JPH01213553A - Method for removing thermal noise of photodetector as well as method for measuring grain size and method for measuring immune reaction - Google Patents

Abstract

PURPOSE:To determine the exact power spectral density from which the thermal noise of a photodetector is removed and to exactly measure grain sizes and immune reaction by counting the output pulse signals of the photodetector at a certain time interval width and substituting the same into the specific equation. CONSTITUTION:A luminous flux 2 from a light source 1 is condensed by a condenser lens 3 and is projected to a transparent container 4 in which a sample contg. fine particles 5 is housed. One component of the light scattered by the particles 5 is inputted through a collimator 6, a photoelectron multiplier 7 and a preamplifier 8 to a pulse height discriminator 9 from which the pulse signals are selectively take out. Further, the photon number is supplied through a pulse generator 10 and a photon counter 11 to a computer 12. The power spectral density Sp(f) is determined by the equation II in accordance with the equation I in the computer 12. In the equations, tau denotes the time width; n(tau) denotes the number of the output pulse signal per tau; Var[n(tau)] denotes the variance of n(tau);(P)r denotes the time average of the number of the output pulse signals.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for removing thermal noise from a photodetector that has an adverse effect on determining power spectral density, and a particle size reduction method using the technique for measuring this power spectral density. and a method for measuring an immune response.

[Conventional technology]

JP-A-61-28866 discloses a conventional technique for measuring the concentration of a substance to be measured using the power spectrum density of scattered light intensity fluctuations. In this system, a photodetector detects scattered light obtained by projecting radiation into an antigen-antibody reaction solution, and the power spectral density is determined from an analog current signal.

In addition, as a conventional technique for determining the average particle size of a composite of IgG and anti-GC using photon correlation spectroscopy, Ann, C.
l1n, Bioche*, 1983:20:pp2
24-226 rt, tght scatterin
g and particle size measurement
element using photon cor
relation 5pec-trosc09V J
[Problems to be solved by the invention] In the conventional technology, the power spectral density is determined from the output signal containing the dark current of the photodetector, and the correlation function is determined, so accurate measurement is difficult. could not. Furthermore, if the scattered light is weak, there is a risk that the true scattered light output signal will be buried in the dark current signal, making it impossible to measure the concentration or particle size of the substance.

The purpose of the present invention is to remove dark current signals caused by thermal noise of a photodetector and obtain accurate power spectral density, and further, to accurately determine particle size from the power spectral density thus obtained. The purpose is to measure or measure the immune response.

[Means and actions for solving problems]

When counting the number of photons incident on a photodetector using the photon counting method, the probability of generating a pulse signal due to thermal noise generated from the photodetector is determined by focusing on the ratio of the zero variance and the average value according to the Poisson process. The ratio of the variance to the average value of the number of output pulse signals of the detector is greater than 1, and the ratio of the variance to the average value of the number of pulse signals caused by thermal noise is l. Therefore, by counting the output pulse signal of the photodetector in a certain time width, subtracting 1 from the ratio of the variance and the average value of this count value, and finding the power spectral density based on this function, the above The purpose is achieved.

This method is effective for various technologies that utilize the power spectral density of light. For example, light from a light source is projected onto a particle, scattered light from the particle is incident on a photodetector, the power spectral density is determined by the method described above, and the particle size of the particle is determined based on this power spectral density. Can be done. In addition, light from a light source is projected into a reaction solution containing a compound produced by the reaction between the substance to be measured and a substance that binds to the substance to be measured, and the scattered light from particles in the reaction solution is It is possible to determine the presence or absence of the substance to be measured or the concentration of the substance to be measured based on the power spectral density by determining the power spectral density using the method described above. It can be applied to diameter measurements and immune reaction measurements.

Examples of the photodetector used in this method include a photomultiplier tube and a photodiode, but any element that converts an optical signal into an electrical signal can be used.

In the measurement of immune reactions, the compound that is the source of some or all of the scattered light that enters the photodetector is a substance produced by a combination such as a ligand-receptor binding reaction or an antigen-antibody reaction. It is produced by the combination of an antigenic substance and an antibody, an immunoglobulin and an antibody of a foreign animal against this immunoglobulin, and a cell membrane protein and an antibody.

