JP2015083922A - Single particle detection device using photometric analysis, single particle detection method and single particle detection computer program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable a single particle to be detected for each kind in a sample solution in which a single particle not emitting in a specific wavelength band of a plurality of kinds is mixed in single particle detection technologies by a scanning molecule counting method individually detecting a single particle using an optical measurement by a confocal microscope or a multiphoton microscope.SOLUTION: A technology detecting a single particle in a sample solution according to the present invention is configured to: detect light of a specific wavelength band from a light detection area while moving a position of the light detection area of a microscope in a sample solution including a single particle of a plurality of kinds not emitting in a mutually different wavelength band, and generate light intensity data in time series; and individually detect fall-off in light intensity occurring when the single particle not emitting in the specific wavelength band in the time series light intensity data enters the light detection area as a signal showing respective existences of the single particle.

Description

  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 or a multiphoton microscope, and uses atoms, molecules or aggregates thereof dispersed or dissolved in a solution ( These are hereinafter referred to as “particles”), 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- More about single particle detection technology that can detect biological particles and obtain useful information in analysis or analysis of their state (interaction, binding / dissociation state, etc.) A single particle detector, a single particle detector that enables various analyzes by detecting a single particle by measuring a change in light intensity due to the presence of a single particle using the optical system as described above. Detection method and single particle detection computer According to the program.

  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. Examples of such an optical analysis technique include fluorescence correlation spectroscopy (FCS; see, for example, Patent Literature 1-3 and Non-Patent Literature 1-3), and fluorescence intensity distribution analysis (Fluorescence-Intensity Distribution Analysis: FIDA, for example, Patent Document 4, Non-Patent Document 4) and 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 11 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 the case of the above-described optical analysis techniques such as FCS and FIDA, in short, from fluorescent molecules floating in a minute region (hereinafter referred to as “light detection region”) that is a light detection region in a sample solution. Statistical calculation processing is executed on the light intensity data obtained by continuously measuring the light, and the concentration and / or other characteristics of the fluorescent molecules are detected. On the other hand, in the novel optical analysis techniques proposed in Patent Documents 9 to 11, the position of the light detection region is moved in the sample solution, that is, the sample solution is scanned by the light detection region. However, when the light detection region includes particles (light-emitting particles) that emit light that randomly moves by being dispersed in the sample solution, the light emitted from the light-emitting particles is individually detected, thereby It is possible to detect each of the luminescent particles one by one and to obtain information on the counting of the luminescent particles and the concentration or number density of the luminescent particles in the sample solution. According to this optical analysis technique (hereinafter referred to as “scanning molecule counting method”), the sample required for the measurement is a very small amount (for example, about several tens of μL) as in the optical analysis techniques such as FCS and FIDA. In addition, the measurement time is short (measurement of time in the order of seconds is repeated several times in one measurement), and the concentration of particles to be observed is the same as that of optical analysis technology such as FCS and FIDA. It is possible to detect the presence of luminescent particles in a sample solution lower than a well measurable level (about 1 nM) and quantitatively detect its concentration, number density or other characteristics. Thus, the “scanning molecule counting method” is used for analysis of rare or expensive samples often used in the field of medical and biological research and development, clinical diagnosis of diseases, screening of bioactive substances, etc. When the number of specimens is large, it is possible to perform experiments or tests at a lower cost or more quickly than conventional biochemical methods, and at a lower concentration so that FCS, FIDA, etc. cannot be performed well. It is expected to be a powerful tool capable of detecting the concentration and / or properties of

JP-A-2005-09876 JP2008-292371 JP 2009-281831 A 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 2010-1119098

Masataka Kaneshiro, Protein Nucleic Acid Enzyme Vol.44, No.9, pp.1431-1438 1999 FJMeyer-Alms, Fluorescence Correlation Spectroscopy, R. Rigler, Springer, Berlin, 2000, 204 -Page 224 Norio Kato, 4 people, Gene Medicine, Vol.6, No.2, pp.271-277 Cask and three others, Bulletin of the American Academy of Sciences 1999, 96, 13756-13761 (P. Kask, K. Palo, D. Ullmann, K. Gall PNAS 96, 13756-13761 (1999))

  In the scanning molecule counting method that individually detects the light of single particles that emit light as described above, the light from the single particles is weak, and is easily affected by stray light or Raman scattering of water. That is, in the case of an analysis method for identifying an increase in light intensity value representing light emitted from a luminescent particle as a signal of the luminescent particle, light due to stray light or water Raman scattering may be mistakenly identified as a signal of the luminescent particle. is there. In the case of a scanning molecule counting method that detects light of a single particle, particles to be observed (observed particles) are limited to luminescent particles. Therefore, when observing particles that do not emit light, it is necessary to attach luminescent labels (fluorescent labels, phosphorescent labels, etc.) to the particles to be observed. It does not always exist (the observation target particle may be denatured by applying a luminescent label).

  In this regard, in general, when optical measurement is performed by an optical system of a microscope, when background light is high, it is hardly affected by stray light or Raman scattering of water. Also, in the case of an optical system of a confocal microscope or a multiphoton microscope, the resolution in the optical axis direction is higher than that of a normal optical microscope, so if a single particle that does not emit light passes through the confocal volume, the confocal volume A decrease in light intensity is observed (Patent Document 12). Therefore, in the Japanese Patent Application No. 2011-184635, the applicant of the present application uses the observation target particle as a single particle that does not emit light, and scans a sample solution that emits a substantially constant background light with a light detection region. The detection of light is performed in the same manner as the `` molecular counting method '', and the presence of a single particle is detected by detecting the shadow of the single particle when a single particle that does not emit light is included in the light detection region. An “inverted scanning molecule counting method” was proposed. According to the “inverted scanning molecule counting method”, stray light or Raman scattered light is not erroneously detected as a signal of an observation target particle, and a single particle that does not emit light at a low concentration can be detected.

  By the way, in general, the particles to be observed of the present invention have a light emission wavelength characteristic that emits light in a specific wavelength band but hardly emits light in another wavelength band. Therefore, when multiple types of particles coexist in the same sample solution, if the wavelength bands that do not emit light of these particles are different, light detection is performed in the “inverted scanning molecule counting method”. By setting the detection wavelength of the light in the region to each of the wavelength bands where there is no light emission of the plurality of types of particles, the specific wavelength of the plurality of types of particles mixed in the sample solution corresponding to the detection wavelength It becomes possible to detect the types of particles separately, and the particles can be detected while identifying the types.

  Thus, the main problem of the present invention is that when a plurality of types of particles are mixed in the same sample solution, the principle of the inverted scanning molecule counting method is used to select a specific type of the plurality of types. It is to provide a single particle detection technique based on a novel principle that makes it possible to detect single particles while detecting particles or identifying types.

  According to one aspect of the present invention, the above-described problem is solved by using a confocal microscope or an optical system of a multiphoton microscope to detect a single particle that is dispersed in a sample solution and randomly moves. In the sample solution, a light detection region moving unit that moves the position of the light detection region of the optical system of the microscope in the sample solution, a light detection unit that detects light from the light detection region, and the sample solution 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, and each single particle in the time-series light intensity data And a signal processing unit that individually detects a signal that represents a first particle in which a single particle does not emit light in the first wavelength band but emits light in a second wavelength band different from the first wavelength band. Single particles of light in the first wavelength band and in the second wavelength band When the wavelength band of light detected by the light detection unit is the first wavelength band, the light from the light detection region is substantially constant in the first wavelength band. The signal representing the presence of each of the single particles, including the background light, is a decrease in the light intensity detected by the light detection unit that occurs when the first single particle enters the light detection region, When the wavelength band of light detected by the light detection unit is the second wavelength band, the light from the light detection region includes a substantially constant background light of the second wavelength band, and each of the single particles The signal representing the presence of is a reduction in the light intensity detected by the light detection unit that occurs when the second single particle enters the light detection region.

  In such a configuration, “single particles that are dispersed in the sample solution and randomly move” are atoms, molecules, or aggregates thereof dispersed or dissolved in the sample solution and fixed to the substrate or the like. Any particle may be used as long as it is a particle that freely performs Brownian motion in the solution. The “light detection area” of the optical system of a confocal microscope or multiphoton microscope is a minute area in which light is detected in those microscopes. When illumination light is given from an objective lens, the illumination light is Corresponds to the focused region (in a confocal microscope, it is determined in particular by the positional relationship between the objective lens and the pinhole). “The light from the photodetection region includes substantially constant background light in the first (second) wavelength band,” means that the wavelength band of the detection light is in the first (second) wavelength band. When set, it means that the intensity value of the detection light (ie background light) when the single particle to be observed does not exist in the light detection region is within the allowable error range. Yes. In other words, the background light is adjusted such that the fluctuation is sufficiently smaller than the decrease in the intensity of the detection light that occurs when a single particle enters the light detection region. On the other hand, a single particle that does not “emit substantially in the first (second) wavelength band” means a single particle whose emission intensity in the corresponding wavelength band is sufficiently smaller than the background light. means. That is, the emission intensity of such single particles is preferably almost zero, but may be acceptable even at a level significantly lower than the intensity of background light. In this specification, the term “single particle signal” refers to a signal indicating the presence of a single particle unless otherwise specified.

