JP2012242141A - Optical analysis device and optical analysis method using optical system of confocal microscope or multiphoton microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To optically connect light from a re-imaging area to a light receiving surface of a photodetector with no light protruded therefrom without using a photodetector having a large light receiving surface, when a confocal volume is enlarged for measuring a sample solution with a low emitting particle concentration in an optical analysis technique using an optical system of a confocal microscope or a multiphoton microscope.SOLUTION: In an optical analysis technique according to the present invention, a multimode optical fiber that optically connects light passing through the re-imaging area where an image of a light detection area in the sample solution of the optical system of a microscope is formed, to a light receiving surface of the photodetector is a tapered optical fiber that has a core diameter in a first end which receives the light passing through the re-imaging area is larger than a core diameter of a second end which emits the light to the light receiving surface of the photodetector.

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- 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.). Specifically, 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. In the present specification, a particle that emits light (hereinafter referred to as “luminescent particle”) is either a particle that emits light itself, or a particle to which an arbitrary luminescent label or luminescent probe is added. The light emitted from the luminescent particles may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, or the like.

  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 apparatuses or methods for detecting characteristics of biomolecules, intermolecular interactions, or binding / dissociation reactions using such a measurement technique of weak light have been proposed. For example, in Fluorescence Correlation Spectroscopy (FCS; see, for example, Patent Documents 1-3 and Non-Patent Documents 1-3), a sample is obtained using an optical system of a laser confocal microscope and a photon counting technique. Fluorescence intensity from fluorescent molecules or fluorescently labeled molecules (fluorescent molecules, etc.) entering and exiting a microscopic area in the solution (focused area where the laser beam of the microscope is focused-called confocal volume) 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, size, and concentration of movement of fluorescent molecules, etc., changes in the structure or size of molecules, molecular binding / dissociation reactions, dispersion / aggregation Of the people of the phenomenon detection is made. In addition, fluorescence-intensity distribution analysis (FIDA, for example, Patent Document 4, Non-Patent Document 4) and photon counting histogram (Photon Counting Histogram: PCH, for example, 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. In addition, 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.

  In particular, according to a method using a micro area fluorescence measurement technique using an optical system of a confocal microscope such as FCS or FIDA and a photon counting technique, 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 μL), 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.

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

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 optical analysis technology using the optical system of confocal microscopes such as FCS and FIDA described above and photon counting technology, the light to be measured is light emitted from one or several fluorescent molecules. Then, 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 optical analysis 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. Therefore, in order to obtain a statistically significant result in these photoanalysis techniques, 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. Must be level. In fact, since the volume of the confocal volume is about 1 fL, 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. On the other hand, the methods for detecting fluorescent molecules 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.

  Therefore, the applicant of the present application handles the concentration or number density of the luminescent particles to be observed in optical analysis technology including statistical processing such as FCS and FIDA 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. In this new optical analysis technology, in short, 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. 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, When 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. To detect information on the concentration or number density of the luminescent particles in the sample solution. According to this new optical analysis technique (hereinafter referred to as “scanning molecule counting method”), the sample required for 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, and the presence of luminescent particles having a lower concentration or number density is detected as compared with the case of photoanalysis techniques such as FCS and FIDA. Other characteristics can be detected quantitatively.

  By the way, 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. In 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. As already mentioned, in the case of FCS, FIDA or PCH, if the concentration of the luminescent particles in the sample solution falls below the extent that one or more luminescent particles are present in the confocal volume during the light measurement time, It becomes difficult to obtain significant results. In the case of the scanning molecule counting method, the lower the luminescent particle concentration in the sample solution, the lower the number of detected particles per unit time during scanning of the confocal volume. Requires a long measurement time.

  As described above, the problem caused by the low concentration of the luminescent particles in the sample solution and the small number of luminescent particles per unit time entering the confocal volume can be obtained by increasing the size of the confocal volume. It can be solved. That is, when the concentration of the luminescent particles in the sample solution is low, the size of the focal region of the excitation light emitted from the objective lens of the confocal microscope or multiphoton microscope or the diameter of the pinhole (in the case of a confocal microscope) ), It is expected that the confocal volume is expanded, thereby expanding the area detected at a time, increasing the amount of detected light, and improving the accuracy of the measurement result.

