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WO2013021984A1 - Dispositif d'analyse optique et procédé d'analyse optique utilisant un système optique de microscope confocal ou de microscope multiphoton - Google Patents

Dispositif d'analyse optique et procédé d'analyse optique utilisant un système optique de microscope confocal ou de microscope multiphoton Download PDF

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WO2013021984A1
WO2013021984A1 PCT/JP2012/070044 JP2012070044W WO2013021984A1 WO 2013021984 A1 WO2013021984 A1 WO 2013021984A1 JP 2012070044 W JP2012070044 W JP 2012070044W WO 2013021984 A1 WO2013021984 A1 WO 2013021984A1
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light
optical system
light detection
sample solution
optical
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Japanese (ja)
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山口光城
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Olympus Corp
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Olympus Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G01N21/6404—Atomic fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N2021/3125—Measuring the absorption by excited molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/3103—Atomic absorption analysis

Definitions

  • 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- It relates to optical analysis technology that can detect light from biological particles and obtain useful information in the analysis or analysis of those states (interaction, binding / dissociation state, etc.).
  • the present invention relates to an optical analysis apparatus and an optical analysis method that enable various optical analyzes by detecting light from particles that emit light using the optical system as described above.
  • a particle that emits light (hereinafter referred to as “luminescent particle”) is either a particle that emits light itself, or a particle to which an arbitrary luminescent label or luminescent probe is added.
  • the light emitted from the luminescent particles may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, or the like.
  • the average residence time (translational diffusion time) of the fluorescent molecules and the like in the minute region determined from the value of the autocorrelation function of the measured fluorescence intensity, and the average number of staying molecules Based on this, acquisition of information such as the speed or size of movement of fluorescent molecules, concentration, concentration, molecular structure or size change, molecular binding / dissociation reaction, dispersion / aggregation, etc.
  • fluorescence intensity distribution analysis Fluorescence-Intensity Distribution Analysis: FIDA.
  • Patent Document 4 Non-Patent Document 4 and Photon Counting Histogram (PCH.
  • Patent Document 5 are measured in the same manner as FCS.
  • a histogram of the fluorescence intensity of the fluorescent molecules entering and exiting the confocal volume generated is generated, and by fitting a statistical model formula to the distribution of the histogram, the average of the intrinsic brightness of the fluorescent molecules etc.
  • the average value of the value and the number of molecules staying in the confocal volume is calculated, and based on this information, changes in the structure or size of the molecule, binding / dissociation state, dispersion / aggregation state, etc. are estimated. It will be.
  • Patent Documents 6 and 7 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.
  • Patent Document 8 describes the presence of fluorescent fine particles in a flow or on a substrate by measuring weak light from fluorescent fine particles distributed in a flow cytometer or fluorescent fine particles fixed on a substrate using a photon counting technique. A signal arithmetic processing technique for detecting the signal is proposed.
  • the sample required for measurement has an extremely low concentration compared with the conventional method. It can be very small (the amount used in one measurement is about several tens of ⁇ L), and the measurement time is greatly shortened (measurement of time on the order of seconds is repeated several times in one measurement). . Therefore, these technologies are particularly useful for analyzing rare or expensive samples often used in the field of medical and biological research and development, for clinical diagnosis of diseases, screening for physiologically active substances, etc. When the number is large, it is expected to be a powerful tool capable of performing experiments or inspections at a lower cost or faster than conventional biochemical methods.
  • the light to be measured is light emitted from one or several fluorescent molecules.
  • statistical processing such as calculation of fluctuation of fluorescence intensity such as calculation of autocorrelation function of fluorescence intensity data measured in time series or fitting to histogram is executed, and light signals from individual fluorescent molecules etc. are processed. It is not individually referenced or analyzed. That is, in these photoanalysis techniques, light signals from a plurality of fluorescent molecules and the like are statistically processed, and statistical average characteristics of the fluorescent molecules and the like are detected.
  • the concentration or number density of the fluorescent molecules or the like to be observed in the sample solution is equal to one second in the equilibrium state.
  • the number of fluorescent molecules that can be statistically processed within the measurement time of the order length enters and exits the micro area, and preferably there is always about one fluorescent molecule in the micro area.
  • the concentration of fluorescent molecules or the like in the sample solution used in the above optical analysis technique is typically about 1 nM or more. When it is significantly lower than 1 nM, a time when the fluorescent molecule or the like is not present in the confocal volume occurs, and a statistically significant analysis result cannot be obtained.
  • the detection methods for fluorescent molecules and the like described in Patent Documents 6 to 8 do not include statistical calculation processing of fluctuations in fluorescence intensity, and even if the fluorescent molecules in the sample solution are less than 1 nM, the fluorescent molecules However, it has not been achieved to quantitatively calculate the concentration or number density of fluorescent molecules that are moving randomly in the solution.
  • the applicant of the present application deals with the optical analysis technique including the statistical processing such as FCS, FIDA, etc. in the concentration or number density of the luminescent particles to be observed in Japanese Patent Application No. 2010-044714 and PCT / JP2011 / 53481.
  • An optical analysis technique based on a novel principle that makes it possible to quantitatively observe the state or characteristics of luminescent particles in a sample solution below a certain level is proposed.