In addition, although this compound is basically formed by the combination of two types of substances, it is also made up of three types of substances that are formed by combining other substances with the composite consisting of these two types of substances. It includes composites, composites formed by combining various substances, and aggregated particles.

In addition, this compound is expected to dramatically increase the intensity of scattered light upon binding, so it is expected that this compound will be effective against blood cells and cells.

A substance that binds to the substance to be measured is immobilized on carrier particles such as gelatin particles, liposomes, and spherical latex particles.
Includes complexes formed by the combination of an insoluble substance and the substance to be measured.

〔Example〕

FIG. 1 is a diagram showing an embodiment of an apparatus for carrying out the method of the present invention. As the radiation, coherent light or incoherent light can be used, and in this embodiment, an argon gas laser (wavelength 514.5 no+) that emits coherent light is provided as the light source 1. A light beam 2 emitted from a light source 1 is condensed by a condenser lens 3 and projected onto a transparent container 4. A sample containing fine particles 5 is contained in the container 4 . One component of the scattered light (for example, 90″ side scattered light) scattered by the fine particles 5 in the container 4 is made to enter the photomultiplier tube 7 through the collimator 6 having a pair of pinholes. The output pulse signal is supplied to a pulse height discriminator (discriminator) 9 via a preamplifier 8 to selectively extract pulse signals caused by scattered light. This pulse signal is sent to a pulse generator 10 for waveform shaping of the pulse signal. After performing this, the number of photons is supplied to the computer 12 via the photon counting device 11.The computer 12 performs signal processing, calculations, etc., which will be described later, and performs estimation of power spectrum density, etc.

Next, a method of processing a photon pulse signal of scattered light will be explained.

FIG. 2(a) shows the output signal of the preamplifier 8. The photon pulse signal of the scattered light and the dark current pulse of the photomultiplier tube 7 are mixed. FIG. 2(b) shows the output signal of the discriminator 9. Pulse signals below the set darkness value are being cut. FIG. 2C shows the output signal of the pulse generator 10, which adjusts the intensity of the input pulse signal to generate a signal pulse of constant current. Photon counting device W111 counts the number of output signal pulses of the pulse generator and provides the count to computer 12.

Next, a method of determining the power spectrum density of scattered light from the fine particles 5 using the photon counting method will be explained. The photomultiplier tube 7 is in a state in which a high voltage is applied to cause a photomultiplying phenomenon and a weak optical signal can be detected. In addition to photon pulse signals of scattered light, dark current pulses caused by random thermal noise are always generated from this photomultiplier tube. This dark current pulse signal is caused by cosmic rays, thermal noise generated from the cathode of a photomultiplier tube, and the like. The dark current pulse signal due to thermal noise fluctuates with time, and the probability of its occurrence follows the Poisson process.At this time, if an input signal such as scattered light is included, the output of the photomultiplier tube 7 will change over a certain time width (time slot τ). Count the pulse signals and calculate the ratio of the variance to the average value (Varia
nce-Mean Ratio (hereinafter referred to as VMR) is l
bigger.

Here, nJ is the number of pulse signals in the first time slot, and 5 is the average value of the number of pulse signals. on the other hand,
The number of dark current pulse signals caused by thermal noise follows the Poisson process, so it is VMR-1.

The pulse signal obtained from the pulse generator 10 is as shown in FIG. Let the detection time be T, and further divide T into short time slots τ. Then, the count number of photons/clueless signals in the third time slot τ is set as n7(
τ), then the intensity function of the scattered light is p (t)
Then, P(t)=<P)t. . Pt (f
) ・exp (2x i f t) df. Here, Pt(f) is the Fourier transform of P(t),
represents the time average.

The VMR of equation (1) can be written as follows.

Here, <n(τ)> is the average value of the number of pulse signals in each time slot from J-1-N. A function φ(τ) obtained by subtracting the VMR of the number of dark current pulse signals caused by thermal noise from the VMR of the number of output pulse signals of the photomultiplier tube 7 is defined by the following equation.