  As understood from the above, in the apparatus of the present invention, basically, as in the “scanning molecule counting method”, the position of the light detection region is moved in the sample solution, that is, the sample. The light is sequentially detected while scanning the inside of the solution with the light detection region. In such a configuration, when the light from the light detection region includes background light having a significant intensity in the wavelength band of the detection light, single particles that do not emit light in the wavelength band of the detection light are light. The light from the light detection region due to the presence of a single particle when entering the detection region or when the light detection region moving within the sample solution includes single particles that do not emit light in the wavelength band of the detection light. The light intensity or light quantity of the background light that reaches the detection unit will decrease. On the other hand, even if a single particle that emits light in the wavelength band of the detection light is present in the light detection region, it is mixed with the background light and almost no decrease in light intensity or light amount is detected. Thus, in the apparatus of the present invention, a single particle does not emit light in the first wavelength band but emits light in the second wavelength band, and light emission in the first wavelength band. When a significant background light is emitted in the first wavelength band when the second single particle that does not emit light in the second wavelength band is included, the wavelength band of the detection light is changed to the first wavelength band. The wavelength band is set, and the decrease in the light intensity or light quantity of the background light is individually detected as a single particle signal, whereby the presence of the particles is detected individually and sequentially one by one. When significant background light is emitted in the wavelength band, the wavelength band of the detection light is set to the second wavelength band, and the decrease in light intensity or light quantity of the background light is individually detected as a single particle signal. Thus, the presence of particles is detected individually and sequentially. Thus, different particles in the sample solution, that is, the first and second particles are detected separately, that is, for each type, and for each type, various information regarding the state of the particles in the solution is obtained. Will be acquired. That is, in the device of the present invention, for each specific wavelength band, by detecting the shadow of a single particle that does not emit light in the specific wavelength band in a region where a certain background light exists, Single particles are detected individually.

  In the above-described apparatus of the present invention, the background light to be included in the light from the light detection region is fluorescence, phosphorescence, chemiluminescence, bioluminescence, or scattered light caused by a substance dispersed in the sample solution. It may be. In this case, when the substance that emits or scatters light is not dispersed in the solution used as the sample solution, the substance that actively emits or scatters light may be dissolved or dispersed in the solution. Further, when the solution used as the sample solution emits autofluorescence, the autofluorescence may be used as the background light. In particular, when a substance that generates background light requires excitation light or illumination light, the microscope apparatus is equipped with a light source and an optical system for illumination light. On the other hand, in the case where a substance that generates background light emits light without excitation light or illumination light, for example, a substance that emits light by chemiluminescence or bioluminescence, illumination light is not required in the microscope apparatus. Furthermore, it should be understood that the background light may be illumination light by transmitted illumination or the like if it is reduced when a single particle is present in the light detection region. In the above-described configuration of the present invention, when the background light reduction due to the presence of a single particle is detected, the degree of background light reduction is determined by the size of the single particle and the size of the light detection region. Depends on the relationship. In this regard, according to the estimation described in detail later, the outer diameter of the single particle to be observed is preferably 15% or more of the diameter of the light detection region, and more preferably, the outer diameter of the single particle. Has been found to be 35% or more of the diameter of the light detection region. (See Japanese Patent Application No. 2011-184635)

  Furthermore, in the above-described configuration of the present invention, when detecting light in a specific wavelength band, single particles that emit light in the wavelength band of the detection light that is not the object of observation have an intensity substantially equal to the intensity of the background light. It is preferable to have. This is because if the difference between the light intensity of a single particle that is not the object of observation and the intensity of the background light is large, it affects the detection of the signal of the single particle that is the object of observation in the light intensity data. (If the brightness of a single particle that emits light in the wavelength band of detection light is excessive, the background light value will be excessively calculated, and the brightness of the single particle that emits light in the wavelength band of detection light. Is too small to be distinguished from a decrease in light intensity corresponding to a single particle to be observed). Therefore, in the present invention described above, preferably, when the wavelength band of the light detected by the light detection unit is the first wavelength band, the intensity of the background light and the light from the second single particle When the intensity is substantially equal and the wavelength band of the light detected by the light detector is the second wavelength band, the intensity of the background light and the intensity of light from the first single particle are substantially equal. The intensity of the background light is adjusted so that Such background light adjustment may be achieved, for example, by dispersing a luminescent material in the sample solution.

  The moving speed of the position of the light detection region in the sample solution in the apparatus of the present invention may be appropriately changed based on the characteristics of single particles or the number density or concentration in the sample solution. In particular, if the moving speed of the light detection region is increased, the degree of decrease in light intensity or light amount due to the presence of a single particle is reduced. Therefore, the decrease in light intensity or light amount due to a single particle is accurate or sensitive. It is preferable that the moving speed of the light detection region can be changed as appropriate so that measurement can be performed.

  Further, the moving speed of the position of the light detection region in the sample solution is preferably set higher than the diffusion moving speed of the single particle to be detected (the average moving speed of the particles due to Brownian motion). As explained above, the device of the present invention detects the reduction of background light due to the presence of a single particle when the light detection region passes through the position where the single particle exists, and individually separates the single particle. To detect. However, when a single particle moves randomly in the solution due to Brownian motion and enters and exits the photodetection region multiple times, the presence of the signal is detected multiple times from one single particle. Therefore, it becomes difficult to make the detected signal correspond to the presence of one single particle. Therefore, as described above, the moving speed of the light detection region is set to be higher than the diffusion moving speed of the single particle, thereby making one single particle correspond to one signal (representing the presence of a single particle). It becomes possible. Since the diffusion movement speed varies depending on the characteristics of the single particle, as described above, the apparatus of the present invention appropriately changes the movement speed of the light detection region according to the characteristics of the single particle (particularly, the diffusion constant). It is preferable that it is configured to be possible.

  The movement of the position of the light detection region in the sample solution may be performed by an arbitrary method. For example, the position of the light detection region may be changed by changing the optical path of the optical system of the microscope by using a galvanometer mirror adopted in a laser scanning optical microscope, or The position of the sample solution may be moved (for example, by moving a microscope stage) to move the relative position of the light detection region in the sample solution. The movement trajectory of the position of the light detection region may be arbitrarily set, and may be selected from, for example, a circle, an ellipse, a rectangle, a straight line, and a curve. In particular, when the position of the light detection region is changed by changing the optical path of the optical system of the microscope, the movement of the light detection region is quick, and mechanical vibrations and hydrodynamic actions are applied to the sample solution. Therefore, it is advantageous in that light can be measured in a stable state without being affected by a dynamic action of a single particle to be detected.

  In the processing of the signal processing unit of the apparatus of the present invention, it is determined whether or not one particle has entered the light detection region from the detection value signal from the sequential light detection unit. May be based on the shape of the signal representing the time-series light detected at. In an embodiment, it is typically determined that a single particle has entered the light detection region when a signal having a light intensity lower than a predetermined threshold, as measured from the intensity of the background light, is detected. Good. More specifically, as will be described later in the section of the embodiment, the signal indicating the presence of a single particle is usually a time-series detection value of the light detection unit, that is, light intensity data. It appears as a downwardly convex bell-shaped pulse signal below a certain intensity, and the noise is not a bell-shaped pulse signal or appears as a high-intensity signal. Therefore, the signal processing unit of the apparatus of the present invention generates a bell-shaped pulse-shaped signal that is lower than a predetermined threshold value measured from the intensity of background light on the time-series light intensity data, and the presence of a single particle. It may be configured to detect as a signal representing. The “predetermined threshold value” can be set to an appropriate value experimentally.

  Furthermore, the light intensity obtained by the apparatus of the present invention is relatively weak and causes a fine increase / decrease, and the fine increase / decrease of the light intensity deteriorates the detection accuracy of a signal indicating the presence of a single particle. Therefore, the signal processing unit smoothes the time-series light intensity data, processes the data so that the fine increase / decrease in the light intensity can be ignored, and then determines the background light intensity in the smoothed time-series light intensity data. It may be configured to detect a downwardly convex bell-shaped pulse signal having an intensity below a predetermined threshold, as a signal representing the presence of a single particle.

  In one of the above embodiments of the present invention, the number of signals may be counted to count the number of single particles included in the light detection region (particle counting). In that case, information on the number density or concentration of the identified single particles in the sample solution can be obtained by combining the number of detected single particles and the amount of movement of the position of the light detection region. Specifically, for example, the number density or concentration ratio of a plurality of sample solutions, or the relative number density or concentration ratio with respect to the standard sample solution serving as a reference for the concentration or number density is calculated, or An absolute number density value or concentration value may be determined using a relative number density or concentration ratio relative to a standard sample solution that is a reference for concentration or number density. Alternatively, if the total volume of the movement locus of the position of the light detection region is specified by any method, for example, by moving the position of the light detection region at a predetermined speed, the number density or concentration of single particles Can be calculated specifically. In particular, in the case of the present invention, it is possible to separately count and / or calculate the concentration of single particles having different wavelength bands that do not emit light and are mixed in the same sample solution.

  By the way, in a typical embodiment of particle counting, the number of single particle signals obtained during an arbitrarily set measurement time is counted. However, in that case, the number of single particle signals detected varies depending on the length of the set measurement time. In particular, when the single particle concentration is low, the single particle value calculated from the number of detection signals is low. The variation of the density value becomes large and the accuracy can be lowered. Therefore, in the above-described apparatus of the present invention, as another aspect of particle counting, measurement is performed until the number of signals of a single particle reaches an arbitrarily set number. One particle concentration value may be calculated. That is, the above-described apparatus according to the present invention allows the position of the light detection region of the optical system by the light detection region moving unit until the number of signals indicating the presence of single particles detected by the signal processing unit reaches a predetermined number. The number representing the presence of a single particle signal is determined in advance by repeating the movement of the light, the detection of light from the light detection region by the light detection unit, and the detection of the signal indicating the presence of a single particle by the signal processing unit. May be configured to determine the concentration of a single particle in the sample solution based on the time taken to reach the number. In this case, shortening of the measurement time for a single particle high-concentration sample solution is expected, and the measurement for a single particle low-concentration sample solution is performed with sufficient time. That is, according to said structure, measurement time is optimized according to the density | concentration of a single particle. In addition, if a predetermined number is set to a number that achieves the accuracy required for the result, the time required to detect the predetermined number of single particles or any result derived therefrom can be obtained. Variations in this are suppressed to a small level, and the accuracy of the result can be satisfied.

