WO2016125278A1 - Optical analysis device using single light-emitting particle detection - Google Patents

Abstract

Provided is a configuration for making it possible to suppress as much as possible the extent of the reduction of the ratio of the light amount of a light-emitting particle to a noise light amount in a scanning molecule counting method using a confocal microscope or multiphoton microscope for optical measurement. An optical analysis device according to the present invention includes a light detection area moving unit for moving the relative position within a sample liquid of a light detection area of an optical system of a microscope, a light irradiation unit for irradiating excitation light onto the light detection area, a light detection unit for detecting light from the light detection area, and a signal processing unit for moving the position of the light detection area within the sample liquid while generating time-series light intensity data for the light from the light detection area detected by the light detection unit and detecting individual light-emitting particle signals in the time-series light intensity data. A beam shaping element for essentially flattening the light intensity distribution of the excitation light within the light detection area is provided in the optical path of the excitation light.

Description

Optical analyzer using single luminescent particle detection

The present invention uses an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope, and uses atoms, molecules or aggregates thereof (hereinafter referred to as these) dispersed or dissolved in a solution. For example, biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, particulate objects such as viruses and cells, or non-biological The present invention relates to an optical analysis technique that can detect light from particles and obtain useful information in analysis or analysis of those states (interaction, binding / dissociation state, etc.). The present invention relates to an optical analysis apparatus that enables various optical analyzes by individually detecting light from a single light emitting particle using the optical system as described above. In the present specification, a particle that emits light (hereinafter referred to as “luminescent particle”) is either a particle that emits light itself, or a particle to which an arbitrary luminescent label or luminescent probe is added. The light emitted from the luminescent particles may be fluorescence, phosphorescence, etc. emitted by irradiation with excitation light.

With the recent development of optical measurement technology, detection of weak light at the level of one photon or fluorescent single molecule using an optical system of a confocal microscope and an ultrasensitive photodetection technology capable of photon counting (one-photon detection)・ Measurement is possible. Thus, various photoanalysis techniques have been proposed for detecting characteristics of biomolecules, intermolecular interactions, or binding / dissociation reactions using such weak light measurement techniques. Such optical analysis techniques include, for example, fluorescence correlation spectroscopy (FCS; see, for example, Patent Document 1-3), fluorescence intensity distribution analysis (Fluorescence-Intensity Distribution Analysis: FIDA, for example, Patent Document 4). ) And photon counting histogram (Photon Counting Histogram: PCH, for example, Patent Document 5) are known. Patent Documents 6 to 8 propose a method for detecting a fluorescent substance based on the passage of time of a fluorescence signal of a sample solution measured using an optical system of a confocal microscope.

Further, the applicant of the present application described in Patent Documents 9 to 12 is an optical analysis technique using an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope. Thus, a new optical analysis technique based on a principle different from optical analysis techniques such as FCS and FIDA has been proposed. In such a novel photoanalysis technique (hereinafter referred to as “scanning molecule counting method”), a micro area (hereinafter referred to as “light detection area”) that is a light detection area in the sample solution is used. In this case, the light detection region is dispersed in the sample solution while moving the position of the sample solution by the light detection region. When the randomly moving luminescent particles are included, the light emitted from the luminescent particles is individually detected, thereby detecting the luminescent particles in the sample solution one by one and counting the luminescent particles or in the sample solution. It is possible to obtain information on the concentration or number density of the luminescent particles.

JP2005-098766 JP2008-292371 JP2009-281831A Patent No. 4023523 International Publication 2008-080417 JP2007-20565 JP2008-116440 JP-A-4-337446 International Publication No. 2011/108369 International Publication No. 2011/108370 International Publication No. 2011/108371 International Publication No. 2012/099234

In the detection of luminescent particles by the above-mentioned “scanning molecule counting method”, in short, the light detection region that moves relative to the sample solution includes one luminescent particle at a time. Is detected, and the presence of the luminescent particles is detected. Therefore, in order to increase the detectable number of luminescent particles per unit time, it is conceivable to increase the moving speed of the light detection region or enlarge the light detection region. However, when the moving speed of the light detection region is increased, the time for which one light emitting particle is included in the light detection region is shortened, the amount of light obtained from the light emitting particle is reduced, and the detection sensitivity or accuracy is reduced. Become. On the other hand, when the photodetection area is enlarged, the amount of light other than the light of the luminescent particles to be observed (such as Raman scattered light of water molecules) (hereinafter referred to as “noise light”) increases. The detection sensitivity or accuracy will be reduced. In this regard, in more detail, typically, the intensity distribution of the excitation light within the light detection region has a substantially bell-shaped profile that has a maximum at the center and decreases toward the periphery. If the size of such an optical detection area (diameter) and the x times, its volume ^tripled x, the total light amount of the excitation light is directly reduces the excitation light intensity per unit volume, in particular, light The light emitted from the luminescent particles in the region close to the periphery of the detection region becomes very weak and the detection accuracy decreases. Therefore, when expanding the photodetection area, it is necessary to increase the total amount of incident excitation light. In that case, since the profile of the light intensity distribution is substantially bell-shaped as described above, the excitation light intensity in the central region of the light detection region is very high. However, even if the photodetection area is enlarged, the number of luminescent particles detected at one time is one, and the luminescent ability of the luminescent particles (the maximum amount of luminescence per unit time) is limited. Even if the intensity of the light that excites the luminescent particles increases, the increase in the intensity of the light from the luminescent particles reaches its peak when the light emission amount of the luminescent particles reaches the saturation range. That is, when the total amount of incident excitation light is increased as the photodetection area is enlarged, if the excitation light intensity exceeds the level at which the light intensity of the luminescent particles saturates, the excitation light intensity is emitted. It does not contribute to the light emission of the particles, but only increases the noise light, and causes only a further decrease in the detection sensitivity or accuracy of the light emitting particles.

By the way, in the time-series light intensity data measured in the scanning molecule counting method, the profile of the time change of the light intensity of the luminescent particles that pass relatively in the light detection region is typically , Having a substantially bell-shaped profile corresponding to the intensity distribution of the excitation light in the light detection region, the central region in the vicinity of the peak may not be sharp, and the region corresponding to the light intensity of the luminescent particles is a specific region. If it has a profile consisting of a significant sequence of light intensity values, the passage of luminescent particles can be detected. Further, as described above, when the emission amount of the luminescent particles reaches a limit at a certain excitation light intensity, the further increase in the excitation light intensity only increases the noise light, and may cause the S / N ratio to deteriorate. It will be. That is, in the scanning molecule counting method, when the photodetection area is enlarged, the excitation light intensity associated with the enlargement of the photodetection area is increased in the central area of the photodetection area where the excitation light intensity is higher than the peripheral area. It can be said that no enhancement is necessary. Such knowledge is used in the present invention.