  • 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, like FCS, FIDA and the like.
  • the photodetection area When used, while moving the position of a minute region (hereinafter referred to as “light detection region”) that is a light detection region in the sample solution, that is, while scanning the sample solution by the light detection region,
  • the photodetection area includes luminescent particles that are dispersed in the sample solution and move randomly, the light emitted from the luminescent particles is detected, and each of the luminescent particles in the sample solution is detected individually.
  • the sample required for measurement is a very small amount (for example, about several tens of ⁇ L) as in the optical analysis techniques such as FCS and FIDA.
  • the measurement time is short, and the presence of luminescent particles having a lower concentration or number density is detected as compared with the case of optical analysis techniques such as FCS and FIDA, and the concentration, number density or Other characteristics can be detected quantitatively.
  • the confocal volume (that is, the light detection region) of the optical system is entered by using the optical system of the confocal microscope or the multiphoton microscope such as the scanning molecule counting method, FCS, FIDA, and PCH as described above.
  • the optical analysis technology that detects light emitted from luminescent particles, when the concentration of luminescent particles in the sample solution is low, the amount of light detected (in the case of photon counting, the number of photons) is reduced, with good accuracy. Obtaining measurement results can be difficult.
  • the problem caused by the low concentration of luminescent particles in the sample solution and the small number of luminescent particles per unit time entering the confocal volume can be solved by expanding the confocal volume. . That is, the region where light is detected at a time by expanding the size of the focal region of the excitation light emitted from the objective lens of the confocal microscope or multiphoton microscope or expanding the diameter of the pinhole (in the case of a confocal microscope) If the confocal volume is expanded, the opportunity for the luminescent particles to enter the confocal volume increases, so the number of detected luminescent particles per unit time increases and the measurement time is relatively short. It is expected that highly accurate measurement results will be obtained.
  • the observed luminescent particle is a particle that is excited by the excitation light and emits light
  • simply increasing the size of the focal region of the excitation light the excitation light density in the confocal volume Decreases, and the amount of light detected per luminescent particle decreases. Therefore, when increasing the number of detections of luminescent particles per unit time by expanding the confocal volume, the emission intensity of the excitation light from the objective lens is increased along with the expansion of the confocal volume. It is preferable to avoid a decrease in the amount of detected light per particle.
  • the intensity of fluorescence or phosphorescence emitted from one luminescent particle increases to a certain extent with an increase in excitation light intensity. Further, it has been found that when the intensity of the excitation light is further increased, it becomes saturated and hardly increases.
  • the background light is scattered light such as stray light of excitation light or Raman scattering of water, it continues to increase without saturation as the excitation light intensity increases. Therefore, if the excitation light intensity becomes higher than a certain level, the background light intensity becomes higher than the light intensity of the luminescent particles in the detection light.
  • the main object of the present invention is to provide the background light intensity in the optical analysis technique using the optical system of the confocal microscope or the multiphoton microscope, such as the above-mentioned scanning molecule counting method, FCS, FIDA, and PCH.
  • the present invention proposes a novel technique capable of increasing the number of detected luminescent particles per unit time by increasing the volume of the confocal volume in a manner that can suppress the increase as much as possible.
  • the diameter of one confocal volume is increased. It has been found that the increase in background light intensity can be suppressed more than when the confocal volume is increased. In the present invention, the above knowledge is adopted.
  • the above problem is an optical analyzer that detects and analyzes light from luminescent particles that are dispersed and randomly moved in a sample solution using an optical system of a confocal microscope or a multiphoton microscope.
  • the optical system of the microscope is achieved by an apparatus having a split optical system that forms at least two light detection regions of the optical system of the microscope in the sample solution.
  • a light-emitting particle dispersed in a sample solution and moving randomly is a particle that emits light, such as atoms, molecules, or aggregates thereof dispersed or dissolved in a sample solution. Any particle may be used as long as it is not fixed to the substrate or the like and freely moves in the solution in Brownian motion.
  • Such luminescent particles are typically fluorescent particles, but may be other particles (such as phosphorescent particles) that emit light upon irradiation with excitation light.
  • the “photodetection area” of the optical system of a confocal microscope or a multiphoton microscope is a confocal volume, that is, a minute area in which light is detected in a microscope, and when excitation light is given from an objective lens. Corresponds to a region where the excitation light is collected.
  • Split optical system means any optical system or optical that splits the excitation light beam propagating from one light source into the excitation light beam so that it is focused on at least two point regions in the sample solution. For example, a wedge-shaped half mirror or a diffractive optical element is employed.
  • the term “signal” refers to a signal representing light from the luminescent particles unless otherwise specified.
  • the optical analyzer which detects the light from a luminescent particle in a sample solution using the optical system of a confocal microscope or a multiphoton microscope, for example, a scanning molecule counting method, FCS, FIDA, PCH, etc.
  • a confocal volume In an apparatus for performing the optical analysis technique, at least two light detection regions, that is, a confocal volume, are formed in the sample solution, thereby increasing the volume of the light detection region. In that case, the intensity of the background light that appears in the detection light of the microscope is relatively low, even if the excitation light density is the same, as compared to the case where the diameter of one light detection region is increased.