  In the apparatus of the present invention described above, in the first or second wavelength band, the position of the light detection region is moved in the sample solution in which single particles having different wavelength bands that do not emit light are mixed. Light detection is performed in the presence of background light, and a decrease in light intensity or light quantity of background light is individually detected as a single particle signal, so that it is single for each type (for each wavelength band that does not emit light). The processing of the single particle detection technique for sequentially detecting the presence of particles one by one can be realized by a general-purpose computer. Therefore, according to another aspect of the present invention, a single particle detection computer program for detecting single particles that are dispersed in a sample solution and randomly move using an optical system of a confocal microscope or a multiphoton microscope. The procedure for moving the position of the light detection area of the microscope optical system in the sample solution and the light from the light detection area are detected while moving the position of the light detection area in the sample solution. The computer executes a procedure for generating time-series light intensity data and a procedure for individually detecting signals representing the presence of each single particle in the time-series light intensity data. A first single particle that emits light in a second wavelength band different from the first wavelength band but does not substantially emit light in the first wavelength band; Substantially no light emission in the wavelength band Two single particles, and when the wavelength band of the detected light is the first wavelength band, the light from the photodetection region includes a substantially constant background light in the first wavelength band, The signal indicating the presence of each particle is a decrease in light intensity in the light intensity data that occurs when the first single particle enters the light detection region. When in the second wavelength band, the light from the light detection region includes a substantially constant background light in the second wavelength band, and a signal representative of the presence of each single particle is There is provided a computer program characterized by a decrease in light intensity in light intensity data generated when entering a light detection area.

  Even in such a configuration, the background light may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, or illumination light by a substance dispersed in the sample solution. The intensity of such background light is preferably such that when the wavelength band of the detected light is the first wavelength band, the intensity of the background light and the light intensity from the second single particle are substantially equal, When the wavelength band of the detected light is the second wavelength band, for example, luminescent particles are placed in the sample solution so that the intensity of the background light is substantially equal to the light intensity from the first single particle. The intensity of the background light is adjusted by dispersing the light. The outer diameter of the single particle is preferably 15% or more of the diameter of the photodetection region, and more preferably 35% or more of the diameter of the photodetection region.

  Also in the above computer program, individual detection of a signal indicating the presence of each single particle may be performed based on the shape of a time-series signal. In an embodiment, typically, in a procedure for individually detecting signals representing the presence of a single particle, a signal having a light intensity lower than a predetermined threshold measured from the background light intensity is detected. It may be determined that one single particle has entered the light detection region. Specifically, in the procedure of individually detecting signals representing the presence of a single particle, a downwardly convex bell having an intensity below a predetermined threshold measured from the intensity of background light in time-series light intensity data. A pulse signal of the type may be detected as a signal representing the presence of a single particle, in which case the time-series light intensity data is smoothed and the downwardly convex bell in the smoothed time-series light intensity data. A type of pulsed signal may be detected as a signal representing the presence of a single particle.

  Furthermore, the moving speed of the position of the light detection region in the sample solution may be appropriately changed based on the characteristics of the single particle, the number density or concentration in the sample solution, and preferably in the sample solution. The moving speed of the position of the light detection region at is set higher than the diffusion moving speed of the single particle to be detected. The position of the light detection region in the sample solution may be moved by an arbitrary method. Preferably, the position of the light detection region is changed by changing the optical path of the optical system of the microscope or moving the position of the sample solution. May be changed. The movement trajectory of the position of the light detection region may be arbitrarily set, and may be selected from, for example, a circle, an ellipse, a rectangle, a straight line, and a curve.

  Furthermore, in the above computer program, a procedure for counting the number of single particles detected during movement of the position of the light detection region by counting the number of individually detected single particle signals and / or Alternatively, a procedure for determining the number density or concentration of single particles in the sample solution based on the number of single particles detected may be included. In the case of the above computer program as well, in particle counting, the number of single particle signals obtained during an arbitrarily set measurement time is typically counted. The measurement may be performed until the number of signals of one particle reaches an arbitrarily set number, and the single particle concentration value may be calculated based on the measurement time. Therefore, the above computer program also moves the position of the light detection region, detects light from the light detection region, and detects single particles until the number of signals indicating the presence of single particles reaches a predetermined number. Detection of a signal representing the presence of a single particle, and determining the concentration of a single particle in the sample solution based on the time taken for the number representing the presence of a single particle signal to reach a predetermined number. A procedure may be configured.

  According to the apparatus or the computer program of the present invention, the first or second wavelength band is moved while moving the position of the light detection region in the sample solution in which single particles having different wavelength bands that do not emit light are mixed. In the presence of background light, the light intensity or light intensity of the background light is individually detected as a single particle signal. ) A novel method of detecting the presence of single particles one after another individually is realized. Thus, according to the present invention, there is further provided a single particle detection method for detecting single particles dispersed and moving randomly in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, The process of moving the position of the light detection area of the optical system of the microscope in the solution, and detecting the light from the light detection area while moving the position of the light detection area in the sample solution A step of generating light intensity data and a step of individually detecting as a signal representing the presence of each of the single particles in the time-series light intensity data, wherein the single particles are in the first wavelength band. A first single particle that does not substantially emit light and emits light in a second wavelength band different from the first wavelength band; and emits light in the first wavelength band and in the second wavelength band. A second single particle that does not substantially emit light, and When the long band is the first wavelength band, the light from the photodetection region includes a substantially constant background light in the first wavelength band, and a signal representative of the presence of each single particle is This is a decrease in light intensity in the time-series light intensity data that occurs when a single particle enters the light detection region. When the wavelength band of the detected light is the second wavelength band, from the light detection region A time series that occurs when the second single particle enters the photodetection region, and the signal representing the presence of each of the single particles includes a substantially constant background light in the second wavelength band. A method is provided that is a reduction in light intensity in light intensity data.

  Even in such a configuration, the background light may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, or illumination light by a substance dispersed in the sample solution. The intensity of such background light is preferably such that when the wavelength band of the detected light is the first wavelength band, the intensity of the background light and the light intensity from the second single particle are substantially equal, When the wavelength band of the detected light is the second wavelength band, for example, luminescent particles are placed in the sample solution so that the intensity of the background light is substantially equal to the light intensity from the first single particle. The intensity of the background light is adjusted by dispersing the light. The outer diameter of the single particle is preferably 15% or more of the diameter of the photodetection region, and more preferably 35% or more of the diameter of the photodetection region.

  Also in the above method, the individual detection of the signal representing the presence of each single particle may be performed based on the shape of the time-series signal. In the embodiment, typically, in the process of individually detecting the signal indicating the presence of a single particle, a signal having a light intensity lower than a predetermined threshold is detected from the background light intensity. It may be determined that one single particle has entered the light detection region. Specifically, in the process of individually detecting signals representing the presence of a single particle, a downwardly convex bell having an intensity below a predetermined threshold measured from the intensity of background light in time-series light intensity data. A pulse signal of the type may be detected as a signal representing the presence of a single particle, in which case the time-series light intensity data is smoothed and the downwardly convex bell in the smoothed time-series light intensity data. A type of pulsed signal may be detected as a signal representing the presence of a single particle.

  Furthermore, the moving speed of the position of the light detection region in the sample solution may be appropriately changed based on the characteristics of the single particle, the number density or concentration in the sample solution, and preferably in the sample solution. The moving speed of the position of the light detection region at is set higher than the diffusion moving speed of the single particle to be detected. The position of the light detection region in the sample solution may be moved by an arbitrary method. Preferably, the position of the light detection region is changed by changing the optical path of the optical system of the microscope or moving the position of the sample solution. May be changed. The movement trajectory of the position of the light detection region may be arbitrarily set, and may be selected from, for example, a circle, an ellipse, a rectangle, a straight line, and a curve.

  Further, in the above method, the process of counting the number of individually detected single particle signals to count the number of single particles detected during the movement of the position of the light detection region and / or A step of determining the number density or concentration of single particles in the sample solution based on the number of single particles detected may be included. In the case of the above method as well, in particle counting, the number of single particle signals obtained during an arbitrarily set measurement time is typically counted. Measurement may be performed until the number of particle signals reaches an arbitrarily set number, and a single particle concentration value may be calculated based on the measurement time. Therefore, the above method also moves the position of the light detection region, detects light from the light detection region, and detects the single particle until the number of signals indicating the presence of a single particle reaches a predetermined number. Configured to repeatedly detect the presence signal and determine the concentration of the single particle in the sample solution based on the time taken for the number of single particle signals to reach a predetermined number May have been.

  The above-described single particle detection technique of the present invention typically involves particulate biological materials such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, viruses, cells and the like. Used for analysis or analysis of the state of a target object in solution, but non-biological particles (eg, atoms, molecules, micelles, liposomes, metal colloids, beads (magnetic beads, polystyrene beads, latex beads, etc. ), Quenchers (azobenzenes (dabcyl, BHQ, etc.), metal particles, etc.) in the state of the solution or analysis, it is understood that such cases also belong to the scope of the present invention. Should.

  In short, the single particle detection technique of the present invention is a reversible scanning molecular counting method that detects single particles that do not emit light dispersed in a solution. Makes it possible to detect. According to the configuration of the present invention, similar to the scanning molecule counting method, the number density or concentration of a single particle to be observed is significantly lower than the level necessary for optical analysis techniques such as FCS, FIDA, PCH, etc. It has the advantage that it can be applied to a solution, and it eliminates the false detection of stray light and Raman scattered light of the inverted scanning molecule counting method as a signal of the observation target particle. Even when one particle is present, it is advantageous in that it can be detected separately if the wavelength bands in which the plurality of types of single particles do not emit light are different. The selection of the wavelength band may be made with reference to the result of a preliminary experiment or the like.