Thus, the main problem of the present invention is that, in the scanning molecule counting method, when the photodetection area is enlarged in order to increase the detectable number of luminescent particles per unit time, An object of the present invention is to provide an optical analyzer for a scanning molecule counting method which is improved so as to reduce the ratio of the light amount, that is, the degree of deterioration of the S / N ratio as low as possible.

According to the present invention, the above problem is an optical analyzer that detects light from luminescent particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope, A light detection region moving unit that relatively moves the position of the light detection region of the optical system of the confocal microscope, a light irradiation unit that irradiates the light detection region with excitation light, and detects light from the light detection region Generate time-series light intensity data of light from the photodetection area detected by the photodetection section while moving the position of the photodetection section and the photodetection area in the sample solution. A signal processing unit that individually detects each signal of the luminescent particles in the data, and in the optical path of the excitation light in the light irradiation unit, in at least a part of the region including the center in the light detection region A beam-shaping element is provided to substantially flatten the light intensity distribution of the excitation light. It is achieved by its dependent device.

In such a configuration, “a light-emitting particle dispersed in a sample solution and moving randomly” is a particle that emits light, such as atoms, molecules, or aggregates thereof dispersed or dissolved in a sample solution. Any particle may be used as long as it is not fixed to the substrate or the like and freely moves in the solution in Brownian motion. Such luminescent particles are typically fluorescent particles, but may be particles that emit light upon irradiation with excitation light such as phosphorescence. The “light detection region” of the optical system of the confocal microscope is a minute region where light is detected in the microscope, and corresponds to a region where excitation light from the objective lens is collected. Also, typically, the light detection unit detects light from the light detection region by photon counting that counts the number of photons that arrive every predetermined measurement unit time (bin time), and in this case, the light intensity in time series The data becomes time-series photon count data. In the present specification, the term “light emitting particle signal” refers to a signal representing light from the light emitting particles unless otherwise specified.

As understood from the above, in the scanning molecule counting method, which is the basic configuration of the present invention, first, the position of the photodetection region is moved in the sample solution, that is, the light is passed through the sample solution. Light is sequentially detected while scanning with the detection region. Then, when the light detection region that moves in the sample solution includes light emitting particles moving at random, the light from the light emitting particles is detected, thereby detecting the presence of one light emitting particle. Therefore, the light signals from the luminescent particles are detected individually in the sequentially detected light intensity data, thereby detecting the presence of the particles individually one by one, and in the solution of particles. Various information related to the state is acquired. As specific data processing, processing described in Patent Documents 9 to 12 or other patent application relating to the scanning molecule coefficient method by the applicant of the present application may be arbitrarily used.

In the above-described configuration, in the apparatus of the present invention, in particular, in the optical path of the excitation light in the light irradiation unit, the light intensity distribution of the excitation light in at least a part of the region including the center in the light detection region. Is provided with a beam shaping element for substantially flattening. When such a beam shaping element is provided, the profile of the light intensity distribution in the light detection region is not substantially bell-shaped with the center being the maximum value, but at least a part of the region including the center with respect to the size of the light detection region Therefore, the maximum intensity of the excitation light with respect to the size of the light detection area is kept lower than when the light detection area of the substantially bell-shaped profile is enlarged. As a result, the amount of excitation light that does not contribute to the light emission of the luminescent particles can be reduced. In addition, since the region where the excitation light intensity is substantially maximum in the light detection region extends in a two-dimensional plane, the light intensity of the substantially bell-shaped profile with the same maximum excitation light intensity. Compared to the case of distribution, a strong signal persists for a longer time while one luminescent particle passes through the light detection region, and the signal of the luminescent particle can be detected more reliably. Be expected.

By the way, in the above configuration, the profile of the light intensity distribution in the light detection region is such that the excitation light intensity is substantially constant over a certain distance in the radial direction from the center to the optical axis. Unlike the cases of Patent Documents 9 to 12, the temporal change in the intensity of light emitted by one luminescent particle passing through the region is no longer substantially bell-shaped. Therefore, in the case of the present invention, the signal processing unit has a profile corresponding to the profile of the light intensity distribution of the excitation light substantially flattened in at least a part of the region including the center in the light detection region. It is possible to individually detect a change in light intensity with a light as an optical signal of one luminescent particle. The profile of the light intensity distribution of the excitation light substantially flattened in at least a part of the region including the center in the light detection region can be determined experimentally or theoretically in advance. The expression of the temporal change profile of the light intensity of the luminescent particles corresponding to the light intensity distribution of the excitation light, which is partially flattened, will be described in the embodiment section.

In the above, the beam shaping element typically uses a diffraction grating to emit a Gaussian beam in a radial direction from the center to the optical axis in a plane perpendicular to the optical axis. An element is employed that substantially constants the light intensity of the beam over a certain distance. In this case, when the position of the beam shaping element on the excitation light optical path is determined so that the light intensity is substantially constant at the focal plane of the objective lens, as will be described later, the focal plane of the objective lens is moved along the optical axis. It has been found that the profile of the light intensity distribution at a certain distance is different from the profile at the focal plane of the objective lens. And the profile of such light intensity distribution is unacceptably different from the substantially flattened profile in at least some areas including the center in the light detection area in the focal plane of the objective lens. When the light emitted from the light-emitting particles that has passed through the area enters the light detection unit, it is highly likely that the light from the light-emitting particles will not be detected as a signal from the light-emitting particles. Will increase and the S / N ratio will deteriorate. Therefore, in the above apparatus, light from a region where the profile of the light intensity distribution is unacceptably different from the profile at the focal plane of the objective lens along the optical axis of the objective lens reaches the light detection unit. It is preferable to limit as much as possible. In other words, the size of the light detection area in the optical axis direction of the objective lens is adjusted so that the light intensity distribution of the excitation light is smaller than the width in the optical axis direction of the objective lens in the area where the light intensity distribution is substantially flattened. It will be preferable to be done. In this regard, the aperture diameter of the pinhole arranged at the conjugate position of the focal position of the objective lens of the optical system is not only the size of the light detection area in the direction parallel to the focal plane of the objective lens, but also the light detection area. The size in the optical axis direction, that is, the range in which the light reaches the light detection unit is also determined. Therefore, the aperture diameter of the pinhole arranged at the conjugate position of the focal position of the objective lens of the optical system is also the width in the optical axis direction of the objective lens in the region where the light intensity distribution of the excitation light is substantially flattened. It may be determined by reference. That is, in the above-described apparatus of the present invention, preferably, the aperture diameter of the pinhole disposed at the conjugate position of the focal position of the objective lens of the optical system is substantially equal to the light intensity distribution of the excitation light. It may be set based on the width in the optical axis direction of the objective lens in the flattened region.