  • the size of one photodetection region may be a size that is set as standard in an optical system of a confocal microscope or a multiphoton microscope (typically, about the wavelength of excitation light and detection light). .
  • light from at least two light detection regions formed by the split optical system may be received by one light detector.
  • a photodiode preferably an APD, is typically used as the photodetector.
  • the split optical system is selectively used when it is desired to enlarge the photodetection area in measurement, and is used when enlargement of the photodetection area is not desired. Therefore, only one photodetection region may be formed.
  • the apparatus can form only one light detection region when the light emitting particles in the sample solution are relatively high, and two or more light detection regions when the light emitting particles in the sample solution are relatively low. Is preferably formed.
  • the above-described apparatus of the present invention has a configuration in which at least two photodetection regions are formed using a split optical system in a microscope optical system, and one photodetection region is formed without using the split optical system. The configuration may be selectable.
  • a split optical system such as a wedge-shaped half mirror or a diffractive optical element may be detachably inserted into the excitation light optical path according to a user's request.
  • the volume of the light detection region can be appropriately selected according to the state of the sample solution to be measured, and the range of the concentration of the luminescent particles in the sample solution that can be measured efficiently and with high accuracy. It will be larger than before.
  • the present invention described above may be applied to an apparatus that executes a “scanning molecule counting method”. Therefore, in one embodiment of the present invention, the above-described apparatus of the present invention further moves the position of the light detection region of the optical system in the sample solution by changing the optical path of the optical system of the microscope.
  • a signal processing unit that individually detects a signal representing light from each of the particles.
  • the position of the photodetection region is moved in the sample solution, that is, while the sample solution is scanned by the photodetection region, sequentially.
  • the light is detected.
  • the moving light detection region includes randomly moving luminescent particles
  • the light from the luminescent particles is detected by the photodetector, thereby detecting the presence of one luminescent particle. It becomes.
  • the signal processing unit of the apparatus detects the signal representing the light from the luminescent particles in the signal from the photodetector that is sequentially detected, thereby individually detecting the presence of the luminescent particles one by one. It detects sequentially and the various information regarding the state in the solution of a luminescent particle will be acquired.
  • the signal processing unit counts the number of signals representing light from the individually detected luminescent particles and moves the position of the light detection region.
  • the number of detected luminescent particles may be counted (counting of luminescent particles).
  • information on the number density or concentration of the luminescent particles in the sample solution can be obtained by combining the number of luminescent particles and the amount of movement of the position of the light detection region.
  • the total volume of the movement locus 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 the luminescent particles is reduced. It can be calculated specifically.
  • the optical detection area is moved to change the position of the photodetection area by changing the optical path of the optical system. Because there is virtually no mechanical vibration or hydrodynamic action, light can be measured in a stable state without being affected by the mechanical action of the luminescent particles.
  • the signal processing unit of the apparatus determines whether one luminescent particle has entered the light detection region from the detection value signal from the sequential light detection unit. May be performed based on the shape of a signal representing time-series light detected by the light detection unit. In the embodiment, typically, when a signal having an intensity greater than a predetermined threshold is detected, it may be detected that one luminescent particle has entered the light detection region. More specifically, as will be described later in the section of the embodiment, the signal representing the light from the luminescent particles is usually the time-series detection value of the light detection unit, that is, the light intensity data.
  • the apparatus of the present invention generates light intensity data composed of detection values of the light intensity sequentially detected by the light detection unit, and the signal processing unit has an intensity exceeding a predetermined threshold on the light intensity data. It may be configured to detect the pulsed signal as a signal representing light from a single luminescent particle.
  • the “predetermined threshold value” can be set to an appropriate value experimentally. According to such a configuration, the light emitting particle signal and the background light or noise are distinguished from each other in the light intensity sequentially measured by the light detection unit, and the individual detection of the light emitting particle signal is advantageously performed. It becomes possible.
  • the detection target of the apparatus of the present invention is light from a single luminescent particle, the light intensity is very weak. Therefore, preferably, the light detection unit detects light from the light detection region by photon counting. In that case, time-series light intensity data becomes time-series photon count data.
  • the light detection unit detects light from the light detection region by photon counting. In that case, time-series light intensity data becomes time-series photon count data.
  • one luminescent particle is a single fluorescent molecule or several molecules
  • the light emitted from the luminescent particle is probabilistically emitted, and the signal value may be lost in a very short time. There is. When such a lack occurs, it becomes difficult to specify a signal corresponding to the presence of one luminescent particle.
  • the signal processing unit smoothes the time-series light intensity data, processes the data so that missing signal values at a minute time can be ignored, and then performs smoothing on the time-series light intensity data.
  • a bell-shaped pulsed signal having an intensity exceeding a predetermined threshold value may be detected as a signal representing light from a single luminescent particle.
  • 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 luminescent particles or the number density or concentration in the sample solution.
  • the manner of light detected from the luminescent particles can vary depending on the properties of the luminescent particles or the number density or concentration in the sample solution.
  • the moving speed of the light detection region increases, the amount of light obtained from one light emitting particle decreases, so that the light from one light emitting particle can be measured with high accuracy or sensitivity.
  • the moving speed is preferably changed as appropriate.