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

FIG. 1 (A) is a schematic diagram of the internal structure of a single particle detection apparatus that executes the inverted scanning molecule counting method according to the present invention. FIG. 1B is a schematic diagram of a confocal volume (light detection 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 and a measurement for explaining the principle of detecting the presence of a single particle that does not emit light in the detection light wavelength band in the inverted scanning molecule counting method in the present invention, respectively. It is a schematic diagram of the time change of the light intensity performed. 2 (C) to 2 (E) are diagrams for explaining how the particles appear when a background light in the detection light wavelength band is given in a solution in which two types of particles having different wavelength bands that do not emit light are mixed. is there. FIG. 3 is a diagram showing, in the form of a flowchart, one embodiment of the processing procedure of the inverted scanning molecule counting method executed in accordance with the present invention. FIGS. 4A and 4B show the case where the single particle crosses the light detection region while performing Brownian motion, and the position of the light detection region in the sample solution is faster than the diffusion movement speed of the single particle. It is a model diagram showing the mode of movement of particles when particles cross the photodetection region by moving at. FIG. 4C shows a signal of a detection signal in a processing procedure for detecting the presence of a single particle from measured time-series light intensity data (time change of photon count) according to the inverted scanning molecule counting method. It is a figure explaining the example of a process. FIG. 5 is a flowchart showing another aspect of the processing procedure of the inverted scanning molecule counting method executed according to the present invention. FIG. 6 is a flowchart showing still another aspect of the processing procedure of the inverted scanning molecule counting method executed according to the present invention. FIG. 7 is a fluorescent image of fluorescent beads. (A) is a case where the wavelength band where the beads emit light is set as the detection light wavelength band, and (B) is a case where the wavelength band where the beads do not emit light is set as the detection light wavelength band. FIG. 8 shows detection of a solution containing no beads (no beads), a solution 1 containing non-fluorescent beads (non-fluorescent beads), and a solution 2 containing fluorescent beads (fluorescent beads) according to the inverted scanning molecular counting method according to the present invention. The average value (bar graph) and standard deviation (error bar) of the number of pulses obtained are shown.

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 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 Single Particle Detection Device A single particle detection device that realizes the single particle detection technology according to the present invention has an FCS, as schematically illustrated in FIG. It may be an apparatus formed by combining an optical system of a confocal microscope capable of executing FIDA and the like and a photodetector. Referring to FIG. 1, single particle detection 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 single particle detection apparatus 1 may be the same as the optical system of a normal confocal microscope, in which laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is transmitted. The light diverges at an angle determined by the specific NA at the emission end of the fiber and is radiated, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, and the reflection mirrors 6 and 7, and is objective. The light enters the lens 8. In the apparatus of the present invention, when background light in a plurality of wavelength bands is detected, the wavelength of the excitation light for generating the background light can be selected. A plurality of light emitting sources (lasers) may be provided. Depending on the mode of measurement, excitation light having different wavelengths may be simultaneously emitted from a plurality of light emission sources and introduced into the objective lens 8.

  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 μL is dispensed is typically arranged is emitted from the objective lens 8. The laser light is focused in the sample solution in the sample container or well 10 to form a region with high light intensity (excitation region). In the sample solution, typically, particles that do not emit light in the detection wavelength band, which is an observation object, and any light-emitting substance that generates background light are dispersed or dissolved, and light is emitted in the detection wavelength band. When the particles that do not enter the excitation region are excited, the luminescent material is excited to emit substantially constant light to become background light, and when particles that do not emit light in the detection wavelength band enter the excitation region, the background light Will be reduced.

Thus, the light (Em) emitted from the excitation region passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter. 14 (here, only a light component in a specific wavelength band is selected), introduced into the multimode fiber 15, reaches the photodetector 16, and is converted into a time-series electrical signal. Thereafter, it is input to the computer 18 and processing for single particle detection is performed in a manner described later. As is known to those skilled in the art, in the above configuration, the pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, and as a result, as shown in FIG. 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 light 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). It has a Gaussian distribution with the center at the apex.The effective volume is the volume of a substantially elliptical sphere bounded by the plane where the light intensity is 1 / e ² )), and is called confocal volume.

  The light that has passed through the pinhole 13 is divided by the wavelength band in such a manner that light in a part of the wavelength band is reflected by the dichroic mirror 14a and light in the remaining wavelength band is transmitted. Each component passes through the corresponding barrier filter 14 (here, only the light component in a specific wavelength band is selected), and is introduced into the multimode fiber 15 to the corresponding photodetector 16. After reaching and being converted into a time-series electrical signal, it is input to the computer 18 and processing for optical analysis is performed in a manner described later. In the present invention, since the decrease in the amount of light due to the presence of a single particle in the background light consisting of weak light from several fluorescent dye molecules is detected, the photo detector 16 is preferably a photon. An ultra-sensitive photodetector that can be used for counting is used. When the light detection is based on photon counting, the light intensity is measured in a manner in which the number of photons arriving at the photodetector is sequentially measured for a predetermined 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 single particle detection apparatus described above, the sample solution is scanned by the light detection region, that is, the focal region, that is, the position of the light detection region is moved in the sample solution. The mechanism 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 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 movement trajectory of the position of the light detection region may be arbitrarily selected from a circle, an ellipse, a rectangle, a straight line, a curve, or a combination thereof (so that various movement patterns can be selected by the program in the computer 18). It may be.) Also, combining the method of moving the absolute position of the light detection region and the method of moving the absolute position of the sample solution, the absolute position of the light detection region can be moved while moving the position of the sample solution. May be executed. In this case, it is avoided that the same single particle is repeatedly detected when the light detection region in the short time passes through the same region. Or, by moving the absolute position of the light detection region, the same region is intentionally repeatedly passed through the light detection region, the same single particle is periodically detected multiple times, and the signal An improvement in accuracy may be achieved. In this case, after the absolute position of the light detection region is moved for a predetermined time, the position of the sample solution moves intermittently, and the same single particle is moved to another place in the sample solution. May be performed to increase the number of single particles detected. Although not shown, the position of the light detection region is moved in the vertical direction by moving the objective lens 8 or the stage up and down, and the locus of the position of the light detection region is three-dimensionally within the sample solution. It may be designed to be deployed.

  When the luminescent material that generates background light emits light by multiphoton absorption, the above optical system is used as a multiphoton microscope. In that case, since there is light emission only in the focal region (light detection region) of the excitation light, the pinhole 13 may be removed. Further, when the luminescent material that generates background light emits light regardless of excitation light due to chemiluminescence or bioluminescence, the optical systems 2 to 5 for generating excitation light may be omitted. When the luminescent material that generates background light emits light by phosphorescence or scattering, the optical system of the confocal microscope is used as it is. Furthermore, the background light may be provided by illumination light. In that case, the sample solution is illuminated from above the objective lens by transmitted illumination (may be Koehler illumination).

Principle of the Single Particle Detection Technology of the Present Invention As described in the “Summary of the Invention” section, the single particle detection technology of the present invention is simply described as a single particle in a specific wavelength band. In a scanning molecule counting method (hereinafter referred to as “inverted scanning molecule counting method”) that detects the shadow of a single particle, that is, in the presence of background light in a specific wavelength band, In a mode in which the position of the light detection region is moved at and a decrease in background light when a single particle that does not emit light in that specific wavelength band is included in the light detection region is detected as a single particle signal, The presence of a single particle is detected individually. By detecting light in a plurality of different wavelength bands, the presence of a plurality of types of single particles is detected for each type, and information regarding the count of those particles or the concentration in the sample solution is provided. To be acquired. Hereinafter, the principle of the inverted scanning molecule counting method according to the present invention will be described.

1. Principle of Inverted Scanning Molecular Counting Method In “scanning molecule counting method” described in Patent Documents 9 to 11, luminescent particles are dispersed in a single particle detector as described above. The light intensity is measured while positioning the light detection region in the sample solution. Then, when the light detection region includes the luminescent particles, the light from the luminescent particles reaches the light detection unit, so that an increase in the measured value of the light intensity occurs. The presence of one luminescent particle will be detected individually. 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, conventional FCS, FIDA, etc. have sufficient accuracy. Even with a sample solution in which the concentration of particles to be observed is so low that the analysis cannot be performed in (3), it is possible to obtain information on the particle concentration or number density.

  In the case of the above-described normal “scanning molecule counting method”, particles to be observed are luminescent particles, and particles that do not emit light in the wavelength band of detection light cannot be detected. Therefore, when particles that do not emit light are to be observed, it is necessary to attach a light-emitting label such as a fluorescent label to the particles. However, depending on the particle, it may be difficult to apply the luminescent label, or the particle may be modified due to the application of the luminescent label. Also, in the case of a format that identifies the light emitted by a particle as a signal indicating the presence of the particle, when the value on the light intensity data increases due to stray light, scattered light, or electrical noise of the photodetector, There may be a case where the increase in the value is mistakenly identified as a signal of the luminescent particles.

  Therefore, the “inverted scanning molecule counting method” of the present invention allows the background light to be emitted from the light detection region (or the light detection region is illuminated with illumination light) in the optical measurement by the scanning molecule counting method described above. ) When a particle to be observed enters the light detection region, a single particle is detected by detecting that the detected background light decreases. Specifically, as in the normal scanning molecule counting method, the optical path is changed by driving a mechanism (mirror deflector 17) for moving the position of the light detection region, or a sample solution is injected. The horizontal position of the container 10 (microplate 9) is moved to move the position of the light detection region CV in the sample solution as schematically illustrated in FIG. That is, light detection is performed while scanning the sample solution by the light detection region CV. As already mentioned, since the luminescent material is dispersed in the sample solution and a large number of luminescent materials are present in the light detection region CV, the light detection region CV is moving (in the figure, time to? t2) Basically, light from these luminescent materials is detected substantially uniformly. However, when the light detection region CV moves through the region where one non-light emitting particle exists (t1), the volume of the region where the light emitting material exists is reduced, so that the light emitted from the light emitting material is reduced. As shown in FIG. 2B, a significant light intensity (Em) in the form of a bell-shaped pulse appears on the time-series light intensity data. Thus, the movement of the position of the light detection region CV and the light detection described above are performed, and a significant decrease in light intensity in the form of pulses as illustrated in FIG. A single particle is detected individually by detecting the signal representing the presence one by one, and by counting the number, the number of single particles present in the measured area, or the concentration or number density The information regarding can be acquired.