In the embodiment, the reason why the light intensity distribution of the excitation light in at least a part of the region including the center in the light detection region is substantially flattened is that the light detection region is enlarged from a certain state. This is because when the incident light quantity of the excitation light is increased, an increase in the intensity of the excitation light in the central area in the light detection area is suppressed. Therefore, in the above configuration, preferably, when the beam shaping element is used, the intensity of the excitation light in the light detection region is the same as the incident light amount of the excitation light and the beam shaping element is not used. The intensity of the excitation light is adjusted so as to be reduced as compared with the case. As described above, the reason why the light intensity distribution of the excitation light is corrected using the beam shaping element is to avoid that the intensity of the excitation light exceeds the limit of the luminous ability of the luminescent particles and becomes unnecessarily high. Therefore, in order to avoid unnecessary excitation light being incident, more preferably, the excitation light intensity in the region where the light intensity distribution in the light detection region is flattened when the beam shaping element is used. Is preferably adjusted to the minimum intensity at which the luminous ability of the luminescent particles reaches a limit, but is not limited thereto. It can be experimentally confirmed whether or not the luminous ability of the luminescent particles is limited at a certain excitation light intensity.

The optical analysis technique of the present invention described above is typically a biological molecule such as a protein, peptide, nucleic acid, lipid, sugar chain, amino acid or aggregate thereof, or a particulate biological object such as a virus or cell. It is used for analysis or analysis of the state of matter in solution, but it may also be used for analysis or analysis of the state of non-biological particles (eg, atoms, molecules, micelles, metal colloids, etc.) in solution. It should be understood that such cases are also within the scope of the present invention. Method and mode of moving the position of the light detection region with respect to the sample solution, method and mode of extracting or detecting the signal of each luminescent particle from the light intensity value in the time-series light intensity data, parameters for determining the absolute concentration value The method and aspect for determining the value may be the same as the method and aspect described in Patent Documents 9 to 12 and the like.

Thus, according to the configuration of the present invention described above, as already mentioned, by using the beam shaping element, the profile of the light intensity distribution in the light detection region has a certain radial direction from the center to the optical axis. Since the excitation light intensity becomes substantially constant over the distance, the intensity of the excitation light in the center area of the light detection area becomes excessively high when the light detection area is enlarged and the excitation light incident light quantity is increased. This can be avoided. And since the light quantity of the excitation light which does not contribute to the increase in the light emission amount of the light emitting particles can be suppressed as compared with the case where the beam shaping element is not used, the deterioration of the S / N ratio is kept low, and the light detection region. By expanding the range, it becomes possible to increase the number of detectable luminescent particles per unit time. In particular, in the scanning molecular coefficient method, until now, when the concentration of the luminescent particles in the sample solution was low, it took a relatively long time to detect the number of luminescent particles necessary for the analysis. For example, it is expected that the optical measurement time by the scanning molecular coefficient method can be shortened in a state where the degree of deterioration of the S / N ratio is kept low.

Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

FIG. 1A is a schematic diagram of the internal structure of an optical analyzer that performs the scanning molecule counting method according to the present invention. FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope). FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 to move the position of the light detection region in the sample solution. FIG. 1D is a schematic diagram of a mechanism for moving the position of the photodetection region in the sample solution by moving the horizontal position of the microplate. FIGS. 2A and 2B are a schematic diagram for explaining the principle of light detection in the scanning molecule counting method to which the present invention is applied, and a schematic diagram of time variation of the measured light intensity, respectively. 3A is a schematic diagram of the light detection region having a diameter d1, and FIG. 3B is an intensity distribution of the excitation light Ex and the fluorescence Em of the luminescent particles in the light detection region of FIG. 3A. FIG. FIG. 3C is a schematic diagram of a light detection region (when no beam shaping element is used) having a diameter d2 (> d1), and FIG. 3D is a diagram illustrating the light detection region of FIG. It is the figure which represented typically the intensity distribution of excitation light Ex in this and fluorescence Em of the light emission particle. FIG. 3E is a schematic diagram of a photodetection region having a diameter d2 in which the central region FR is flattened using a beam shaping element. FIG. 3F is a diagram illustrating the photodetection region in FIG. It is the figure which represented typically the intensity distribution of excitation light Ex in this and fluorescence Em of the light emission particle. FIG. 4A is a diagram for explaining a change in the light intensity distribution in the light detection region in the optical axis direction of the objective lens when there is no beam shaping element in the illumination light optical system and when it exists. . FIG. 4B is a diagram for explaining the action of the pinhole arranged at the focal position of the objective lens. FIG. 4C is a diagram for explaining that most of light emitted from a position away from the focal position of the objective lens in the optical axis direction of the objective lens cannot pass through the pinhole. FIG. 5 is a flowchart showing the procedure of the scanning molecule counting method executed in accordance with the present invention. FIGS. 6A and 6B show the case where the luminescent particles traverse the light detection region while performing Brownian motion and the position of the light detection region in the sample solution at a speed faster than the diffusion movement speed of the luminescent particles. It is a model figure showing the mode of movement of particles when luminous particles cross a photodetection region by moving. FIG. 6C shows the signal processing process of the detection signal in the processing procedure for detecting the presence of the luminescent particles from the measured time-series light intensity data (time change of the photon count) according to the scanning molecule counting method. It is a figure explaining an example.

1 ... Optical analyzer (confocal microscope)
DESCRIPTION OF SYMBOLS 2 ... Light source 3 ... Single mode optical fiber 4 ... Collimator lens 5 ... Dichroic mirror 6, 7, 11 ... Reflection mirror 8 ... Objective lens 9 ... Microplate 10 ... Well (sample solution container)
DESCRIPTION OF SYMBOLS 12 ... Condenser lens 13 ... Pinhole element 13 ... Pinhole aperture 14 ... Barrier filter 14a ... Dichroic mirror or polarizing beam splitter 15 ... Multimode optical fiber 16 ... Photo detector 17 ... Mirror deflector 17a ... Stage position change device 18 …Computer

Hereinafter, preferred embodiments of the present invention will be described in detail.