  • the moving speed of the position of the light detection region in the sample solution is preferably the diffusion moving speed of the luminescent particles to be detected (the average moving speed of the particles due to Brownian motion). ) Is set higher.
  • the apparatus of the present invention detects light emitted from the luminescent particles when the light detection region passes through the position where the luminescent particles exist, and emits light. Detect particles individually. However, if the luminescent particles move randomly in the solution due to Brownian motion and enter and exit the photodetection region multiple times, a signal (representing the presence of the luminescent particles) is emitted multiple times from one luminescent particle.
  • the moving speed of the light detection region is set to be higher than the diffusion moving speed of the luminescent particles, so that one luminescent particle can correspond to one signal (indicating the presence of the luminescent particle). It becomes. Since the diffusion movement speed varies depending on the luminescent particles, as described above, according to the characteristics of the luminescent particles (particularly, the diffusion constant), the apparatus of the present invention can appropriately change the movement speed of the light detection region. It is preferable to be configured.
  • the change of the optical path of the optical system for moving the position of the light detection area may be performed by an arbitrary method.
  • the position of the light detection region may be changed by changing the optical path using a galvanometer mirror employed in a laser scanning optical microscope.
  • 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.
  • an optical analysis method for detecting and analyzing light from luminous particles dispersed and randomly moving in a sample solution using an optical system of a confocal microscope or a multiphoton microscope.
  • a method characterized by using a split optical system that forms at least two photodetection regions of a microscope optical system in a sample solution.
  • the optical system of the microscope a configuration in which at least two photodetection regions are formed using a split optical system and a configuration in which one photodetection region is formed without using the split optical system. It may be selectable, and preferably, light from at least two photodetection regions formed by the split optical system is received by one photodetector.
  • the method of the present invention moves the position of the optical detection region of the optical system in the sample solution by changing the optical path of the optical system.
  • the process of detecting the light from the light detection area while moving the position of the light detection area in the sample solution, and the process of individually detecting the light signal from each luminescent particle from the detected light is characterized by including.
  • the number of light signals from the individually detected light emitting particles is counted and the number of light emitting particles detected during the movement of the position of the light detection region is counted and / or detected.
  • a step of determining the number density or concentration of luminescent particles in the sample solution based on the number of luminescent particles may be included. Further, in the process of moving the position of the light detection region, the position of the light detection region may be moved at a predetermined speed or at a speed faster than the diffusion movement speed of the luminescent particles. The moving speed of the position of the detection region may be set based on the characteristics of the luminescent particles or the number density or concentration in the sample solution. Further, in the signal processing process, the fact that one luminescent particle has entered the light detection region is, for example, an optical signal having an intensity greater than a predetermined threshold based on the shape of the detected time-series optical signal. It may be determined when is detected.
  • the fact that one luminescent particle has entered the light detection region is determined based on the shape of the detected time-series optical signal, for example, when an optical signal having an intensity greater than a predetermined threshold is detected.
  • a bell-shaped pulse-like signal having an intensity exceeding a predetermined threshold on the light intensity data may be detected as a signal representing light from a single luminescent particle.
  • the time-series photon count data is used in the analysis process.
  • the light intensity data is smoothed, and a bell-shaped pulse signal having an intensity exceeding a predetermined threshold is detected as a signal representing light from a single light emitting particle in the smoothed time-series light intensity data. Good.
  • the optical analysis technique according to the present invention is typically used for biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, and particulate biological objects such as viruses and cells. Used for analysis or analysis of conditions in solution, but may be used for analysis or analysis of conditions in solution of non-biological particles (eg, atoms, molecules, micelles, metal colloids, etc.) It should be understood that such a case also belongs to the scope of the present invention.
  • non-biological particles eg, atoms, molecules, micelles, metal colloids, etc.
  • the present invention by forming a plurality of light detection regions in a photoanalysis technique such as a scanning molecule counting method using a confocal microscope or a multiphoton microscope optical system, FCS, FIDA, PCH, etc.
  • a photoanalysis technique such as a scanning molecule counting method using a confocal microscope or a multiphoton microscope optical system, FCS, FIDA, PCH, etc.
  • FCS confocal microscope
  • FIDA FIDA
  • PCH multiphoton microscope optical system
  • the split optical system for forming a plurality of light detection regions can be achieved with a relatively inexpensive and simple configuration.
  • the measurement time can be shortened by increasing the light detection area (confocal volume), and the accuracy of the measurement result can be achieved at a low cost. It becomes.
  • FIG. 1A is a schematic diagram of the internal structure of an optical analyzer that realizes the present invention.
  • FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope).
  • FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 to move the position of the light detection region in the sample solution.
  • FIG. 2A is a schematic diagram of an optical system in a microscope when a wedge-shaped half mirror is used as a splitting optical system
  • FIG. 2B is a schematic diagram of an example of a wedge-shaped half mirror.
  • FIG. 2C is a schematic diagram of an optical system in the microscope in the case of using a splitting optical system that splits one light beam into a plurality of beams and combines the plurality of light beams into one.
  • 2D to 2E are schematic diagrams of an optical system in a microscope in the case where an element that divides excitation light such as a diffractive optical element is used as the dividing optical system.
  • FIGS. 3A and 3B are a schematic diagram for explaining the principle of light detection in the scanning molecule counting method constituting a part of the present invention, and a schematic diagram of temporal change in measured light intensity, respectively. is there.