The degree of decrease in the light intensity can be estimated from the relationship between the diameter of a single particle that does not emit light and the diameter of the light detection region. Typically, the light intensity distribution in the light detection region has a bell-shaped profile f (r) having a maximum intensity Imax at the center and decreasing toward the radius r. Therefore, using the radius a of the photodetection region where f (r) is substantially 0, the total amount α of light emitted from the photodetection region when there is no non-emitting particle in the photodetection region is
α = 4π∫r ² f (r) dr [Integration interval is 0 to a] (1)
Given in On the other hand, when a single particle that does not emit light having a radius b enters the photodetection region and is positioned at the center of the photodetection region, the luminescent material in that region is excluded. The corresponding amount of light will be reduced. The amount of light corresponding to the excluded luminescent material, that is, the decrease amount β is
β = 4π∫r ² f (r) dr [Integral interval is 0 to b] (2)
Given in Thus, the rate of decrease in light intensity can be estimated by β / α. Here, when f (r) is a Gaussian function and α = 1 and a = 1,
f (r) = 0.684exp (-2r ² ) (3)
It becomes. Typically, when the fluctuation rate of the background light is about 1% and the rate of decrease in light intensity due to a single particle is 1% or less, signal detection becomes impossible. When calculated, the ratio b / a of the single particle radius to the radius of the photodetection region should be 0.15 or more. When the rate of decrease in light intensity due to single particles is 10% or more, the ratio b / a of the detectable single particle radius to the radius of the photodetection region is 0.35. If the single particle to be observed is a quencher or a fluorescence energy transfer acceptor, the single particle absorbs ambient light (for example, 10 nm). It can be reduced from the illustrated radius.

2. Inverted Scanning Molecular Counting Method for Measuring Light in Multiple Wavelength Bands As mentioned in the “Summary of the Invention” section, the particles to be observed in the present invention usually have a wavelength band that emits light (emission wavelength). Band) and a wavelength band that substantially does not emit light (non-emission wavelength band). The emission wavelength band and the non-emission wavelength band are different from each other depending on the type of particles, and It is possible to prepare. In the inversion scanning molecule counting method as described above, if the wavelength band of the detection light, that is, the wavelength band of the background light is set within the non-emission wavelength band of the particle, the shadow of the particle is formed, and the particle The presence of can be detected. On the other hand, light emitted by particles having an emission wavelength band in the detection light wavelength band is indistinguishable from background light, and such particles are not detected. That is, as shown in FIG. 2C, when there are particles a that emit light in the first wavelength band and particles b that emit light in the second wavelength band, as shown in FIG. Under the condition in which background light in the wavelength band is detected, the particle a emits light, so that the background light does not decrease and the particle a is not detected, but the particle b does not emit light, resulting in a decrease in background light. Thereby, the presence is detected. On the other hand, as shown in FIG. 2E, under the condition in which the background light in the second wavelength band is detected, the particle b emits light, so that the background light does not decrease and the particle b is not detected. Does not emit light, the background light is reduced, and its presence is detected.

  Thus, in the present invention, a single particle having a non-emission wavelength band in the detected wavelength band by detecting light in a plurality of wavelength bands and detecting a decrease in background light for each wavelength band. Is detected. As already described, the non-emission wavelength band varies depending on the type of particles. Therefore, when a plurality of types of particles are mixed in the same sample solution, the non-emission wavelength band is different if the non-emission wavelength band is different. It is possible to detect a single particle while discriminating.

Processing Operation Process of Inverted Scanning Molecule Counting Method In the embodiment of the inverted scanning molecule counting method according to the present invention using the single particle detector 1 illustrated in FIG. (1) Preparation of a sample solution containing a single particle and a luminescent substance that generates background light, (2) Measurement processing of the light intensity of the sample solution, and (3) Analysis processing of the measured light intensity The FIG. 3 shows processing in this embodiment expressed in the form of a flowchart.

(1) Preparation of sample solution The particles to be observed in the single particle detection technique of the present invention are particles that are dispersed in the sample solution and move randomly in the solution, and the particle size is the light detection region. Preferably, the diameter may be 15% or more, more preferably 35% or more, such as protein, peptide, nucleic acid, lipid, sugar chain, amino acid, or an aggregate thereof. Particulate biological objects such as biomolecules, viruses, cells, non-biological particles (eg, atoms, molecules, micelles, liposomes, metal colloids, beads (magnetic beads, polystyrene beads, latex beads, etc. ), Quenchers (azobenzenes (dabcyl, BHQ, etc.), metal particles, etc.) The luminescent substance that gives the background light can be any luminescent molecule, such as a fluorescent molecule, phosphorescent molecule, chemical, etc. It may be a bioluminescent molecule, and the luminescent substance is dissolved or dispersed in the sample solution at a concentration that is always present in the light detection region at a few or more molecules, which is typically an aqueous solution. The organic solvent and other arbitrary liquids may be used.

  In particular, in the single particle detection technique of the present invention, as already mentioned, background light is given in the non-emission wavelength band and the specific emission wavelength band of the single particle to be observed. Sample preparation is performed. For example, in a sample solution, a first single particle that does not emit light in a first wavelength band and emits light in a second wavelength band different from the first wavelength band; A light emitting material that provides background light in the first wavelength band and a background in the second wavelength band if the second single particle that emits light and does not emit light in the second wavelength band is included. A luminescent material that provides light may be dispersed. In this regard, when detecting light in the emission wavelength band of the particles, the luminance of the background light and the emission luminance of the particles are completely the same, the emission luminance in the particles is uniform, Ideally, there should be no uneven brightness between the particles, but it is not always necessary to adjust the brightness of the particles and the background light, and there is a slight difference in brightness and a decrease in background light. However, it is sufficient if it can be identified in the analysis. For example, the particle | grains which have the outer shell which shows fluorescence in a non-fluorescent core, the particle | grains with which the fluorescent substance was covered by the non-fluorescent shell, etc. may be sufficient, for example. The brightness difference between the background light and the particles is ideally a brightness difference equal to or lower than the CV of the background light. Since the CV value of typical background light is 10%, it is desirable that the luminance difference between the light emitting particles and the background light is 10% or less, and preferably, the sample solution achieves such a state. It is prepared as follows. The background light may be fluorescence, autofluorescence, scattering (Raman scattering (solvent (water) carbon disulfide, isoprene, transition metal complex)), light from a luminescent material, or illumination light from a uniform light source. .

(2) Measurement of light intensity of sample solution (FIG. 3-Step 100)
The light intensity in the optical analysis by the inverted scanning molecule counting method of the present embodiment is measured by driving the mirror deflector 17 or the stage position changing device 17a during the measurement, and the position of the light detection region in the sample solution. Other than performing the above movement (scanning in the sample solution), it may be executed in the same manner as the light intensity measurement process in FCS or FIDA. 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 the measurement, under the control of the processing operation according to the program of the computer 18, the mirror deflector 17 or the stage position changing device 17a drives the mirror 7 (galvanomirror) or the microplate 9 on the stage of the microscope, and the wells. 10, the position of the light detection region is moved, and at the same time, the light detector 16 sequentially converts the detected light into an electric signal and transmits it to the computer 18. In this manner, time-series light intensity data is generated from the transmitted signal and stored. In the present embodiment, each of the plurality of photodetectors 16 detects light in different wavelength bands, and time-series light intensity data is generated for each detected different wavelength band. Measurement of light in different wavelength bands may or may not be performed simultaneously. Typically, the photodetector 16 is an ultra-sensitive photodetector that can detect the presence or absence of the arrival of one photon. Therefore, when the light is detected by photon counting, the time-series light intensity data is It may be a series of photon count data.

  The moving speed of the position of the light detection region during the measurement of the light intensity may optionally be a predetermined speed set, for example, experimentally or to suit the purpose of the analysis. When acquiring information on the number density or concentration based on the number of detected single particles, the size or volume of the region through which the light detection region has passed is required, so that the moving distance is grasped. The movement of the position of the light detection region is executed in the manner. 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.

  By the way, with respect to the moving speed of the position of the light detection region, in order to carry out quantitatively accurate detection of single particles or counting of the number of single particles from the measured time-series light intensity data. For this reason, it is preferable that the moving speed is set to a value faster than the moving speed by the random movement of the single particle, that is, the Brownian movement. Since the observation target particles of the single particle detection technique of the present invention are particles that are dispersed or dissolved in a solution and move 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 in the light detection region decreases outward with the center of the region as the apex), corresponding to each single particle. It becomes difficult to identify a significant change in light intensity. Therefore, preferably, as shown in FIG. 4B, the particle crosses the light detection region in a substantially straight line, so that in the time-series light intensity data, the uppermost stage of FIG. As shown in the example, the light intensity change profile corresponding to each single particle becomes substantially uniform (if the single particle passes through the light detection region substantially linearly, the light intensity change profile is It is almost the same as the profile obtained by inverting the excitation light intensity distribution.) The moving speed of the position of the light detection region is the average due to the Brownian motion of the particles so that the correspondence between individual particles and light intensity can be easily identified Is set to be faster than the moving speed (diffusion moving speed).

Specifically, the time Δt required for a particle having a diffusion coefficient D to pass through a photodetection region (confocal volume) having a radius Wo by Brownian motion is expressed by a relational expression of mean square displacement (2Wo) ² = 6D · Δt (4)
From
Δt = ( ² Wo) ² / 6D (5)
Therefore, the speed at which the particles move due to Brownian motion (diffusion movement speed) Vdif 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 of light intensity When time-series light intensity data is obtained by the above-described processing, the computer 18 detects a single particle signal by processing according to a program stored in a storage device, Various analyzes such as particle counting and concentration calculation are performed.