Configuration of Optical Analysis Apparatus An optical analysis apparatus that realizes the optical analysis technique according to the present invention has a basic configuration described in Patent Documents 9 to 12, as schematically illustrated in FIG. The apparatus may be a combination of an optical system of a confocal microscope capable of executing a scanning molecule counting method and a photodetector. Referring to FIG. 1, optical analysis apparatus 1 includes optical systems 2 to 17 and a computer 18 for controlling the operation of each part of the optical system and acquiring and analyzing data. The optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber. The light is emitted as a divergent light at an angle determined by a specific NA at the outgoing end of the light, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, the reflection mirrors 6, 7, and the objective lens 8. Is incident on. As will be described later, in the apparatus of the present invention, a beam shaping element 100 is inserted between the collimator 4 and the dichroic mirror 5 (for example, AdolOptica Optical Systems (Germany) Systems GmbH) Focal-πShaper etc. can be used. Above the objective lens 8, a microplate 9 in which sample containers or wells 10 into which a sample solution of 1 to several tens of μL is dispensed is arranged is typically disposed from the objective lens 8. The emitted laser light is focused in the sample solution in the sample container or well 10 to form a region (excitation region) with high light intensity. In the sample solution, light-emitting particles that are the observation target, typically particles to which fluorescent labels such as fluorescent particles or fluorescent dyes are added are dispersed or dissolved, and these light-emitting particles enter the excitation region. In the meantime, the luminescent particles are excited and light is emitted. The emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter 14. (Here, only the light component of a specific wavelength band is selected.), Introduced into the multimode fiber 15, reaches the photodetector 16, is converted into a time-series electrical signal, and then to the computer 18. Input and processing for optical analysis is performed in a manner described later.

In the above configuration, the pinhole 13 is arranged at a position conjugate with the focal position of the objective lens 8 as known to those skilled in the art. Only the light emitted from the focal region of the laser beam, that is, the excitation region as schematically shown, passes through the pinhole 13, and the light from other than the excitation region is blocked. The focal region of the laser beam illustrated in FIG. 1B is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL (typically, the light intensity is in the region). The distribution is a substantially bell-shaped distribution (Gaussian distribution) with the center at the apex, and the effective volume is the volume of a substantially elliptical sphere with the boundary where the light intensity is 1 / e ² of the center light intensity. It is called a confocal volume. The dimensions of the light detection region in the optical axis direction and the focal plane direction are determined by the diameter of the pinhole 13. In this regard, as will be described in detail later, in the present invention, light from a region where the profile of the light intensity distribution of the excitation light is unacceptably different from the profile at the focal plane of the objective lens is detected. The condition of the aperture diameter of the pinhole 13 is set so as to adjust the range in the optical axis direction of the photodetection region so that it can be limited as much as possible.

Further, in the present invention, light from one luminescent particle, for example, faint light from one fluorescent dye molecule is detected. Therefore, the photodetector 16 is preferably an ultra-high light that can be used for photon counting. A sensitive photodetector is used. When the light detection is based on photon counting, the light intensity is measured in a mode in which the number of photons arriving at the light detector is sequentially measured for each measurement unit time (BIN TIME) over a predetermined time. Executed. Therefore, in this case, the time-series light intensity data is time-series photon count data. Further, a stage position changing device 17a for moving the horizontal position of the microplate 9 may be provided on a microscope stage (not shown) in order to change the well 10 to be observed. The operation of the stage position changing device 17a may be controlled by the computer 18. With this configuration, it is possible to achieve quick measurement even when there are a plurality of specimens.

Further, in the optical system of the above optical analyzer, a mechanism for scanning the sample solution with the light detection region, that is, for moving the position of the focal region, that is, the light detection region in the sample solution. Is provided. As a mechanism for moving the position of the light detection region, for example, a mirror deflector 17 that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG. A method of moving the absolute position of the light detection area). Such a mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope. Alternatively, as another embodiment, as illustrated in FIG. 1D, the horizontal position of the container 10 (microplate 9) into which the sample solution is injected is moved to detect light in the sample solution. The stage position changing device 17a may be operated to move the relative position of the region (a method of moving the absolute position of the sample solution). In addition, the optical detection area is moved around the scanning trajectory by changing the optical path and moving the absolute position of the optical detection area. The position of the scanning trajectory of the photodetection area in may be moved along a predetermined movement path. In any case, the mirror deflector 17 or the stage position changing device 17a cooperates with the light detection by the light detector 16 under the control of the computer 18 in order to achieve a desired movement pattern of the position of the light detection region. Driven. The scanning trajectory of the position of the light detection region may be a closed circulation path such as a circle or an ellipse, and the movement path of the position of the sample solution is arbitrarily selected from a circle, an ellipse, a straight line, a curve, or a combination thereof (Various movement patterns may be selected in the program in the computer 18). Although not shown, the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 or the stage up and down.

Furthermore, in the optical analyzer 1, a plurality of excitation light sources 2 may be provided as shown in the figure, and the wavelength of the excitation light may be appropriately selected according to the excitation wavelength of the luminescent particles. Similarly, a plurality of photodetectors 16 may be provided, and when a plurality of types of light emitting particles having different wavelengths are included in the sample, light can be separately detected based on the wavelengths. It's okay.

The computer 18 includes a CPU and a memory, and the CPU executes various arithmetic processes to execute the procedure of the present invention. Each procedure may be configured by hardware. All or a part of the processing described in the present embodiment may be executed by the computer 18 using a computer-readable storage medium storing a program for realizing the processing. That is, the computer 18 may realize the processing procedure of the present invention by reading a program stored in a storage medium and executing information processing / calculation processing. Here, the computer-readable recording medium may be a magnetic disk, a magneto-optical disk, a CD-ROM, a DVD-ROM, a semiconductor memory, or the like. Alternatively, the above-mentioned program is distributed to a computer via a communication line. The computer that has received the distribution may execute the program.

As described in the “Summary of Invention” section of the principle of the optical analysis technique of the present invention, in the optical analysis apparatus of the present invention, the excitation light Ex is passed through the beam shaping element 100 as described above. Thus, the light intensity distribution of the excitation light in the light detection region has a flattened profile in at least a part of the region including the center, that is, the central region, instead of a substantially bell-shaped profile. As a result, when the size of the light detection region and the amount of incident light of the excitation light Ex are increased, an unnecessary increase in the excitation light intensity in the central region is avoided, and the S / N ratio (the intensity of the luminescent particles relative to the intensity of the noise light) is avoided. The degree of deterioration of the light intensity ratio) can be kept as low as possible. Hereinafter, the principle of the scanning molecule counting method and a novel configuration using the beam shaping element according to the present invention will be described.