  • FIG. 4 is a flowchart showing the processing procedure of the scanning molecule counting method to which the present invention is applied.
  • FIG. 5A and 5B respectively show the case where the luminescent particles cross the light detection region while performing Brownian motion and the position of the light detection region in the sample solution in the scanning molecule counting method. It is a model figure showing the mode of movement of particles when luminous particles cross the photodetection region by moving at a speed faster than the moving speed.
  • FIG. 5C shows the signal processing process of the detection signal in the processing procedure for detecting the presence of the luminescent particles from the measured time-series light intensity data (time change of the photon count) according to the scanning molecule counting method. It is a figure explaining an example.
  • FIG. 6 shows an actual measurement example (bar graph) of measured photon count data, a curve (dotted line) obtained by smoothing the data, and a Gaussian function (solid line) fitted in the pulse existence region.
  • a signal labeled “noise” is ignored as it is a signal due to noise or foreign matter.
  • FIG. 7 (A) shows a per-molecule amount with respect to the excitation light intensity in the FCS measurement when one photodetection region having a radius of about 0.5 ⁇ m is formed as schematically depicted in FIG. 7 (B).
  • the fluorescence intensity (CCP) and the background light intensity (BG) are shown.
  • FIG. 8A shows the background light intensity (BG) in the FCS measurement when one light detection region having a radius of about 2.0 ⁇ m is formed as schematically illustrated in FIG. 8B. ing.
  • the fluorescence intensity (CCP) per molecule could not be measured.
  • FIG. 9 (A) shows the per-molecule amount relative to the excitation light intensity in the FCS measurement when two photodetection regions having a radius of about 0.5 ⁇ m are formed as schematically depicted in FIG. 9 (B).
  • the fluorescence intensity (CCP) and the background light intensity (BG) are shown.
  • optical analyzer for realizing the optical analysis technique according to the present invention can execute FCS, FIDA, etc. as schematically illustrated in FIG. 1A in the basic configuration. It may be an apparatus that combines an optical system of a confocal microscope and a photodetector. Referring to FIG. 1, optical analysis apparatus 1 includes optical systems 2 to 17 and a computer 18 for controlling the operation of each part of the optical system and acquiring and analyzing data.
  • the optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber.
  • the light is emitted as a divergent light at an angle determined by a specific NA at the outgoing end of the light, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, the reflection mirrors 6, 7, and the objective lens 8. Is incident on.
  • a microplate 9 in which a sample container or well 10 into which a sample solution of 1 to several tens of ⁇ L 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).
  • luminescent particles that are the object of observation typically particles to which luminescent labels such as fluorescent particles or fluorescent dyes have been added are dispersed or dissolved, and these luminescent particles enter the excitation region.
  • the luminescent particles are excited and light is emitted.
  • the emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter 14. (Here, only the light component of a specific wavelength band is selected.), Introduced into the multimode fiber 15, reaches the photodetector 16, is converted into a time-series electrical signal, and then to the computer 18.
  • Input and processing for optical analysis is performed in a manner described later.
  • 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 beam illustrated in FIG. 1B is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL (typically, the light intensity is in the region).
  • the photodetector 16 is preferably an ultra-high light that can be used for photon counting.
  • a sensitive photodetector is used.
  • 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.
  • the optical path of the optical system is further changed to detect the inside of the sample solution.
  • a mechanism is provided for scanning by the region, that is, for moving the position of the focal region (ie, the light detection region) within the sample solution.
  • a mirror deflector 17 that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG.
  • Such a mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope.
  • the mirror deflector 17 is driven in cooperation with light detection by the light detector 16 under the control of the computer 18.
  • the movement path 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.)
  • the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 up and down.
  • a stage (not shown) of the microscope is provided with a stage position changing device 17a for moving the horizontal position of the microplate 9 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.
  • 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.
  • the optical system of the confocal microscope is used as it is.
  • a plurality of excitation light sources 2 may be provided, and the wavelength of the excitation light can be appropriately selected according to the wavelength of the light for exciting the luminescent particles. Good.
  • FIG. 2 schematically shows an example of an optical system (divided optical system) for forming a plurality of confocal volumes.
  • a wedge-shaped half mirror 34 is inserted in the optical path where the excitation light optical path Ex and the detection light optical path Em overlap.
  • a wedge-shaped half mirror reflects a part of the incident light on the surface, and the other part is refracted to determine the incident angle of the incident light.
  • the wedge-shaped half mirror 34 reflects part of the incident light on the surface and the other part.
  • the wedge-shaped half mirror 34 used in the later embodiment reflects 36% (R1) of incident light on the incident-side surface.
  • the remaining light beam (R2) transmitted through the side surface is reflected by the surface (back surface) on the side opposite to the incident side without being transmitted, and is about 36% of the incident light beam from the incident side surface with respect to R1. It was configured to emit with a 1.2 degree offset.
  • two confocals are displaced from each other on the focal plane of the objective lens 8 as shown in FIG. Volume CV is formed (in the case of FIG. 2B, when the objective lens is a 40-times water immersion lens, two confocal volumes CV were formed at a distance of about 100 ⁇ m).