(I) Individual detection of single particle signal In time-series light intensity data, the trajectory of one particle passing through the light detection region is substantially linear as shown in FIG. In some cases, the change in light intensity in the signal corresponding to the particle has a downwardly convex, generally bell-shaped profile that reflects the light intensity distribution in the light detection region (determined by the optical system) (see FIG. 4 (C) Refer to the top row). Therefore, in the scanning molecule counting method, basically, when the time width in which the decrease in the light intensity that falls below the appropriately set threshold value measured from the background light is within a predetermined range, a profile of the decrease in the light intensity is obtained. It may be determined that the signal it has corresponds to that one particle has passed through the light detection region, and one particle may be detected. Then, a signal in which the time width in which the decrease in light intensity below the threshold continues is not within the predetermined range is determined as a noise or foreign matter signal. Further, the light intensity distribution in the light detection region has a Gaussian distribution that protrudes downward from the background light Ibg:
I = Ibg−A · exp (−2t ² / a ² ) (7)
Can be assumed that the intensity A and the width a calculated by fitting the equation (7) to a significant light intensity decrease profile (a profile that can be clearly determined not to be fluctuation of background light) are predetermined. When it is within the range, it is determined that the light intensity profile corresponds to the passage of one particle through the light detection region, and one particle may be detected. (A signal whose intensity A and width a are outside the predetermined range is determined as a noise or foreign object signal and may be ignored in the subsequent analysis or the like.)

  As an example of a processing method for collectively detecting single particles from time-series light intensity data, first, time-series light intensity data (FIG. 4C, uppermost “detection result (unprocessed)” ) Is subjected to smoothing (smoothing) (FIG. 3-step 110, upper part “smoothing” in FIG. 4C). The light emitted by the luminescent material is probabilistically emitted, and the light intensity is relatively weak. Therefore, the fine increase / decrease occurs. The detection accuracy of a signal indicating the presence of the signal is deteriorated. The smoothing process makes it possible to ignore the fine increase and decrease on the data. The smoothing process may be performed, for example, by a moving average method (for example, an adjacent average method, an algorithm of Savinsky-golay method), a percentile filter method, or an FFT filter method. Parameters for performing 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 speed) of the position of the light detection region when acquiring light intensity data. , BIN TIME may be set as appropriate. For example, the moving average method may be used.

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

Thereafter, a significant pulse signal is sequentially detected on the time-series light intensity data, and it is determined whether or not the detected signal is a signal corresponding to a single particle. Specifically, first, on the time-series time differential value data of the time-series light intensity data, with reference to the time differential value sequentially, the start point and the end point of one pulse signal are searched and determined, A pulse presence region is identified (step 130). When one pulse existence area is specified, a downward convex bell-shaped function fitting is performed on the smoothed time-series light intensity data in the pulse existence area (see FIG. Bell-shaped function fitting ”), peak-shaped intensity of peak of bell-shaped function (maximum decrease from background light) Ipeak, pulse width (full width at half maximum) Wpeak, correlation coefficient (of least squares method) in fitting, etc. A parameter is calculated (step 140). The bell-shaped function to be fitted is typically a Gaussian function, but may be a Lorentz-type function. Whether or not the calculated bell-shaped function parameter is within a range assumed for the bell-shaped profile parameter drawn by the pulse signal detected when one single particle passes through the light detection region. That is, whether the peak intensity of the pulse (A in equation (7), the maximum value of the decrease in background light), the pulse width, and the correlation coefficient are within predetermined ranges, for example,
The following conditions:
20 μs <pulse width <400 μs peak intensity> 4.0 [pc / 10 μs] (A)
Correlation coefficient> 0.95
Whether or not the condition is satisfied is determined (step 150). Thus, the signal determined that the calculated bell-shaped function parameter is within the assumed range in the signal corresponding to one particle is determined to be the signal corresponding to one particle. On the other hand, a pulse signal whose calculated bell-shaped function parameter is not within the assumed range is ignored as noise.

  The search and determination of the pulse signal in the processing of steps 130 to 150 may be repeatedly executed over the entire area of the time-series light intensity data (step 160). In particular, in the present embodiment, the above steps 110 to 170 are executed for each of time-series light intensity data in different wavelength bands, and the number of pulse signals corresponding to particles in each wavelength band is counted. May be. In addition, the process which detects the signal of a single particle individually from time series light intensity data is not restricted to said procedure, You may perform by arbitrary methods.

(Ii) Determination of particle concentration Furthermore, the number of particles detected may be counted to determine the number of particles (particle counting). If the total volume of the region through which the light detection region has passed is calculated by an arbitrary method, the number density or concentration of particles in the sample solution is determined from the volume value and the number of particles (step 170). ).

The total volume of the region through which the light detection region has passed may be theoretically calculated based on the wavelength of the excitation light or detection light, the numerical aperture of the lens, and the adjustment state of the optical system. For a solution having a known concentration (control solution), the light intensity measurement, particle detection and counting described above are performed under the same conditions as the measurement of the sample solution to be examined. It may be determined from the number and the concentration of particles in the control solution. Specifically, for example, for a control solution with a particle concentration C, if the number of detected single particles in the control solution is N, the total volume Vt of the region through which the light detection region has passed is
Vt = N / C (8)
Given by. In addition, as a control solution, a plurality of solutions having different concentrations of single particles are prepared, and measurement is performed on each of them. The calculated average value of Vt is adopted as the total volume Vt of the region through which the light detection region passes. You may be supposed to. Then, given Vt, the particle concentration c of the sample solution with a single particle counting result of n is
c = n / Vt (9)
Given by. The volume of the light detection region and the total volume of the region through which the light detection region has passed are given by any method, for example, using FCS or FIDA, without depending on the above method. It's okay. In the apparatus of the present embodiment, information on the relationship between the concentration C and the number N of various standard particles (formula (6)) is assumed for the assumed movement pattern of the light detection region. Information stored in advance in the storage device of the computer 18 may be used so that the user of the device can appropriately use the stored information when performing single particle detection.

  Thus, in the inverted scanning molecular counting method, in which the particles are individually detected by scanning in the sample solution using the light detection area, the counting and concentration of particles in the sample solution can be determined by the above processing procedure. It becomes. In particular, in the case of the present invention, particle counting is performed for each detection wavelength band, and a single particle concentration is determined, thereby making it possible to determine the concentration for each type of particle.

(4) Single particle detection process for detecting a certain number of signals In the single particle detection process described above, after performing light measurement over a set time, the obtained light intensity data is displayed on the obtained light intensity data. Single particle signal is detected. In that case, when the particle concentration in the sample solution is unknown, when measuring the light intensity over a fixed measurement time, the measurement time is sufficient to prepare for the case where the particle concentration is low. Will be set longer. On the other hand, when the concentration of the particles in the sample solution is high, the measurement of the light intensity is continued for more than the time necessary to determine the characteristics such as the concentration with an acceptable or satisfactory accuracy. Further, when the particle concentration in the sample solution is lower than the concentration assumed by the experimenter and the set measurement time is insufficient, the error of the result becomes large. Therefore, as another aspect of the single particle detection process, measurement of light intensity while moving in the light detection region and detection of single particle signals are repeated until the number of signals reaches a predetermined number, The time required for the number of particles to reach a predetermined number is measured, and based on the time required for the number of such single particle signals to reach a predetermined number, the particle concentration is determined. Good. According to this configuration, when the particle concentration in the sample solution is high, the time required for measuring the light intensity is shortened, and when the particle concentration in the sample solution is low, the result (ie, particle concentration) is required. The light intensity measurement can be continued until the number of particles that achieve the desired accuracy is obtained. And by setting the predetermined number that the number of single particle signals should reach to the number of particles that achieves the accuracy required for the result, the number of single particle signals is a predetermined number. The time taken to reach the value reflects the number of particles that achieve the required accuracy in the results, so the particle concentration value determined based on that time is acceptable or satisfactory. It is expected to have accuracy. Even in such a configuration, in the case of the present invention, from the optical measurement to the calculation of the particle concentration is executed for each of a plurality of detection wavelengths.

(I) Basic Principle The concentration value of particles and the time required for the number of signals to reach a predetermined number are related as follows. That is, in a sample solution having a certain particle concentration C, when the photodetection region is moved at the scanning speed u over time τ, the detected particle is represented by S as the cross-sectional area of the photodetection region. The number of signals X is
X = CSuτN _A ... (10)
It becomes. Here, _{N A} is Avogadro's number. Therefore, if time T is required for the number of signals to reach a predetermined number XE, the particle concentration C is
_{C = XE / (STuN A)} ... (11)
Is given as a function of time T. In the equation (11), the particle detection speed V per unit time is based on the time T required for the number of signals to reach a predetermined number XE and the number of detected particles XE: V = XE / T (12)
The particle concentration C is given by
_{C = V / (SuN A)} ... (13)
It is expressed. In this equation (13), the particle concentration C is linearly proportional to the detection speed V, and the correspondence between the particle concentration C and the detection speed V is easy to understand. Therefore, in an actual experiment, the particle concentration C May be determined using the detection speed V.

(Ii) Processing operation procedure The single particle detection processing for detecting a certain number of signals may be executed, for example, by the processing procedure shown in the flowchart of FIG. In the example of the figure, to put it briefly, a series of processes of moving the position of the light detection region, detecting light from the light detection region, detecting a single particle signal and counting the detected particle signal. Are repeatedly executed at every analysis time interval t (predetermined time interval) until the detected particle number X reaches the end particle number XE (a predetermined number that a single particle number should reach). The It should be understood that the series of processes and configurations described below are realized by the processing operation of the computer 18.