1. Principle of Scanning Molecule Counting Method “Scanning Molecule Counting Method” (Patent Documents 9 to 12) basically drives a mechanism (mirror deflector 17) for moving the position of a light detection region to drive an optical path. Or by moving the horizontal position of the container 10 (microplate 9) into which the sample solution is injected, as schematically shown in FIG. In this case, light detection is performed while moving the position of the light detection region CV, that is, while scanning the sample solution by the light detection region CV. Then, for example, when the light detection region CV moves (time t0 to t2 in the figure) and passes through the region where one light emitting particle exists (t1), light is emitted from the light emitting particle, A pulse-like signal having a significant light intensity (Em) appears on the time-series light intensity data as depicted in FIG. Thus, the movement of the position of the light detection region CV and the light detection are executed, and one pulse-like signal (a significant change in light intensity with time) as illustrated in FIG. By detecting each of them, the luminescent particles are individually detected, and by counting the number thereof, information on the number, concentration, or number density of the luminescent particles present in the measured region can be acquired. In the principle of the scanning molecule counting method, statistical calculation processing such as calculation of fluctuations in fluorescence intensity is not performed, and luminescent particles are detected one by one. Therefore, FCS, FIDA, etc. have sufficient accuracy. Even with a sample solution in which the concentration of particles to be observed is so low that analysis is impossible, information on the concentration or number density of particles can be obtained. As described in detail below, in the present invention, the light intensity distribution in the plane perpendicular to the optical axis of the excitation light in the photodetection region is usually obtained using a beam shaping element. From the substantially bell-shaped shape, the central region (region including the center and extending to a certain radius in the radial direction from the optical axis) has a flattened shape. Therefore, the temporal change (pulse-shaped signal) of the light intensity value from the luminescent particles is not a normal substantially bell-shaped profile (dotted line in FIG. 2B), but the luminescent particles are in the central region of the light detection region. A profile (thick solid line in FIG. 2B) in which the corresponding region is flattened while passing through is drawn.

2. Formation and operation effect of light intensity distribution with flattened central region Referring to FIG. 3 (A) and FIG. 3 (B), as mentioned in the “Summary of the invention” column, the light of the confocal microscope The intensity distribution of the excitation light Ex in the detection region (unless otherwise specified, the intensity distribution in the radial direction with respect to the central optical axis direction) is typically on the optical axis as shown in the figure. And has a substantially bell-shaped profile (dotted line Ex) that decreases toward the periphery. Therefore, when the scanning molecule counting method is executed, the emission intensity Em (thick solid line) of the luminescent particle when the luminescent particle moves in the photodetection region is usually at the position of the luminescent particle in the photodetection region. Accordingly, it changes so as to draw a substantially bell-shaped profile similar to the intensity distribution of the excitation light Ex.

By the way, in the above scanning molecule counting method, in order to increase the detectable number of luminescent particles per unit time, it is conceivable to enlarge the light detection region. For example, if the diameter of the light detection region is increased from the diameter d1 in FIG. 3A to the diameter d2 (= X · d1) in FIG. 3C, the cross-sectional area perpendicular to the moving direction of the light detection region is X. because the ^2-fold, it is expected that the number of light-emitting particles encompassed per unit time is ^doubled X. At this time, if the total amount of excitation light incident on the light detection region, that is, the total energy is the same before and after the diameter is increased, the excitation light in the light detection region when the diameter is increased The energy density is reduced, and especially in the region near the periphery, the light emitted from the luminescent particles becomes very weak, so the detection sensitivity of the luminescent particles is reduced (light detection without increasing the incident light quantity of excitation light). When the area is enlarged, it becomes difficult to distinguish the light and noise of the luminescent particles near the periphery, and eventually the luminescent particles cannot be detected, so the meaning of expanding the light detection area is lost.) Therefore, when the photodetection area is expanded, the total amount of excitation light is increased.

As described above, when the photodetection area is expanded and the total amount of excitation light is increased, the light intensity distribution Ex (dashed line) of the excitation light is substantially bell-shaped as illustrated in FIG. The height and width are increased while maintaining the profile. Accordingly, the fluorescence intensity of the luminescent particles passing therethrough is expected to increase as the excitation light intensity increases, as depicted by Em ′ (dotted line: light quantity S2 ′) in FIG. Is done. However, in practice, the luminous ability of the luminescent particles is limited, and when the excitation light intensity reaches a certain level, the increase in the fluorescence intensity of the luminescent particles is saturated even if the excitation light intensity further increases. (It becomes a peak.) The fluorescence intensity of the luminescent particles passing through the light detection region is such that, as depicted by the thick solid line in the figure, a profile in which the central region of the substantially bell-shaped shape is flattened is drawn. Become. That is, the amount of excitation light irradiated at an intensity higher than the excitation light intensity that reaches the limit of the luminous ability of the luminescent particles (the amount of excitation light indicated by v in the figure) is the emission of the luminescent particles. It can be said that the amount of light is unnecessary. Further, when excitation light is irradiated into the light detection region, noise light such as scattered light of water molecules is also emitted in proportion to the amount of light. Therefore, the amount of excitation light that does not contribute to the light emission of the luminescent particles is not only unnecessary, but also increases noise light, and reduces the ratio of the amount of luminescent particles to the amount of noise light, that is, deteriorates the S / N ratio. Will be triggered. For example, in the case where the diameter of the light detection region is X times, assuming that the profile of the light intensity distribution of the excitation light is maintained, the incident light amount of the excitation light is X ⁴ times, so the light amount of the noise light is also X ⁴ times. On the other hand, if it is assumed that the luminous capacity of the luminescent particles has not reached the limit, the excitation light intensity becomes X times and the movement distance becomes X times, so that it becomes X ² times. Therefore, when the diameter of the photodetection region is increased by X times, the decrease in the S / N ratio is expected to be 1 / X ² times. However, when the light-emitting ability of the light-emitting particles reaches the limit, the amount of light emitted from the light-emitting particles, will be less than ² times X, so that the S / N ratio is reduced more than expected. For example, if X = 1.2, the S / N ratio is about 0.7 times that before the enlargement of the light detection region if the emission intensity of the emission particles is not saturated, but the emission intensity of the emission particles. If there is saturation, the S / N ratio is further lowered.

