luminous flux of a predetermined wavelength output from the spectroscope 90 is generally output with a certain spread angle. When the light quantity of this light beam is small, the light beam is converged by the lens 87 and is incident on a certain small area of the entrance window 12 of the photodetector 10. On the other hand, when the light amount of the light beam output from the spectroscope 90 is large, the light beam is incident on substantially the entire surface of the entrance window 12 of the photodetector 10 by the lens 87. As described above, this lens 87 is provided between the spectroscope 90 and the photodetector 10 along the optical axis so that the area where the light beam enters the entrance window 12 of the photodetector 10 can be variably set in accordance with the amount of the light beam. Preferably, it is movable along. When fluorescent light enters the photoelectric conversion surface 13 of the photodetector 10, photoelectrons are emitted in accordance with the amount of light, and photoelectrons enter the APDs 15a to 15d, respectively, and the number of avalanche-multiplied electron-hole pairs increases. A corresponding current signal is output. APD 15a to 15d
Each current signal output from each is integrator,
Amplifier, sample hold circuit, multiplexer, AD
The signal is input to a signal processing unit 100 including a converter, a history memory, and an arithmetic unit.

Hereinafter, the operation of the signal processing section 100 will be described together with the amount of pulse laser light (excitation light), the amount of fluorescent light, and the time change of each signal. First, APD 15a
The processing of the current signal output from the CPU and its timing will be described. FIG. 5 is an explanatory diagram of the operation of the photometric device according to the present invention, and FIG. 6 is a diagram illustrating a pulse laser beam light amount, a fluorescent light amount, and a time change of each signal. In FIG.
The pulse laser light (excitation light) generation time is used as a reference (FIG. 6A).

The amount of fluorescence (FIG. 6 (b)) generated from the fluorescent substance in the sample 83 is generated from the pulse laser beam irradiation time and then gradually decreases. Photodetector 1 that has received this fluorescence
The current signal output from the APD 15a of 0 (FIG. 6)
(C)) changes according to a change in the amount of fluorescent light. That is, the photoelectrons emitted from the photoelectric conversion surface 13
Since it takes some time until a current signal corresponding to the number of avalanche-multiplied electron-hole pairs incident on a is output, the magnitude of the current signal output from the APD 15 a It gradually increases from the time when it is performed, and then gradually decreases like the amount of fluorescent light.

The integrator 20a integrates the current signal output from the APD 15a. The integration time Tw starts at or immediately before the pulse laser light generation time and is set to be almost completed (for example, about 4 to 5 times the fluorescence lifetime) of the fluorescence emission. The control unit 91 generates and outputs a gate signal (FIG. 6D) for instructing that the switch 21 is opened only during the integration time Tw based on the laser light output timing signal. An electric charge obtained by integrating the current signal (FIG. 6C) output from the APD 15a during the integration time Tw is stored in the capacitor 22a. The potential at the point Pa in FIG. 5 (FIG. 6 (e)) gradually increases, but eventually becomes saturated and becomes a substantially constant value as the amount of fluorescent light gradually decreases. The potential at the point Pa at which the constant value is obtained is expressed by AP
This corresponds to the total number of electron-hole pairs generated in D15a.

The potential at the point Pa amplified by the amplifier 30a is sampled and held by the sample and hold circuit 40a. The timing is immediately before the end of the integration time Tw in the integrator 20a, that is, immediately before the gate signal is turned off. A signal instructing this operation (FIG. 6)
(F)) is also output from the control unit 91.

The current signals output from each of the other APDs 15b to 15d also correspond to the AP described above.
Processing is performed in the same manner as the processing for the current signal output from D15a. The operations in integrators 20a to 20b are performed at the same timing, and the operations in sample hold circuits 40a to 40d are also performed at the same timing. That is, APD
For each of the current signals output from each of 15a to 15d, an integration result from the same time to the same time after the generation of the pulsed laser light can be obtained.

Sample hold circuits 40a to 40d
The signals output from each are sequentially selected by the multiplexer 45, input to the AD converter 50, and are converted into digital values. The selection operation of the multiplexer 45 is performed after the elapse of the settling time of each of the sample / holds 40a to 40d (the time required until the output value is determined after the start of the sample / hold operation) and the address output from the control unit 91. Signal (APD1
5a to 15d). The operation of the AD converter 50 is performed after each output value from the multiplexer 45 is determined.
This is also performed according to a control signal output from control section 91.

The digital value output from the AD converter 50 is input to the history memory 60, and a predetermined value is cumulatively added to an address corresponding to the address signal and the digital value.
A peak height distribution n (h) for each of the APDs 15a to 15d is generated.