  • the light emitted from the two confocal volumes CV is reflected by the wedge-shaped half mirror 34 and propagates toward the photodetector 16.
  • one light beam provided between the dichroic mirror 5 and the objective lens 8 is divided into a plurality of light beams, which are provided between the dichroic mirror 5 and the objective lens 8, regardless of the wedge-shaped half mirror.
  • any optical system capable of combining a plurality of light beams traveling from the objective lens 8 toward the dichroic mirror 5 may be used as the splitting optical system 34.
  • a splitting optical system 31 that splits the excitation light beam into a plurality of paths is inserted in the excitation light optical path.
  • a plurality of light beams divided by the dividing optical system 31 are reflected by the dichroic mirror 5 and transmitted through the objective lens 8, a plurality of confocal focal points are displaced from each other on the focal plane of the objective lens 8.
  • a volume CV is formed. The light emitted from the plurality of confocal volumes CV passes through the dichroic mirror 5 and travels toward the photodetector 16.
  • the light emitted from the plurality of confocal volumes CV forms an image at positions shifted from each other after passing through the condenser lens 12, as shown in FIG. A pinhole 13 is provided. Further, the light that has passed through the pinhole 13 corresponding to each confocal volume CV may be received by a separate photodetector (FIG. 2 (D)) or as shown in FIG. 2 (E).
  • the relay lens 12a may be used to overlap and be connected to a single multimode optical fiber 15. The relay lens 12a is adjusted so that each of the light passing through the plurality of pinholes 13 becomes parallel light and is received by the core 15a of the multimode optical fiber. Note that a diffraction grating (diffractive optical element) or the like can be used as the splitting optical system 31 that splits the excitation light beam into a plurality of parts.
  • the split optical system (wedge half mirror, diffraction grating) may be detachably inserted into the optical path as appropriate when formation of a plurality of confocal volumes CV is desired.
  • the wedge-shaped half mirror may be arranged in place of the mirror 6 or 7 in FIG.
  • the diffraction grating may be inserted at a position denoted by reference numeral 4a in FIG.
  • the spectroscopic analysis technology such as FCS and FIDA is superior to the conventional biochemical analysis technology in that it requires a very small amount of sample and can perform inspection quickly. ing.
  • the characteristics of the luminescent particles are calculated based on the fluctuation of the fluorescence intensity. Therefore, in order to obtain an accurate measurement result, the luminescence in the sample solution is obtained.
  • the concentration or number density of the particles is a level at which about one luminescent particle is always present in the light detection region CV during the measurement of the fluorescence intensity, and it is required that a significant light intensity is always detected during the measurement time.
  • the concentration or number density of the luminescent particles is lower than that, and if the luminescent particles are at a level that only occasionally enters the light detection region CV, a signal with a significant light intensity is only part of the measurement time. Therefore, it is difficult to calculate the fluctuation of the light intensity with high accuracy. Also, if the concentration of the luminescent particles is much lower than the level at which about one luminescent particle is always present in the light detection area during measurement, the light intensity fluctuation is affected by the background. The measurement time is increased in order to obtain significant light intensity data sufficient for calculation.
  • FIG. 2 As a process executed in the scanning molecule counting method, in brief, a mechanism (mirror deflector 17) for moving the position of the light detection region is driven to change the optical path, and FIG. As schematically depicted in FIG. 2, light detection is performed while moving the position of the light detection region CV in the sample solution, that is, while scanning the sample solution by the light detection region CV. . Then, for example, as shown in FIG. 3A, when the light detection region CV moves (time to to t2 in the figure) and passes through the region where one luminescent particle exists (t1), Light is emitted from the luminescent particles, and a pulse-like signal having a significant light intensity (Em) appears on the time-series light intensity data as illustrated in FIG.
  • a pulse-like signal having a significant light intensity (Em) appears on the time-series light intensity data as illustrated in FIG.
  • the movement of the position of the light detection region CV and the light detection are performed, and pulse-like signals (significant light intensity) appearing in the meantime illustrated in FIG. 3B are detected one by one.
  • the luminescent particles are individually detected, and information on the characteristics of the luminescent particles can be acquired.
  • statistical calculation processing such as calculation of fluctuations in fluorescence intensity is not performed, and luminescent particles are detected one by one. Therefore, FCS, FIDA, etc. have sufficient accuracy. Information on the characteristics of the particles can be obtained even in a sample solution in which the concentration of particles to be observed is so low that analysis is impossible.
  • FIG. 4 shows a processing process in the present embodiment expressed in the form of a flowchart.
  • the particles to be observed in the present invention may be any particles as long as they are dispersed in the sample solution and move randomly in the solution, such as dissolved molecules.
  • it may be a biomolecule such as a protein, peptide, nucleic acid, lipid, sugar chain, amino acid or aggregate thereof, virus, cell, metal colloid, or other non-biological particle (
  • the sample solution is typically an aqueous solution, but is not limited thereto, and may be an organic solvent or any other liquid.
  • the particles to be observed may be particles that emit light themselves, or may be particles to which a light emitting label (fluorescent molecule, phosphorescent molecule) is added in an arbitrary manner.
  • the mirror deflector 17 is driven to move the position of the light detection region in the sample solution (sample The light intensity is measured while scanning the solution) (step 100 in FIG. 4).