Therefore, in the present invention, as already described, the beam shaping element 100 is disposed between the collimator 4 and the dichroic mirror 5 on the excitation light path, and is schematically shown in FIGS. 3 (E) and 3 (F). As shown in the figure, it is modified to have a profile in which a region FR in which the excitation light intensity distribution is flattened is formed in the central region in the light detection region. According to such a configuration, as shown in FIG. 3 (F), in the excitation light intensity distribution Ex (one-dot chain line), a substantially bell-shaped profile is shown as indicated by a white arrow. The intensity of the central region will be distributed to the surrounding region and will be relatively reduced (compared to the case of a substantially bell-shaped profile). As a result, in the peripheral region in the light detection region, excitation light intensity sufficient for detection of light of the luminescent particles is given, and in the central region, the excitation light intensity is unnecessarily high. It can be avoided. Then, the excitation light intensity in the central region in the light detection region is reduced as compared with the case where the same amount of excitation light is incident without using a beam shaping element and the light intensity distribution has a substantially bell-shaped profile, Since the excitation light intensity in the surrounding area increases, the amount of excitation light that does not contribute to the light emission of the light-emitting particles is reduced while the light-emitting particles are in the central region in the light detection region, During the passage from the outside of the central region to the peripheral region, the light intensity of the luminescent particles is increased. In other words, when the beam shaping element 100 is modified so that the excitation light intensity distribution has a flattened profile in the central region in the light detection area, compared to the case where the beam shaping element 100 is not used. The incident excitation light is more effectively used for light emission of the luminescent particles, and the S / N ratio is improved. Preferably, when the profile of the excitation light intensity distribution in the light detection region is corrected by the beam shaping element 100, the distribution profile in the light detection region is flattened so as not to increase the amount of noise light unnecessarily. The excitation light intensity in the region is adjusted to a level below which the light emission ability of the light emitting particles reaches the limit. Since the emission intensity of the luminescent particles with respect to the excitation light intensity can be experimentally measured in advance, the excitation light intensity at a level at which the luminous ability of the luminescent particles reaches the limit can also be determined in advance experimentally. For example, as described above, in the case where the diameter of the light detection region is X times, if the excitation light intensity distribution is flattened by the beam shaping element 100 and the emission intensity of the luminescent particles is not saturated, the S / N ratio is It is expected to recover to about 0.7 times before the expansion of the light detection area.

3. As described above, when a beam shaping element that flattens the excitation light intensity distribution in the central region in the light detection region is used, in the focal plane (Z = 0 μm) of the objective lens, When the excitation light intensity distribution in the central region is adjusted so as to be flattened, unlike the case of having a substantially bell-shaped profile depicted on the left of FIG. 4A, from the focal plane to the optical axis direction of the objective lens. (The propagation direction of the beam. Hereinafter, the profile of the excitation light intensity distribution at a position along the optical axis direction is flat in the central region as depicted on the right side of FIG. 4A. It turns out that it changes from the state which was made into. This is because the intensity distribution obtained by flattening the profile realized in the beam shaping element is generally obtained by transmitting the Gaussian beam through the diffraction grating and adjusting the optical path length so that interference unevenness does not occur on a predetermined surface. This is because interference unevenness occurs when the position deviates greatly from a predetermined plane. For example, in the case of a certain beam shaping element, as shown in the drawing, the excitation light intensity distribution profile greatly changes from the focal plane profile when it is separated from the focal plane (Z = 0 μm) by ± 40 μm in the optical axis direction. That is, when the beam shaping element as described above is used in the apparatus of the present invention, a surface changed from a state in which the central region of the excitation light intensity distribution is flattened is included in the range of the light detection region. When the luminescent particles pass along the inside, the change in the light intensity of the luminescent particles is significantly different from the case of passing through the vicinity of the focal plane.

By the way, as will be described in detail later, in the case of the apparatus of the present embodiment, the detection of the signal of the luminescent particles is performed when the time-varying profile of the light intensity on the time-series light intensity data is a predetermined profile. More specifically, when the luminous particle has a profile assumed when it passes through the light detection region, a temporal change in the light intensity is detected as a signal of the luminous particle. In this case, in the present embodiment, the profile of the temporal change in the light intensity assumed when the luminescent particles pass through the light detection region is typically the excitation light intensity distribution in the central region. A profile assumed for the luminescent particles that have passed near the flattened focal plane is employed. Then, the change in the light intensity of the luminescent particles that has passed through the surface where the central region of the excitation light intensity distribution has changed significantly from the flattened state is not detected as a signal of the luminescent particles, but the light intensity is not detected as noise light. Since it appears on the time-series light intensity data, the noise light is eventually increased, and the S / N ratio is deteriorated. Therefore, in the case of the apparatus of the present embodiment, it is preferable that the range in the optical axis direction of the light detection region is limited to a region where the excitation light intensity distribution profile substantially coincides with the vicinity of the focal plane that is substantially flattened. . The range in the optical axis direction of the photodetection region is determined by the hole diameter of the pinhole along with the range in the direction perpendicular to the optical axis direction. The requirement is set based on the width in the optical axis direction of the objective lens in the region where the intensity distribution is substantially flattened.

As a requirement for the opening diameter of the pinhole, specifically, referring to FIG. 4B, the excitation light intensity distribution was substantially flattened in the vicinity of the focal plane (Z = 0). If the range is ± Zx μm, it can be considered that A% of the light from the region above + Zx μm and below −Zx μm cannot pass through the pinhole 13 along the optical axis direction. Therefore, first, the position of the image Pi formed by the objective lens 8 (focal length F1 [mm]) and the condenser lens 12 (focal length F2 [mm]) at a point Pr shifted from the focal plane in the Zx μm optical axis direction. The distance Zi [mm] from the pinhole is
Zi = Zx · (F2 / F1) ² (μm) (1)
Given by. When the pupil diameter of the objective lens is W [mm], the area of light passing through the condenser lens 12 on the pinhole 13 (shaded area in FIG. 4C) is
π (W / 2 · Zi / (Zi + F2)) ² (2)
Therefore, the diameter D of the opening 13a when A% cannot pass through the pinhole 13 is
D = 2 [{(100-A) / 100} ² (W / 2 · Zi / (Zi + F2))] (3)
It becomes. Here, if F1 = 4.5 mm, F2 = 180 mm, W = 10 mm, and Zx = 10 μm, then Zi = 16 mm, and if A% = 90%, D is 260 μm. For example, when the diameter of the light detection region in the direction perpendicular to the optical axis direction is set to 5 μm, the pinhole opening diameter is 200 μm. Therefore, in the above example, the light intensity distribution of the excitation light is substantially flattened. The requirement is satisfied on the basis of the width of the objective lens in the optical axis direction in the region thus formed.