The arithmetic unit 70 calculates the peak height distribution n k (h) and the peak height distribution p _k (h) of each of the single photoelectron event and the k-photoelectron event separately obtained from the equations (5) and (6). )
(K = 1, 2, 3,..., K) and the value N obtained by counting the number of output pulses of the pulse laser light by the control unit 91, the log likelihood expressed by the equation (7) is obtained. Find the maximum λ value. In this manner, the average value of the number of photoelectrons incident on each of the APDs 15a to 15d is estimated, and the light amount of the light beam incident on the incident window 12 of the photodetector 10 is obtained from the sum of these.
Then, the selected wavelength of the spectroscope 90 is scanned, and the light amount of each of the wavelength components of the fluorescence is obtained in the same manner as described above, and the fluorescence spectrum is measured.

When the spectrum is measured as described above, the light intensity range that can be accurately detected by the photodetector is expanded by providing a plurality of APDs, so that the peak intensity difference of the spectrum of the measured light beam is large. Even in this case, the spectrum can be accurately measured.

In FIG. 6, the fluorescence lifetime and the integration time Tw are shown.
It was necessary to mention the relationship between the fluorescent light amount and the amount of fluorescent light.
The time change of the current signal output from 0 is illustrated.
However, when the amount of the fluorescent substance is very small and the number of fluorescent photons is about several, the time change of the amount of fluorescent light does not correspond to the gradually decreasing curve shown in FIG. 6B, but corresponds to the generation time of each fluorescent photon. It will be pulsed. Even in such a case, the photometric device according to the present invention can be used effectively.

In the above embodiment, the photodetector has 4
Although one APD is arranged in a one-dimensional array, the present invention is not limited to this. Even in a one-dimensional array, a larger number of APDs may be arranged, and for example, 4 ×
4, more generally m × n (m ≧ 2, n ≧ 2) A
The PDs may be arranged in a two-dimensional array, and the two-dimensional array arrangement is not limited to the arrangement in a rectangular shape, and may be arbitrarily arranged in a fixed-shaped area. . In any case, it is important that each of the plurality of APDs has an arrangement and an electric potential such that a light beam is incident on the photoelectric conversion surface and emitted photoelectrons are accelerated and incident.

Further, the photometric device according to the present invention can be applied to not only the above-described measurement of the fluorescence generated in a pulse shape but also other application modes. For example, it is also possible to measure the light amount of a continuously generated light beam. In this case, the integrator of the photometric device repeatedly performs integration for a fixed time, and the sample-hold circuit and the AD converter operate in synchronization with the operation of the integrator.
Based on the wave height distribution generated in the history memory at the time when the integration operation in the integrator is performed a predetermined number of times, the calculation unit estimates the number distribution of the photoelectrons emitted from the photodetector in the same manner as described above, and The light quantity of the measured light beam can be obtained based on the estimated value.

The distribution of the number of photoelectrons is most preferably estimated by the maximum likelihood method, but other methods, for example, the least square method can also be used.

[0076]

As described above in detail, in the photometric device according to the present invention, first, when the light beam to be measured enters the photoelectric conversion surface of the photodetector, photoelectrons are emitted, and the photoelectrons are emitted to a predetermined position in the photodetector. A current signal is output after being incident on one of the number multiplication units and multiplied. Here, the number of photoelectrons incident on each of the multipliers follows a photoelectron number distribution according to the amount of incident light flux. Each of the current signals output from each of the predetermined number of multipliers is integrated for a predetermined time by the integration means to become a voltage signal, and this voltage signal is converted to a digital value by the AD conversion means, and this digital value is converted into a peak value. The signal is input to the distribution generating means, and a peak distribution of the voltage signal is generated for each multiplication unit. The wave height distribution generated by the measured light beam incident on the photodetector, the wave height distribution of a single photoelectron event obtained by the single photoelectron event wave height distribution generating means, and the wave height distribution calculated by the k-photoelectron event wave height distribution generating means Height distribution of k-photoelectron events (k = 2,3,
..), The photoelectron number distribution estimating means estimates the photoelectron number distribution of each multiplication unit when the light beam to be measured enters the photodetector, and further calculates the light quantity of the light beam to be measured. It is preferable to use an avalanche photodiode as a multiplier of the photodetector used in the photometric device. In estimating the number distribution of photoelectrons, the maximum likelihood method is used assuming a Poisson distribution. Further, a lens system may be provided in front of the entrance window of the photodetector so that the beam diameter of the beam to be measured is enlarged so as to be incident on the entrance window.

With such a configuration, even when the measured light beam is incident on the photoelectric conversion surface of the photodetector and the number of emitted photoelectrons is large, the light amount of the measured light beam can be accurately measured. Can be. Therefore, when the present apparatus is used for spectrum measurement, the spectrum can be measured accurately even when the peak intensity difference is large when measuring the spectrum of the measured light flux. Further, for example, in the field of biochemistry and the like, the present invention can be suitably used as a photometric device for photon counting fluorescence generated from a sample excited by a pulsed 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 photometric device according to the present invention.