=(τ/ (P > t) 5df(1/T) < l
Pt (f) l earthquake > 6 (sin” (gfr) 1
/(xfr)” where 〈〉 is the unsample average.

If the signal fluctuates over time, φ(τ)>0.

The power spectral density S p (f) can be determined from the following equation using φ(τ).

When f=(2τ)−1, 5p(f)= −<Pay τ ta (φ (
τ) / τ ) /d τ −・−・・−−−−
・(3) By determining the power spectral density using equation (3), the influence of thermal noise can be removed, and the power spectral density of the photon pulse signal of scattered light buried in the dark current pulse can be accurately estimated. .

Figure 4 shows the power spectrum density obtained by equation (3) and the power spectrum density including thermal noise. (3)
The equation was evaluated by computer simulation using a random pulse signal containing an intensity function P (t) with a Lorentzian power spectral density. Minimum time slot τ, -0,05sec, total number of time slots 3
0.000. <Pat = 1.200 and time slot T = 0.05 to 50 sec. In the figure, the curve (a) is the power spectral density determined. Curve 8 (b), which is significantly affected by thermal noise in the high frequency wI range after dHz, is the power spectral density determined by equation (3). Thermal noise components are removed from the low frequency range to the high frequency range, and the power spectrum density is obtained particularly accurately in the high frequency range.

As described above, the power spectral density can be calculated based on the function φ(τ) obtained by subtracting the dark ' due to thermal noise from the VMR of the output pulse signal of the photomultiplier tube by the VMR of the number of pulse signals. In addition to removing the thermal noise of the doubler tube,
The power spectral density can be determined accurately.

Furthermore, in this embodiment, an effective S/N ratio can be obtained even when the scattered light intensity is weak.

The above-mentioned method can be applied to the measurement of the particle size of various fine particles. The particle size is measured using the apparatus shown in FIG. A sample containing fine particles is placed in a container 4.

The concentration of fine particles in the sample is preferably adjusted by a photomultiplier tube 7 to a range where the scattered light intensity can be recognized as the number of current pulses generated by the incidence of photons, and optionally a liquid medium in which the fine particles are suspended or Dilute the sample in the same gaseous medium in which the particulates are suspended. The number of photons obtained by measuring the scattered light from the container 4 is supplied to the computer 12, and the power spectrum density from which thermal noise of the photomultiplier tube 7 has been removed is determined by equations (2) and (3).

Computer 12 also performs smoothing processing and particle size measurements.

Since the power spectral density determined by equations (2) and (3) contains many small fluctuation components as shown in Figure 5 (a), smoothing processing is performed to express the power spectral density with a smooth curve. .

The smoothing process can be performed, for example, in the following manner.

(1) Sequential power spectral density data stored in the memory of the combiator 12 al+alt・−a□−・
-..., a2,. For example, all-+ +
a n + 8.1 ii 1'-” a lt-1
'-'-+ a! Data a after performing smoothing processing once by calculating in order like N6”aENi1
', ~a'EN11 is obtained. Note that the same process is performed again and l, ~t! Obtaining MD allows approximation to a smoother curve.

(ii) Fourier transform the sequential power spectral density data to obtain a signal as shown in FIG. 5(b). Furthermore, when this is passed through a low-pass filter consisting of a digital filter that passes signals below frequency fc, and then subjected to inverse Fourier transform, a smooth power spectrum density is obtained as shown in FIG. 5(C).

(in) After smoothing φ(τ) by multiplying φ(τ) in equation (2) by cosine weighting (K-π/L, L is the number of moving averages + Xj is data), equation (3) By calculating the power spectral density using , a power spectral density with a very smooth curve can be obtained.

It should be noted that all the smoothing methods used in boat fishing can be used for smoothing processing, for example,
Needless to say, the proximal method using the first-order least squares method and the second-order least squares method described in the No. 041 publication can be applied.

For example, the particle size can be determined from the smoothed power spectrum density using the following method.

(i) The half width r of the power spectral density and the diffusion constant r have the following relationship.