(A) Initial setting Referring to FIG. 5, in the operation process, specifically, first, the sample solution is injected into the well 10 of the microplate 9 and placed on the microscope stage, and then the user. When the computer 18 inputs an instruction to start light intensity measurement and particle detection / counting processing to the computer 18, the computer 18 sets the end particle number XE (step 10) and the analysis time interval t as initial settings. Is set (step 20). The number of end particles XE and the analysis time interval t may be arbitrarily set by the user. The number XE of end particles can be appropriately determined with reference to the result of a preliminary experiment using a solution having a known particle concentration so that the accuracy required for the result value of the particle concentration can be achieved. The analysis time interval t is an arbitrary time interval that is sufficiently shorter than the time from the start of processing until the number of particles (X) reaches the number of end particles (XE). It may be set as appropriate in consideration. In addition, as the end particle number XE and the analysis time interval t, values preliminarily determined with reference to the result of a preliminary experiment using a solution having a known particle concentration are stored in the apparatus 1, and such storage is performed. The set value may be used automatically or by user selection.

(B) Detection of the number of particles When the number XE of the end particles and the analysis time interval t are set as described above, the light by the scanning molecule counting method over the analysis time interval t is analyzed at each analysis time interval t as follows. Intensity measurement processing, detection of particle signals from the measured light intensity data and detection of the number of particles x (step 30), and accumulation of the number of particles x detected in step 30 results in the total number of particles X (tn) (Step 40) is repeatedly executed until the total number of particles X (tn) reaches the final particle number XE (step 50). Prior to the repeated execution of the processes in steps 30 to 50, the start time Ts of a series of processes may be stored (step 25).

  The light detection / particle number detection process in step 30 may be the same as the process shown in FIG. In short, the light intensity is measured over the analysis time interval t while moving the position of the light detection region in the sample solution (scanning in the sample solution), and then the obtained analysis is performed. In the time-series light intensity data at the time interval t, the computer 18 performs processing according to a program stored in the storage device to detect a signal indicating the presence of a single particle and count the detected number. Executed.

Thus, when the number of particles x in the time-series light intensity data over the analysis time interval t is detected, the total number X (t _n ) of single particles detected is X (t _n ) = X (t _n−1 ). + X (14)
(Step 40 in FIG. 5). X (t _n-1 ) is the total number of particles detected up to the previous analysis time interval t, and its initial value is zero. Steps 30 to 40 are performed until the total number X (tn) of single particles reaches the end particle number XE, that is,
X (t _n ) ≧ XE (15)
Is repeated at each analysis time interval t until the above is established (step 50). And if Formula (15) is materialized while repeating step 30-50, the process of the measurement of the light intensity of a sample solution, and the detection and count of particle | grains will be complete | finished. When the iterative process of steps 30 to 50 ends, the end time TE may be stored (step 60).

(C) Display of the number of particles and the measurement end time By the way, in the repeated execution period of steps 30 to 50 at every analysis time interval t (until the expression (15) is established), a display on the monitor of the computer 18 The total number of detected particles X (t _n ) and / or the measurement end time TE or the remaining measurement time Tr may be displayed. According to such a configuration, it is advantageous in that the user can predict when the measurement being performed will end by looking at the display.

In the case of executing the display as described above, when the formula (15) is not satisfied in the determination in step 50 of FIG. 5, each process indicated by a dotted line in the drawing is executed. Specifically, first, the latest total number of detected particles X (tn) calculated in step 40 is displayed on the display (step 52). Note that if the repeated execution of steps 30 to 50 has already been performed, the value of the total number of detected particles X (tn) so far is updated. Next, in order to calculate the measurement end time TE or the measurement remaining time Tr, the particle detection speed v after the start of the processing of Steps 30 to 50 is calculated (Step 54). The particle detection speed v up to now is
v = X (t _n ) / (Tp−Ts) (16)
May be given by Here, Tp is the current time. Thus, using the particle detection speed v, the measurement remaining time Tr (time until the processing of steps 30 to 50 is finished) is
Tr = (XE−X (t _n )) / v (17)
And the measurement end time TE (the time at which the processing of steps 30 to 50 is completed)
TE = Tp + Tr (18)
(Step 56). Then, the estimated measurement end time TE or the remaining measurement time Tr is displayed on the display (step 58). If the repeated execution of steps 30 to 50 has already been performed, the value already displayed is updated. Further, when X (t _n ) = 0, Expression (17) or (18) may not be calculated, and Tr and TE may be displayed as unknown.

  The processing of steps 30 to 50 in FIG. 5 is repeated every analysis time interval t as already described. In this regard, the measurement of the light intensity in step 100 of FIG. 3 may be continuously performed during the execution of the signal processing steps other than step 100 from the start to the end of the measurement. That is, in the light detection / particle number detection process, when the measurement of the light intensity over the analysis time interval t of one cycle is completed, the measurement of the light intensity over the analysis time interval t of the next cycle is performed. At the same time, the computer 18 executes processing for detecting and counting the particle signal from the light intensity data acquired over the analysis time interval t of the completed cycle. Become. Thereby, detection / counting of particles is achieved in real time.

(D) Analysis of concentration calculation etc. Thus, when the number of particles reaches the number of end particles, the time T (= TE−Ts) until the number of particles reaches the number of end particles or other signals obtained from the detected particle signal Analysis such as concentration calculation may be executed using the information (step 70). As described above, the particle concentration is calculated from the time T until reaching the end particle number and the end particle number XE using the equation (12), and the particle detection speed V is calculated. , Determined using the relationship of equation (13).

In addition, the cross-sectional area S of the passage region of the light detection region in the equations (10) to (13) is theoretically calculated based on the wavelength of the excitation light or detection light, the numerical aperture of the lens, and the adjustment state of the optical system. However, experimentally, for example, for a solution having a known particle concentration (control solution), measurement of light intensity and particle detection described above under the same conditions as the measurement of the sample solution to be examined. It may be determined from the number of particles detected by performing the counting and the concentration of a single particle in the control solution. Specifically, for example, assuming that the number of detected particles in the measurement of light intensity performed for a certain time τo at a moving speed uo for a control solution having a particle concentration C is N The cross-sectional area S of the passage area of the detection area is
_{S = N / (C · N} A · uo · τo) ... (19)
Given by. In addition, a plurality of solutions having different concentrations of particles are prepared as a control solution, and measurement is performed on each of them. The calculated average value of S is adopted as the cross-sectional area S of the light detection region. Good.

(E) Modified example of measurement of light intensity of sample solution and detection / counting process of particles In the measurement of light intensity of sample solution and detection / counting process of single particle, analysis is performed as another aspect. The time interval t is not a fixed value but may be corrected according to the detection state of a single particle. FIG. 6A is a flowchart of the measurement of the light intensity of the sample solution and the particle detection / counting process configured to include a process (step 20 ′) for correcting the analysis time interval t according to the particle detection state. FIG. 6B shows the calculation processing of the analysis time interval t in step 20 ′ in the form of a flowchart. In FIG. 6A, the same step number is assigned to the same process as in FIG.

Referring to FIG. 6, in the process of FIG. 6, every time the measurement of the light intensity over the analysis time interval t is completed, the analysis time interval t is corrected (step 20 ′). In addition, the process of the example shown in the drawing is referred to as a predetermined number N (hereinafter referred to as “scheduled number of updates”) in one measurement from the start until the number of particles reaches the end particle number XE. ) Only the light intensity measurement and particle detection / counting processing cycles are executed. Specifically, as an initial setting, first, after setting the number XE of end particles (step 10) and storing the start time Ts (step 25), first the light intensity measurement and particle detection / counting processing are executed. In other words, when the number of executions k of the light intensity measurement and particle detection / counting processing cycle is 0, an initial value to which can be arbitrarily set is given to the analysis time interval t (FIG. 6B ) See steps 200 and 210). Then, the number of executions k of the processing cycle is incremented by 1 (step 270), and the light intensity measurement and particle detection / counting processing over the analysis time interval t are executed (step 270) (step 270). 30-50). Thus, when the number x (= X (t ₁ )) of particles in the first cycle is obtained, the particle detection speed v (step 54) and the remaining measurement time Tr (step 56) are calculated in order. As in the case of FIG. 5, the total number of detected particles X (t _n ) and / or the measurement end time TE or the remaining measurement time Tr are displayed on a display such as the monitor of the computer 18. (Steps 52 and 58). If the number of particles has reached the end particle number XE in the first processing cycle, the light intensity measurement and particle detection / counting processes are terminated (step 50).

After the first processing cycle, the analysis time interval t is corrected, and the light intensity measurement and particle detection / counting processing cycle (steps 20 ′, 30 to 58) is the same as in FIG. Repeat until particle number XE is reached. At this time, in step 20 ′ for correcting the analysis time interval t, it is first determined whether or not the number of particles X (tn) detected so far is 0 (step 220). . When X (tn) = 0, the analysis time interval t in the immediately preceding cycle may be multiplied by m (m is a positive number of 1 or more). When X (tn)> 0, the analysis time interval t is determined by using the remaining measurement time Tr, the scheduled update count N, and the execution count k of the processing cycle.
t = Tr / (N−k) (20)
(Step 240). Note that a lower limit may be set for the calculated analysis time interval t, and when the analysis time interval t falls below the lower limit value tmin, the analysis time interval t may be set to the lower limit value tmin (step 250, 260). As described above, according to the aspect in which the analysis time interval t is corrected, the measurement remaining time Tr reflects the detection state of particles in the sample solution to be observed. It will be optimized according to the detection status of such particles.

  In order to verify the effectiveness of the present invention described above, the following experiment was conducted. It should be understood that the following examples illustrate the effectiveness of the present invention and do not limit the scope of the present invention.

  It was verified by a confocal fluorescence microscope image that a signal indicating the presence of a single particle that does not emit light can be detected in time-series light intensity data obtained by light measurement by the inverted scanning molecule counting method.