FIG. 2 is a diagram showing a wave height distribution p _k (h) of a _k -photoelectron event.

FIG. 3 is a diagram showing a wave height distribution (broken line) obtained by actually measuring a light flux to be measured and a wave height distribution (solid line) calculated based on a λ value estimated by the maximum likelihood method. is there.

FIG. 4 is an explanatory diagram of fluorescence spectroscopy measurement using the photometric device according to the present invention.

FIG. 5 is an explanatory diagram of an operation of the photometric device according to the present invention.

FIG. 6 is a diagram showing a pulse laser beam light amount, a fluorescent light amount, and a time change of each signal.

[Explanation of symbols]

10 photodetector, 11 vacuum chamber, 12 entrance window, 13
... photoelectric conversion surfaces, 15a, 15b, 15c, 15d ... avalanche photodiodes (APD), 16a, 16
b, 16c, 16d ... Anode, 17a, 17b, 17
c, 17d: cathode, 18: reverse bias power supply, 19 ...
High-voltage power supply, 20a, 20b, 20c, 20d ... integrator,
21a, 21b, 21c, 21d ... switch, 22a,
22b, 32c, 22d ... capacitors, 30a, 30
b, 30c, 30d ... amplifiers, 40a, 40b, 40
c, 40d: sample and hold circuit, 45: multiplexer, 50: AD converter, 60: history memory, 70:
Arithmetic operation unit, 80: pulse laser light source, 81, 82: lens, 83: sample, 84: lens, 85: barrier filter, 86, 87: lens, 90: spectroscope, 91: control unit, A: light beam to be measured, B: Photoelectrons.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01J 1/02 G01J 1/44 G01N 21/64

Claims (5)

(57) [Claims] 1. A photoelectric conversion surface that emits a number of photoelectrons according to a distribution according to a light amount of a light beam to be measured, and a number of photoelectrons according to a photoelectron number distribution are input and multiplied to output a current signal. A photodetector comprising a predetermined number of two or more multipliers, and a vacuum vessel having an entrance window through which the measured light beam is transmitted and including the photoelectric conversion surface and the predetermined number of multipliers therein; Integrating means for integrating each of the current signals output from each of the predetermined number of multipliers for a predetermined time to convert the current signal into a voltage signal; AD converting means for converting the current signal into a digital value corresponding to the voltage signal; A pulse height distribution generating means for cumulatively adding a predetermined value to each of the multipliers and the address corresponding to the digital value, thereby generating a pulse height distribution of the voltage signal for each of the predetermined number of multipliers; Multiplication section of each A single photoelectron event wave height distribution generating means for acquiring a wave height distribution generated by the wave height distribution generating means as a single photoelectron event wave height distribution when only one photoelectron is incident on the photomultiplier; Number of incident photoelectrons k
The k-photoelectron event crest distribution generating means for calculating the k-photoelectron event crest distribution in each case of 2 or more and a certain number or less based on the single photoelectron event crest distribution by a convolution calculation based on the recurrence, The photoelectron emitted when the light beam to be measured is incident on the photoelectric conversion surface is incident on one of the predetermined number of multipliers, and the wave height distribution generated by the wave height distribution generating means and the single photoelectron event Crest height distribution, based on the k-photoelectron event crest distribution in each case of the number k of the photoelectrons, based on the k-photoelectron event crest distribution, for each of the predetermined number of multipliers when the measured light flux is incident on the photoelectric conversion surface. A photoelectron number distribution estimating means for estimating the photoelectron number distribution to obtain the light quantity of the measured light flux. 2. A predetermined number of multiplying sections, to each of which a reverse bias voltage is applied between an anode and a cathode, and a portion facing the photoelectric conversion surface has a higher potential than a potential of the photoelectric conversion surface. An avalanche photodiode that avalanche multiplies the electron-hole pairs generated by inputting the photoelectrons and outputs the current signal according to the number of avalanche-multiplied electron-hole pairs. The photometric device according to claim 1, wherein: 3. The photometric device according to claim 1, wherein said photoelectron number distribution estimating means estimates said photoelectron number distribution by a maximum likelihood method. 4. The photometric device according to claim 1, wherein said photoelectron number distribution estimating means estimates said photoelectron number distribution on the assumption that said photoelectron number distribution is a Poisson distribution. 5. The light detector is provided in front of the entrance window of the photodetector, and the diameter of the incident light beam to be measured is enlarged or reduced, and the enlarged or reduced light beam to be measured is incident on the entrance window. The photometric device according to claim 1, further comprising a lens system for causing the light to be emitted.