The refractive index, θ, is the scattering angle. Further, according to the Stokes-Einstein relationship, the diffusion constant has the following relationship with the particle diameter d.

T D Snow 3 π η d Here, k is Boltzmann's constant, T is the absolute temperature, and η is the viscosity coefficient of the medium.

From the above formula, the particle diameter d has the following relationship with the half width r.

Therefore, after determining the half width of the power spectral density, the particle size or average particle size d can be determined from equation (4).

(ii) Figure 6 shows the white level S and particle size of the power spectral density S p (f) of a standard sample in which spherical latex particles of known particle size are suspended in an aqueous liquid medium at 0.4 wt%. It is a graph showing the relationship between. The white level S of the power spectral density changes in proportion to the particle size. This shows that the particle size of fine particles whose particle size is unknown can be measured from the white level of the power spectrum density.

Next, a method for determining the white level from the smoothed power spectrum density will be explained.

First, frequencies fo, 'f+, ft・-・ separated by a predetermined interval Δ are
・・−・・・−+r＊−・−・−・・−・−, (f
−−f−+・Δ) corresponding to the power spectral density 5
p(fo), 5p(f+).

5p (f snow), 5p (fs), -・-・・・・・
・, 5p(fa), ・・・・・・・−・−・− are compared sequentially, and when several or more than ten predetermined pieces of data decrease continuously, the data that starts to decrease first is The data immediately before is used as the 0th-order average level S, which is the frequency rp at the bending point.

Next, in order to measure the particle size of the fine particles contained in the sample, a calibration line is created to determine the particle size from the white level. First, prepare multiple standard samples with different particle sizes adjusted to a constant weight concentration.Next, the scattered light intensity of each of the standard samples with different particle sizes was measured using the apparatus shown in Figure 1, and the method described above was carried out. Calculate the white rail of the power spectral density at the 0th order, and from the obtained multiple white levels, calculate the particle size-white level relationship, φ (x, s) = a N + b −
−−−−−−−−(5) is obtained. Here, φ(X, S
) is the particle size at a certain weight concentration Xwt%, S is the white level, a is the slope, and b is the initial value.

Equation (5) has been created in advance for multiple weight concentrations to facilitate sample measurement (.

When measuring the particle size, first measure the weight concentration of fine particles contained in the sample, and adjust the weight concentration by diluting with a suspending medium so that the weight concentration is equal to the one at which the calibration line has been created. do. A sample adjusted to a predetermined weight concentration is placed in a container 4, set in the apparatus shown in FIG. 1, and the number of photons of scattered light from fine particles in the sample is measured, (2)
The power spectral density is determined by equation (3), the white level S of the smoothed power spectral density is calculated, and the particle size of the fine particles in the sample is determined from equation (5).

The particle size measurement method described in this example is applicable to spherical and non-spherical particles. In the case of non-spherical microparticles and a mixture of microparticles with different particle sizes, the average particle size can be determined.

As described above, the particle size measurement method described above can be used to measure the particle size in the process of manufacturing latex particles, and to measure the particle size of various fine particles in atmospheric dust and factory wastewater.

of.

The immune response is measured using the apparatus shown in FIG. A suspension of spherical latex particles having a substance that binds to the substance to be measured is immobilized on the surface is contained in the container 4.Next, when a sample containing the substance to be measured is added to the container 4,
The substance immobilized on the latex particles binds to the substance to be measured, and the latex particles aggregate together via the substance to be measured. latex particles 4x1
By storing 10 pl of a suspension containing 0' particles/Cta and adding 1 Opl of a sample containing CRP into this container, agglomerated particles can be formed.The substance to be measured is divalent particles. Examples include the above-mentioned antigens, immunoglobulins, cell membrane proteins, and other components involved in the ligand-receptor reaction. The substances used immobilized on carrier particles include the other component involved in the ligand-receptor reaction or the substance that binds to the ligand-receptor complex, that is, when the antigen to be measured is a multivalent antigen, its antigen-antibody. Also included are antibodies that bind to the complex.

It is also known that cells themselves serve as carrier particles and other components involved in ligand-receptor reactions.