The pinhole aperture diameter is set according to the requirements determined by the diameter in the direction perpendicular to the optical axis direction of the light detection region and the optical axis direction of the objective lens in the region where the light intensity distribution of the excitation light is substantially flattened It may be made so that the smaller one of the requirements is satisfied based on the width of.

Processing Operation Process The embodiment of the optical analysis processing operation using the optical analysis device 1 according to the present invention illustrated in FIG. 1A is basically described in Patent Documents 9 to 12 or other cases by the applicant of the present application. The processing described in the patent application relating to the scanning molecular coefficient method may be arbitrarily used. However, as will be described later, when detecting the signal of the luminescent particles on the time-series light intensity data obtained by measuring the light intensity, the function of the excitation light intensity distribution is not a function having a substantially bell-shaped profile. Fitting is performed using a function having a profile similar to the profile. Specific processing operations typically include (1) preparation of a sample solution containing luminescent particles, (2) measurement processing of the light intensity of the sample solution, and (3) analysis processing of the measured light intensity. Is executed. FIG. 5 shows processing in the present embodiment expressed in the form of a flowchart.

(1) Preparation of sample solution The particles to be observed in the optical analysis technique of the present invention are arbitrary as long as they are dispersed particles in the sample solution and move randomly in the solution, such as dissolved molecules. For example, biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, viruses, cells, metal colloids, other non-biological molecules, etc. It's okay. When the particle to be observed is not a particle that emits light, a particle in which a luminescent label (fluorescent molecule, phosphorescent molecule) is added to the particle to be observed in an arbitrary manner is used. The sample solution is typically an aqueous solution, but is not limited thereto, and may be an organic solvent or any other liquid.

(2) Measurement of light intensity of sample solution (Figure 5-Step 100)
The light intensity in the optical analysis by the scanning molecule counting method of the present embodiment is measured by driving the mirror deflector 17 and / or the stage position changing device 17a during the measurement, and the position of the light detection region in the sample solution. Is carried out while moving (scanning in the sample solution). In the operation process, typically, after injecting the sample solution into the well 10 of the microplate 9 and placing it on the stage of the microscope, the user inputs an instruction to start measurement to the computer 18. Then, the computer 18 stores a program stored in a storage device (not shown) (procedure for moving the position of the light detection region in the sample solution, and from the light detection region during movement of the position of the light detection region). In accordance with a procedure for detecting light and generating time-series light intensity data, irradiation of excitation light and measurement of light intensity in the light detection region in the sample solution are started. During such measurement, the mirror deflector 17 and / or the stage position changing device 17a drive the mirror 7 (galvano mirror) and / or the microplate 9 on the stage of the microscope under the control of the processing operation according to the program of the computer 18. Then, the movement of the position of the light detection region is executed in the well 10, and at the same time, the light detector 16 converts the light sequentially detected into an electric signal and transmits it to the computer 18 to transmit it to the computer. In 18, time-series light intensity data is generated and stored from the transmitted signal in an arbitrary manner. Typically, the photodetector 16 is an ultra-sensitive photodetector that can detect the arrival of one photon, and therefore when the light detection is based on photon counting, the time-series light intensity data is a time-series data. It may be photon count data.

The moving speed of the position of the light detection region during the measurement of the light intensity may be a predetermined speed that is arbitrarily set, for example, experimentally or so as to suit the purpose of analysis. When acquiring information on the number density or concentration based on the number of detected luminescent particles, the size or volume of the region through which the light detection region has passed is required, so that the movement distance can be grasped. The movement of the position of the light detection region is executed at. In addition, since it is easier to interpret the measurement result when the elapsed time during measurement and the movement distance of the position of the light detection region are in a proportional relationship, it is preferable that the movement speed is basically a constant speed. However, the present invention is not limited to this.

In order to quantitatively and accurately execute the individual detection of the luminescent particles from the measured time-series light intensity data or the counting of the number of luminescent particles with respect to the moving speed of the position of the light detection region, it is necessary. The movement speed is preferably set to a value faster than the random movement of the luminescent particles, that is, the movement speed due to the Brownian movement. Since the observation target particle of the optical analysis technique of the present invention is a particle that is dispersed or dissolved in a solution and moves freely and randomly, the position moves with time by Brownian motion. Accordingly, when the moving speed of the position of the light detection region is slower than the movement of the particle due to Brownian motion, the particle moves randomly within the region as schematically illustrated in FIG. As a result, the light intensity changes randomly (as already mentioned, the excitation light intensity of the light detection region decreases outward with the center of the region as the apex), and it corresponds to the individual luminescent particles. It is difficult to specify a change in light intensity. Therefore, preferably, as shown in FIG. 6 (B), the particles cross the light detection region in a substantially straight line, so that in the time-series light intensity data, the uppermost stage in FIG. 6 (C). As shown in the example, the light intensity change profile corresponding to each light emitting particle becomes substantially uniform (when the light emitting particle passes through the light detection region substantially linearly, the light intensity change profile is excited). It is almost the same as the light intensity distribution.), So that the correspondence between each light emitting particle and light intensity can be easily identified, the moving speed of the position of the light detection region is the average moving speed (diffusion due to the Brownian motion of the particle) It is set faster than (moving speed).

Specifically, the time Δt required for the luminescent particles having the diffusion coefficient D to pass through the light detection region (confocal volume) having the radius Wo by Brownian motion is expressed by a relational expression (2Wo) ² = 6D · Δt (4)
From
Δt = ( ² Wo) ² / 6D (5)
Therefore, the speed (diffusion movement speed) Vdif at which the luminous particles move by Brownian motion is approximately
Vdif = 2Wo / Δt = 3D / Wo (6)
It becomes. Therefore, the moving speed of the position of the light detection region may be set to a value sufficiently faster than that with reference to the Vdif. For example, when the diffusion coefficient of the observation target particle is expected to be about D = 2.0 × 10 ⁻¹⁰ m ² / s, if Wo is about 0.62 μm, Vdif is 1.0 × Since 10 ⁻³ m / s, the moving speed of the position of the light detection region may be set to 15 mm / s or more, which is 10 times or more. In addition, when the diffusion coefficient of the observation target particle is unknown, the profile of the change in the light intensity by setting the moving speed of the position of the light detection region in various ways is expected (typically, the excitation light intensity distribution). A preliminary experiment for finding a condition that is substantially the same as that described above may be repeatedly performed to determine a suitable moving speed of the position of the light detection region.