  • 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 starts irradiation of excitation light and measurement of light intensity in the light detection region in the sample solution according to a program stored in a storage device (not shown).
  • the photodetector 16 When the measurement is started, first, under the control of the processing operation according to the program of the computer 18, light having the excitation wavelength of the luminescent particles in the sample solution is emitted from the light source 2, and the mirror deflector 17 is moved to the mirror 7. (Galvanomirror) is driven to move the position of the light detection region in the well 10, and at the same time, the photodetector 16 sequentially converts the detected light into an electric signal.
  • the computer 18 generates and stores time-series light intensity data from the transmitted signal in an arbitrary manner.
  • the photodetector 16 is an ultra-sensitive photodetector that can detect the arrival of one photon, so that the light detection is performed sequentially over a predetermined period of time at a predetermined unit time (BIN).
  • TIME for example, photon counting executed in a mode in which the number of photons arriving at the photodetector every 10 ⁇ sec is measured, and the time-series light intensity data is time-series photo
  • the moving speed of the position of the light detection region in order to carry out the individual detection of the luminescent particles from the measured time-series light intensity data quantitatively and accurately in the scanning molecule counting method, preferably
  • the moving speed of the position of the light detection region during the measurement of the light intensity is set to a value faster than the random movement of the luminescent particles, that is, the moving speed due to the Brownian movement.
  • 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.
  • the moving speed of the position of the light detection region may be set to 15 mm / s or the like that is approximately 10 times or more.
  • various movement speeds of the position of the light detection region are set, and the profile of the change in light intensity is expected to be expected (typically, the excitation light intensity distribution and Preliminary experiments for finding conditions that are substantially the same) may be repeatedly performed to determine a suitable moving speed of the position of the light detection region.
  • the concentration of the luminescent particles in the sample solution the lower the number of light signals from the luminescent particles detected per unit time. Then, for example, when it is intended to accurately measure the concentration or number density of the luminescent particles in the sample solution, it is necessary to acquire a signal of a certain number of luminescent particles, and the concentration of the luminescent particles in the sample solution is When it is low, the measurement time required to acquire signals of such a number of luminescent particles becomes long. Therefore, in the present invention, as described above, the total volume of the light detection area is increased by increasing the number of confocal volumes (light detection areas) in order to shorten the measurement time.
  • the locus of one luminescent particle passing through the light detection region is a substantially straight line as shown in FIG.
  • the light intensity change in the signal corresponding to the particle has a substantially bell-shaped profile reflecting the light intensity distribution in the light detection region (determined by the optical system) (FIG. 5 ( C) See top row). Therefore, in the scanning molecule counting method, basically, when the time duration in which the light intensity exceeds an appropriately set threshold value is within a predetermined range, a signal having the light intensity profile is detected as one particle. It may be determined that it corresponds to having passed through the region, and one luminescent particle may be detected.
  • a signal whose duration of light intensity exceeding the threshold is not within a predetermined range is determined as a noise or foreign matter signal.
  • time-series light intensity data (FIG. 5C, uppermost “detection result (unprocessed)”)
  • smoothing smoothing processing
  • FIG. 4-step 110 upper part “smoothing” in FIG. 5C.
  • the smoothing process may be performed by, for example, a moving average method.
  • the parameters for executing the smoothing process are the moving speed (scanning of the position of the light detection region at the time of acquiring the light intensity data Speed) and BIN TIME may be set as appropriate.
  • 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 the 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.
  • 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 the luminescent particles. 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 bell-shaped function fitting is performed on the smoothed time-series light intensity data in the pulse existence area (see FIG. 5C, “bell-shaped function fitting”).
  • the bell-shaped function to be fitted is typically a Gaussian function, but may be a Lorentz-type function.
  • 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 luminescent particle passes through the light detection region, That is, whether the peak intensity, pulse width, and correlation coefficient of the pulse are within predetermined ranges, for example, The following conditions: 20 ⁇ sec ⁇ pulse width ⁇ 400 ⁇ sec peak intensity> 1.0 [pc / 10 ⁇ s] (A) Correlation coefficient> 0.95 Whether or not the condition is satisfied is determined (step 150).
  • the signal determined that the calculated bell-shaped function parameter is within the range assumed in the signal corresponding to one luminescent particle is one luminescent particle.
  • 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 the above steps 130 to 150 may be repeatedly executed over the entire area of the time series light intensity data (step 160).
  • the process which detects the signal of a luminescent particle separately from time series light intensity data is not restricted to said procedure, You may perform by arbitrary methods.
  • the number of luminescent particles may be determined by counting the number of detected luminescent particle signals (counting of luminescent particles). Further, if the total volume of the region through which the light detection region passes is calculated by an arbitrary method, the number density or concentration of the luminescent particles in the sample solution is determined from the volume value and the number of luminescent particles ( Step 170).
  • the total volume of the region through which the light detection region passes 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.
  • control solution a plurality of solutions having different concentrations of luminescent particles are prepared, measurement is performed for each, and the calculated average value of Vt is adopted as the total volume Vt of the region through which the light detection region has passed. It may be like this.
  • 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.