  As a sample solution, a 20% polyethylene glycol solution containing 200 μM fluorescent dye ATTO488, 50 μM fluorescent dye ATTO633 and 10 fM fluorescent beads (FP-4052-2: spherotec (D: 4000 nm)) was prepared. The sample solution was observed using a confocal laser scanning microscope FV10i (Olympus) to obtain a confocal fluorescence microscope image. The measurement was performed using excitation light 473 nm (120 μW) and excitation light 635 nm (47 μW). The fluorescent beads used are particles that emit light when illuminated with excitation light at 473 nm, and hardly emit light when illuminated with excitation light at 635 nm. ATTO 488 and ATTO 633 are luminescent materials for emitting background light when fluorescence is observed with excitation light 473 nm and excitation light 635 nm, respectively.

  FIG. 7 shows confocal fluorescence microscope images observed at (A) excitation light 473 nm and (B) excitation light 635 nm, respectively. As can be seen from the figure, (A) in the case of excitation light 473 nm, the shadow of the beads was hardly detected, whereas in the case of (B) excitation light 635 nm, the shadow of each bead was observed. . This means that, as in the present invention, the presence of particles that emit light in a specific wavelength band and hardly emit light in another wavelength band can be detected in the presence of background light in the non-emission wavelength band of the particle, This indicates that the light is not detected in the presence of background light in the emission wavelength band.

  In accordance with the inverted scanning molecule counting method, the beads were counted in the sample solution containing the beads. As a sample solution, fluorescent solution was obtained with solution 1 and 10 fM in which non-fluorescent beads (CP-40-10: spherotec (D: 4000 nm)) were dispersed at 10 fM in a 20% polyethylene glycol solution containing 0.5 μM fluorescent dye ATTO488 Solution 2 in which beads (FP-4052-2: spherotec (D: 4000 nm)) were dispersed was prepared. In the measurement of light, a single molecule fluorescence measurement device MF20 (Olympus Corporation) equipped with an optical system of a confocal fluorescence microscope and a photon counting system is used as an optical analysis device, and the above-mentioned “(2) Sample solution In accordance with the embodiment described in “Measurement of Light Intensity”, time-series light intensity data (photon count data) was obtained for the above two sample solutions. At that time, 50 μW of 488 nm laser light was used as the excitation light, and light in the wavelength band of 650-690 nm was measured with an optical filter (615LP and 670BP) to generate time-series photon count data. The moving speed of the position of the light detection region in the sample solution was 9000 rpm (67.5 mm / second), the BIN TIME was 50 μsec, and the measurement for 100 seconds was performed three times for each solution. Note that, under the above conditions, the beads in the solution 2 fluoresce, so that the light intensity is hardly lowered.

In the data processing after optical measurement, first, in the acquired time-series photon count data, “(i) Individual detection of single particle signal” and steps 110 to 160 in FIG. According to the procedure, smoothing processing is performed on the time-series photon count data, and after determining the start point and end point of the pulse signal in the smoothed data, a Gaussian function is fitted to each pulse signal by the least square method, The peak intensity (in the function) (maximum amount of decrease from background light), pulse width (full width at half maximum), and correlation coefficient were determined. And the following conditions:
20 μsec <pulse width <400 μsec Peak intensity ≧ 20% of the average value of background light (A)
Correlation coefficient> 0.95
Only pulse signals satisfying the above were extracted as single particle signals.

  FIG. 8 shows the number of pulses detected in the time-series photon count data for the solution containing no beads, the solution 1 containing non-fluorescent beads, and the solution 2 containing fluorescent beads in the above procedure. . Referring to the figure, about 2700 pulses were detected in solution 1 containing non-fluorescent beads, whereas the number of pulses was greatly reduced in solution 2 containing fluorescent beads. . This suggests that the light emitted from the fluorescent beads is less likely to reduce the light intensity from the background light. This result indicates that it is possible to detect a specific type of particles in the sample solution depending on whether or not light similar to background light is emitted. (In the solution 2 containing fluorescent beads, the reason why the number of pulses was detected is considered to be due to variations in the emission luminance of the fluorescent beads. That is, in the beads used as fluorescent beads, This is because some of them have low emission brightness.)

  Thus, as can be seen from the results of the above examples, the inverted scanning molecule counting method in multiple wavelength bands in accordance with the teachings of the present invention allows for emission wavelength bands and non-emissions dispersed in the sample solution. Detection of single particles having different wavelength bands and acquisition of information on their concentrations are possible for each type of particle. In particular, since the present invention individually detects single particle signals, particles can be detected even if the particle concentration in the sample solution is lower than the concentration range required by optical analysis techniques such as FCS. Yes, such a feature may be advantageous when performing analysis on rare or expensive samples often used in the field of medical and biological research and development.

Claims (12)

A single particle detection device for detecting single particles dispersed and moving randomly in a sample solution using an optical system of a confocal microscope or a multiphoton microscope,
A light detection region moving unit that moves the position of the light detection region of the optical system in the sample solution;
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 signals representing the presence of each of the single particles in the data;
A first single particle that emits light in a second wavelength band different from the first wavelength band, wherein the single particle does not substantially emit light in the first wavelength band; and A second single particle that emits light in a wavelength band and does not substantially emit light in the second wavelength band, and a wavelength band of light detected by the light detection unit is the first wavelength band The light from the light detection region includes a substantially constant background light in the first wavelength band, and a signal indicative of the presence of each of the single particles is When the light intensity detected by the light detection unit is reduced when entering the light detection region, and the wavelength band of light detected by the light detection unit is the second wavelength band, The light from the light detection region comprises a substantially constant background light in the second wavelength band; The signal indicating the presence of each single particle is a decrease in light intensity detected by the light detection unit that occurs when the second single particle enters the light detection region. Device to do.   The apparatus according to claim 1, wherein when the wavelength band of light detected by the light detection unit is the first wavelength band, the intensity of the background light and the light intensity from the second single particle Are substantially equal, and the wavelength band of the light detected by the light detection unit is the second wavelength band, the intensity of the background light and the light intensity from the first single particle are substantially The intensity of the background light is adjusted to be equal to   3. The apparatus according to claim 2, wherein the intensity of the background light is adjusted by dispersing luminescent particles in the sample solution.   4. The apparatus according to claim 1, wherein the signal processing unit counts the number of signals indicating the presence of the individually detected single particles and moves the position of the light detection region. An apparatus for counting the number of detected single particles. A single particle detection method for detecting single particles dispersed in a sample solution and moving randomly using an optical system of a confocal microscope or a multiphoton microscope,
Moving the position of the light detection region of the optical system in the sample solution;
Detecting light from the light detection region while moving the position of the light detection region in the sample solution to generate time-series light intensity data;
Individually detecting a signal representing the presence of each of the single particles in the time-series light intensity data,
A first single particle that emits light in a second wavelength band different from the first wavelength band, wherein the single particle does not substantially emit light in the first wavelength band; and A second single particle that emits light in a wavelength band and does not substantially emit light in the second wavelength band, and when the wavelength band of the detected light is the first wavelength band, The light from the light detection region includes a substantially constant background light in the first wavelength band, and a signal representative of the presence of each of the single particles indicates that the first single particle is in the light detection region. A decrease in light intensity in the time-series light intensity data that occurs when the light enters, and when the wavelength band of the detected light is the second wavelength band, light from the light detection region is Including a substantially constant background light in the second wavelength band, and the presence of each of the single particles. How to signal, wherein said second single particles are reduced in in light intensity to the time-series light intensity data generated when entering into the light detection region.   6. The method according to claim 5, wherein when the wavelength band of the detected light is the first wavelength band, the intensity of the background light and the light intensity from the second single particle are substantially equal. Equally, when the wavelength band of the detected light is the second wavelength band, the intensity of the background light is substantially equal to the intensity of light from the first single particle. A method characterized in that the intensity is adjusted.   7. The method of claim 6, wherein the intensity of the background light is adjusted by dispersing luminescent particles in the sample solution.   The method according to any one of claims 5 to 7, further comprising: counting the number of signals representing the presence of the individually detected single particles to detect the position of the light detection region during movement. A method comprising the step of counting the number of single particles. A computer program for single particle detection that detects single particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope or a multiphoton microscope,
A procedure for moving a position of a light detection region of the optical system in the sample solution;
A procedure for generating light intensity data in time series by detecting light from the light detection region while moving the position of the light detection region in the sample solution;
Causing the computer to execute a procedure of individually detecting signals representing the presence of each of the single particles in the time-series light intensity data,
A first single particle that emits light in a second wavelength band different from the first wavelength band, wherein the single particle does not substantially emit light in the first wavelength band; and A second single particle that emits light in a wavelength band and does not substantially emit light in the second wavelength band, and when the wavelength band of the detected light is the first wavelength band, The light from the light detection region includes a substantially constant background light in the first wavelength band, and a signal representative of the presence of each of the single particles indicates that the first single particle is in the light detection region. A decrease in light intensity in the light intensity data that occurs when the light enters, and when the wavelength band of the detected light is the second wavelength band, the light from the light detection region is A signal representing the presence of each of the single particles, including substantially constant background light in two wavelength bands. But the computer program, wherein the second single particles are reduced in in light intensity in said light intensity data generated when entering into the light detection region.   10. The computer program according to claim 9, wherein when the wavelength band of the detected light is the first wavelength band, the intensity of the background light and the light intensity from the second single particle are substantially equal. When the wavelength band of the detected light is the second wavelength band, the background light is set to be substantially equal to the intensity of the background light and the light intensity from the first single particle. A computer program characterized in that the intensity of the program is adjusted.   11. The computer program according to claim 10, wherein the intensity of the background light is adjusted by dispersing luminescent particles in the sample solution. The computer program according to any one of claims 9 to 11, further comprising: detecting a number of signals indicating the presence of the individually detected single particles while detecting the position of the light detection region. A computer program comprising a procedure for counting the number of single particles.