After all, if the substance forms a complex or aggregate due to an immune reaction, it is possible to determine the presence or absence of an immune reaction and the concentration of the given substance by the measurement method shown in this example.

Based on the number of photons that changes depending on time obtained by measuring the scattered light from particles before reaction or aggregated particles formed after reaction contained in container 4, Equations (2) and (3) are calculated. The power spectrum density from which the thermal noise of the photomultiplier tube 7 has been removed is determined, and then, after the above-mentioned smoothing process is performed, the presence or absence of an immune reaction and the concentration of the predetermined substance are determined as follows. Can be done.

i) Determine the relaxation frequency (frequency at a position 3 dB below the white level) after the reaction between the sample and reagent, and determine whether there is an immune reaction or not and the specified substance in the sample from the calibration curve between the concentration and the relaxation frequency determined in advance. Determine the concentration of

ii) Calculate the white level value or integral value after the reaction between the sample and the reagent, and determine the presence or absence of an immune reaction and the concentration of the specified substance in the sample from a calibration curve between the predetermined concentration and the white level value or integral value. Determine.

1ii) The ratio of relaxation frequencies before and after the reaction between the sample and the reagent is determined, and the presence or absence of an immune reaction and the concentration of a predetermined substance in the sample are determined from a calibration curve of the concentration determined in advance and the relaxation frequency ratio.

iv) Calculate the white level value or the ratio of the integral value before and after the reaction between the sample and the reagent, and use the calibration curve between the predetermined concentration and the white level value or integral value ratio to determine the presence or absence of an immune reaction and the predetermined concentration in the sample. Determine the concentration of a substance.

(2) The ratio of relative fluctuation before and after the reaction between the sample and the reagent is determined, and the presence or absence of an immune reaction and the concentration of a predetermined substance in the sample are determined from a calibration curve of the concentration determined in advance and the relative fluctuation ratio.

The method of determining the concentration of a predetermined substance from the power spectral density is already known and does not require further detailed explanation, so it will be omitted. For example, JP-A-61-28866, JP-A-61-175549, and JP-A-62
Using the method described in Japanese Patent No. 291547, those skilled in the art can easily determine the presence or absence of an immune reaction and the concentration of the substance to be measured based on the smoothed power spectrum density described above.

According to this embodiment, the presence or absence of an immune reaction, that is, the presence or absence of a substance to be measured, or the concentration of a substance to be measured is determined based on the power spectrum density from which thermal noise of the photomultiplier tube 7 has been removed. It is possible to accurately determine the presence or absence of the substance to be measured or quantify the substance to be measured from among the detection signals buried in the detection signal.

In the above-mentioned examples of particle size measurement and immune reaction measurement, the apparatus shown in FIG. Although the power spectral density is measured by detecting photons of light scattered by fine particles contained in the reaction solution using a photomultiplier tube, the method of the present invention can be applied to any device that can detect photons of light scattered by fine particles. All can be used. - An example is shown in FIG.

FIG. 7 is a diagram showing an embodiment of an apparatus for carrying out the method of the present invention. Components that are the same as those in FIG. 1 are designated by the same reference numerals, and explanations thereof will be omitted. Polarizing plates 13 and 14 are installed on the optical axis of the light beam 2 so as to sandwich the container 4, and a photomultiplier tube 7 is placed on the optical axis of the light beam 2 to convert the 0° forward scattered light by the particles in the container 4. The point to be detected is different from the first rgJ. The polarizing plate 14 is provided so that only the polarized light component at 90° with respect to the polarization direction of the polarizing plate 13 passes through. Therefore, the orthogonal polarization of the linearly polarized light incident on the container 4
The O° scattered light of the 0 spherical particle, which the photomultiplier tube 7 detects only ion), has the same polarization component as the incident light and is therefore cut by the polarizing plate 14, resulting in multiple scattered light having a polarization component different from the incident light. Only the light passes through the polarizing plate 14. The transmitted light is polarized F
Completely cut by i13.14. In addition, non-spherical particles and aggregated particles exhibit optical anisotropy, and the 0° scattered light by these particles has a polarization component that is different from the polarization direction of the linearly polarized light incident on the container 4, so it is polarized light. It passes through plate 14. Therefore, the power spectral density is determined by the photon counting method using the orthogonal polarization components of the forward scattered light, and the particle size and immune reaction can be measured based on this power spectral density.