  • the relationship between the concentration C of various standard luminescent particles and the number N of luminescent particles (formula (5)) with respect to the assumed movement pattern of the light detection region. ) May be stored in advance in the storage device of the computer 18 so that the user can use the information stored as appropriate when the user of the device performs optical analysis.
  • a phosphate buffer (containing 0.1% Pluronic F-127) and a solution in which the fluorescent dye ATTO633 was dissolved in a phosphate buffer so as to have a concentration of 1 nM were prepared.
  • 30 ⁇ L of the prepared sample solution was dispensed into the wells of the microplate.
  • the single molecule fluorescence measuring apparatus MF20 (Olympus Corporation) provided with the optical system of the confocal fluorescence microscope and the photon counting system was used as an optical analyzer.
  • the excitation light was a laser beam of 633 nm
  • the detection wavelength band was a wavelength band of 660-710 nm using a bandpass filter.
  • the objective lens was a 40 ⁇ water immersion type lens.
  • the radius of the confocal volume was adjusted by adjusting the diameter of the incident laser beam. Further, when the number of confocal volumes is increased, the wedge-shaped half mirror illustrated in FIG. 2B is replaced with the mirror 7 in FIG. And the measurement of light was performed by photon counting over 10 seconds, changing various excitation light outputs.
  • the average value of the photon count value per second in the photon counting data is divided by the average number of particles obtained by analyzing the photon counting data according to FCS, thereby obtaining one luminescent particle.
  • the per unit fluorescence intensity (CCP) was calculated.
  • the average value of photon counts per second was calculated as the background light intensity (BG).
  • FIGS. 7 to 9 (A) show that when one confocal volume with a radius of 0.5 ⁇ m is formed (normal setting—FIG. 7B), one confocal volume with a radius of 2.0 ⁇ m is formed.
  • FIGS. 7B show that when one confocal volume with a radius of 0.5 ⁇ m is formed (normal setting—FIG. 7B), one confocal volume with a radius of 2.0 ⁇ m is formed.
  • CCP And BG are plotted against the pumping light power density in the confocal volume (lower part of the horizontal axis in FIGS. 7 to 9A) (the horizontal axis in FIGS. 7 to 9A).
  • the upper row shows the output intensity of the objective lens.
  • the excitation light power density was calculated by [the output intensity of the objective lens / the confocal volume cross-sectional area].
  • the output intensity of the objective lens was measured using a power meter at the tip of the objective lens.
  • CCP increases with increasing pumping light power density in a region where pumping light power density is relatively low (60 kW / cm 2 or less). In the region where the pumping light power density is relatively high (60 kW / cm 2 or more), the pump light was saturated.
  • the size of BG is about 1/100 of CCP, but it increased with increasing pumping light power density even in a region where CCP was saturated. This indicates that the S / N ratio deteriorates simply because the BG continues to increase relatively when the pumping light power density is increased.
  • the BG when the confocal volume radius is four times the normal setting, the BG is 200 to 300 as compared with the case of FIG. (When the pumping light power density was 76 kW / cm 2 , the BG was 0.2 to 0.3 kHz in FIG. 7 and about 60 kHz in FIG. 8.) could not be measured (the calculation result was an abnormally small value). Since the volume of the confocal volume in FIG. 8 is about 60 times that in the case of FIG. 7, when the volume of the confocal volume increases, BG increases more than the increase in the volume.
  • the translational diffusion time, the number of particles, and the CPP obtained by analyzing the photon count data acquired in each of the configurations of FIGS. 7B and 9B according to FCS are as follows. (Excitation light intensity was 100 ⁇ W). Referring to the results in Table 1 above, since the translational diffusion times are substantially equal in FIGS. 7 and 9, the size of one confocal volume is substantially equal, but the number of particles in FIG. The total volume of the confocal volume shown in FIG. 9 is about twice the total volume of the confocal volume shown in FIG. 7, and two confocal volumes are formed. It has been confirmed. (The reason why the CCP in FIG. 9 is relatively low is considered to be a loss due to reflection by the wedge-shaped half mirror, as already mentioned.)

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Abstract

La présente invention porte sur une technique d'analyse optique utilisant un système optique d'un microscope confocal ou d'un microscope multiphoton, à l'aide duquel, afin de mesurer une solution d'échantillon ayant une faible concentration de particules d'émission lumineuse, il est possible d'augmenter le nombre de particules d'émission lumineuse détectées par unité de temps, par agrandissement du volume confocal d'une façon telle qu'une augmentation de l'intensité optique d'arrière-plan peut être réduite autant que possible. Dans la technique d'analyse optique de la présente invention, le système optique du microscope comporte un système optique divisé formant dans la solution d'échantillon au moins deux régions de détection de lumière de système optique. De cette façon, lorsque la capacité de volume confocal est agrandie par augmentation du nombre de volumes confocaux, il devient possible de réduire une augmentation de l'intensité optique d'arrière-plan dans une plus grande mesure que lorsque la capacité de volume confocal est agrandie par augmentation du diamètre d'un seul volume confocal.
PCT/JP2012/070044 2011-08-08 2012-08-07 Dispositif d'analyse optique et procédé d'analyse optique utilisant un système optique de microscope confocal ou de microscope multiphoton Ceased WO2013021984A1 (fr)

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