(3) Analysis process of light intensity When the time-series light intensity data is generated, the light intensity value in the time-series light intensity data is used to detect the light-emitting particle signal and Various analyzes such as counting and concentration calculation are executed.
(I) Individual detection of signal of luminescent particles (FIG. 5-steps 110 to 160)
As already mentioned, when the locus of one luminescent particle passing through the light detection region is substantially linear as shown in FIG. 6B, the light intensity in the signal corresponding to the particle. The change in the light intensity on the data has a profile in which the central area is flattened reflecting the excitation light intensity distribution in the light detection area determined by the optical system. Such a profile is represented by a step function having the height A and the width T as parameters, or an expansion formula of an odd term of a sine wave as described below.
f (t) = A {1 + sin (t / T) + (1/3) sin (3t / T) + (1/5) sin (5t / T) +…} (7)
Therefore, in the time-series light intensity data, the height A and the width T calculated by fitting the expression (7) to a significant light intensity profile (profile that can be clearly determined not to be background) are as follows. When the light intensity profile is within the predetermined range, it is determined that the light intensity profile corresponds to the passage of one particle through the light detection region, and one light emitting particle may be detected. A signal whose height A and width T are out of a predetermined range is determined as a noise or foreign matter signal, and may be ignored in subsequent analysis or the like.

In one example of processing for detecting a signal on light intensity data, first, smoothing (smoothing) is performed on the light intensity data (FIG. 6C, “detection result (unprocessed)” at the top stage). ) Is performed (FIG. 5—Step 110, FIG. 6C, “Upper Smoothing”). The light emitted by the luminescent particles is probabilistically emitted, and data values may be lost in a very short time. Therefore, such a data value loss can be ignored by the smoothing process. The smoothing process may be performed by a moving average method, for example. It should be noted that the parameters for executing the smoothing process, such as the number of data points averaged at a time in the moving average method and the number of moving averages, are the moving speed (scanning of the position of the light detection region at the time of acquiring the light intensity data) Speed) and BIN TIME may be set as appropriate.

Next, in the light intensity data after the smoothing process, in order to detect a time region (pulse existing region) where a significant pulse-like signal (hereinafter referred to as “pulse signal”) exists, A primary differential value with respect to time of the light intensity data is calculated (step 120). The time differential value of the light intensity data has a large change in the value at the time of change of the signal value as illustrated in the lower “time differential” in FIG. Thus, the significant signal start and end points can be advantageously determined.

Thereafter, significant pulse signals are sequentially detected on the light intensity data (steps 130 to 160). Specifically, first, the start and end points of one pulse signal are searched and determined sequentially on the time differential value data of the light intensity data to identify the pulse existence region. (Step 130). Once a single pulse presence region is identified, a function similar to the profile of the excitation light intensity distribution (“distribution shape function”) is fitted to the smoothed light intensity data in that pulse presence region. (FIG. 6C, “Distribution shape function fitting” in the lower part), parameters such as the intensity A and width T of the flat area of the distribution shape function, and the correlation coefficient (of the least square method) in the fitting are calculated. (Step 140). Then, whether the calculated function parameter is within an assumed range for the profile parameter drawn by the pulse signal detected when one luminescent particle passes through the light detection region, that is, the distribution shape function It is determined whether the intensity A, the width T, and the correlation coefficient of the flat area are within predetermined ranges (step 150). Thus, the signal determined that the calculated parameter of the distribution shape function is within the range assumed in the signal corresponding to one luminescent particle is determined to be a signal corresponding to one luminescent particle, Thereby, one luminescent particle is detected. On the other hand, a pulse signal whose calculated distribution shape function parameter is not within the assumed range is ignored as noise. Note that the counting of the number of signals, that is, the counting of the luminescent particles may be performed simultaneously with the detection of the signal of the luminescent particles.

The search and determination of the pulse signal in the processing of the above steps 130 to 150 may be repeatedly executed over the entire area of the light intensity data (step 160). When optical measurement is performed using excitation light of different wavelength bands, and multiple time-series light intensity data are generated corresponding to the excitation light of each wavelength band, each time-series light intensity data The processing of steps 130 to 150 may be executed. In addition, the process which detects the signal of a luminescent particle separately from light intensity data is not restricted to said procedure, You may perform by arbitrary methods.

In addition, when the number of detected luminescent particles is counted and the number of luminescent particles is determined, the total volume of the region through which the photodetection region passes is further calculated by an arbitrary method. Then, the concentration of the luminescent particles in the sample can be determined from the volume value and the number of the luminescent particles. The total volume of the region through which the light detection region has passed may be determined, for example, in the manner described in Patent Documents 9 to 12.

Thus, according to the configuration of the present embodiment described above, the light intensity distribution profile in the light detection region has a substantially constant excitation light intensity over a certain distance in the radial direction from the center to the optical axis. To be corrected. When the excitation light intensity distribution has a substantially bell-shaped profile, the amount of excitation light that has passed through the central area of the light detection area with excessive light intensity is distributed to the area outside the central area. Therefore, it is possible to increase the emission intensity of the luminescent particles in the region outside the central region when the photodetection region is enlarged. Therefore, compared with the case where the excitation light intensity distribution has a substantially bell-shaped profile, the emission intensity of the luminescent particles in the peripheral region of the light detection region is increased. It is also expected that the increase in total light quantity can be reduced.

Claims (4)

An optical analyzer that detects light from luminescent particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope or a multiphoton microscope,
A light detection region moving unit that relatively moves the position of the light detection region of the optical system in the sample solution;
A light irradiation unit for irradiating the light detection region with excitation light;
A light detection unit for detecting light from the light detection region;
Generate time-series light intensity data of light from the light detection area detected by the light detection unit while moving the position of the light detection area in the sample solution, and the time-series light intensity A signal processor for individually detecting each of the signals of the luminescent particles in the data,
A beam shaping element for substantially flattening a light intensity distribution of the excitation light in at least a part of a region including a center in the light detection region in an optical path of the excitation light in the light irradiation unit; Equipment provided. 2. The apparatus according to claim 1, wherein an opening diameter of a pinhole arranged at a conjugate position of a focal position of the objective lens of the optical system is a region where the light intensity distribution of the excitation light is substantially flattened. A device that is set based on the width of the objective lens in the optical axis direction. 3. The apparatus according to claim 1, wherein the signal processing unit has a light intensity distribution of the excitation light substantially flattened in at least a part of a region including a center in the light detection region. An apparatus for individually detecting a temporal change in light intensity having a profile corresponding to the profile as an optical signal of one luminescent particle. 4. The apparatus according to claim 1, wherein the maximum intensity of the excitation light irradiated into the light detection region is reduced as compared with a case where the beam shaping element is not used.

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