〔Effect of the invention〕

In the method described in claim 1.2, it is possible to remove dark current signals caused by thermal noise of the photodetector and obtain accurate power spectral density.

In the method according to claim 3, the particle size can be accurately measured.

In the method according to claim 4.6.7, an immune reaction can be accurately measured.

According to the method described in claim 5, a power spectral density having a smooth curve can be obtained from a power spectral density containing many small fluctuation components.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the configuration of an embodiment of an apparatus for carrying out the method of the present invention, and FIGS. 2(a), (b), and (c) each show a preamplifier 8. 3 is a diagram showing the output pulse signal of the discriminator 9° pulse generator IO; FIG. 3 is a diagram showing the relationship between the output pulse signal obtained from the pulse generator 10, the detection time T, and the time slot τ; FIG. The figures are graphs showing the power spectral density obtained by the method of the present invention and the power spectral density including thermal noise.
B). (C) is a graph for explaining the method of smoothing the power spectral density curve, Figure 6 is a graph showing the relationship between the particle size of spherical latex particles and white level S, and Figure 7 is a graph for explaining the method of smoothing the power spectrum density curve.
The figure is a diagram showing the configuration of an embodiment of an apparatus for carrying out the method of the present invention. 1- Light source 2.- Luminous flux 3 ・- Condensing lens 4..- Container 5 .
−・・Fine particles 6 ・−・Collimator 7−・
・−Photomultiplier tube 8 ・−・Preamplifier 9−・
・・Discriminator 10・・・Pulse generator 11−
・・Photon counting device 12・−・−Combiator 13
．． 14-...Polarizing plate (0) a machine = (C) Shearing 2 Mini frequency [Hzl ぢ 4 ゛((+) fc f (b) (C) , ↓56:

Claims (1)

[Claims] 1. In a method for determining the power spectrum density from the number of photons incident on a photodetector, the output pulse signal of the photodetector is counted in a certain time width, and the ratio of the variance and the average value of the counted values is calculated. 1. A method for removing thermal noise from a photodetector, characterized in that a function is obtained by subtracting 1 from , and a power spectrum density is obtained based on this function. 2. The above function is expressed as φ(τ)={Var[n(τ)]}/(<P>r・τ)
Based on this φ(τ), the formula Sp(f)=-<P>r・τ^2d{φ(τ)/τ)/
2. The thermal noise removal method for a photodetector according to claim 1, wherein the power spectral density Sp(f) is determined by dτ. Explanation of symbols in the formula: τ is the time width; n(τ) is the number of output pulse signals per τ: Var[n(τ)] is the variance of n(τ); <P>r is the number of output pulse signals The time average of; is shown respectively. 3. Project the light from the light source onto the particles, make the scattered light from the particles incident on the photodetector, determine the power spectral density by the method according to claim 1 or claim 2, and determine the power spectral density based on the power spectral density. A method for measuring particle size, characterized by determining the particle size of. 4. Project the light from the light source into the reaction solution containing the compound produced by the reaction between the substance to be measured and the substance that binds to the substance to be measured, and collect the scattered light from the particles in the reaction solution. The method is characterized in that the power spectrum density is determined by the method according to claim 1 or 2, and the presence or absence of the substance to be measured or the concentration of the substance to be measured is determined based on the power spectrum density. A method for measuring immune responses. 5. The measuring method according to claim 3 or 4, characterized in that smoothing processing is performed on the function φ(τ) or the power spectral density Sp(f). 6. The method for measuring an immune reaction according to claim 4, wherein the complex is generated by a reaction between a substance to be measured and an insoluble substance that binds to the substance to be measured. 7. The method for measuring an immune reaction according to claim 6, wherein the insoluble substance is a substance that binds to the substance to be measured and is immobilized on spherical fine particles.