  However, in this regard, when the confocal volume is increased simply by increasing the size of the focal region of the excitation light or the diameter of the pinhole, depending on the configuration of the optical analyzer, the confinement in the sample solution may be increased. In some cases, the amount of detected light cannot be increased in response to an increase in focal volume. As will be described in more detail in the section of the embodiment described later, typically, in an optical analyzer that performs the optical analysis technique as described above using an optical system of a confocal microscope or a multiphoton microscope, The light emitted from within the confocal volume passes through the objective lens, and is then collected again and imaged at a certain position (re-imaging region-in the case of a confocal microscope, re-imaging Pinholes are placed in the area.) And then introduced into the photodetector. As the photodetector, a photodiode having a light-receiving surface having a finite size or a diameter, preferably an APD (avalanche photodiode) is preferably employed. In general, an optical fiber is used to guide the detection light from the re-imaging region to the light receiving surface of the photodetector. At this time, when the confocal volume is enlarged, the width of the light beam from the re-imaging region to the incident end of the optical fiber is also enlarged, so it is necessary to use an optical fiber having a large core diameter on the light receiving surface. In that case, the normal optical fiber has the same core diameter at the light receiving surfaces at both ends, and therefore the width of the light beam emitted from the optical fiber having a large light receiving surface is also expanded. Therefore, in order to detect all the light beams emitted from the optical fiber, It becomes necessary to employ a photodiode having a larger light receiving surface. However, photodiodes, particularly those having a large light receiving surface in an APD, are generally very expensive, which greatly increases the cost of the device. In addition, the lens used for optical coupling from the reimaging area to the end of the optical fiber is exchanged according to the size of the confocal volume, so that the light beam from the reimaging position toward the end of the optical fiber can be changed. Although it is possible to reduce the width, it is necessary to adjust the optical axis every time the lens is replaced, and the measurement operation becomes troublesome. Therefore, it is possible to increase the confocal volume so that a sufficiently accurate measurement result can be obtained in a relatively short measurement time even for a sample solution having a low concentration of luminescent particles. If there is a technique that can be achieved without adopting the method and without repeating troublesome optical axis adjustment, it is possible to avoid a significant increase in the cost of the apparatus.

  Thus, one object of the present invention is to provide a larger light receiving surface in optical analysis technology using the optical system of a confocal microscope or a multiphoton microscope, such as the above-described scanning molecule counting method, FCS, FIDA, and PCH. Even in the measurement of a sample solution with a low concentration of luminescent particles, it is possible to obtain a result in a relatively short time without employing an expensive photodiode having a large amount of light, and without repeating troublesome optical axis adjustment. Is to do so.

  A further object of the present invention is to provide an optical analysis technique such as that described above, even if a photodiode having a normal or smaller light receiving surface is used, the light from an enlarged confocal volume can be obtained. The present invention proposes a new technique for optically coupling the light beam after passing through the re-imaging region without protruding from the light receiving surface of the photodiode.

  According to the present invention, the above-described problem is an optical analyzer that detects light from luminescent particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope or a multiphoton microscope, Including a multimode optical fiber for optically coupling a light beam passing through a re-imaging region where an image of the photodetection region in the sample solution of the microscope optical system is imaged to the light receiving surface of the photodetector; The optical fiber is a tapered optical fiber in which the core diameter at the first end that receives light passing through the re-imaging region is larger than the core diameter at the second end that emits light to the light receiving surface of the photodetector. This is achieved by a device characterized in that. In such a configuration, “a light-emitting particle dispersed in a sample solution and moving randomly” is a particle that emits light, such as atoms, molecules, or aggregates thereof dispersed or dissolved in a sample solution. Any particle may be used as long as it is not fixed to the substrate or the like and freely moves in the solution in Brownian motion. Such luminescent particles are typically fluorescent particles, but may be 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. As already mentioned, the “re-imaging region” is a region where an image of the light detection region is imaged after passing through the objective lens in the optical system of a confocal microscope or a multiphoton microscope, typically The light emitted from the light detection region is a region conjugate with the focal region of the objective lens, and is introduced into the photodetector through the “re-imaging region”. In particular, in a confocal microscope, a pinhole is arranged in the re-imaging region, so that substantially only light emitted from the re-imaging region, ie, the light detection region, passes through the pinhole. However, by blocking light emitted from outside the light detection region, it is possible to detect substantially only light from within the light detection region. That is, in the case of a confocal microscope, the diameter of the confocal volume is determined by the diameter of the pinhole. As the photodetector, a photodiode, preferably an APD, is typically used. In the present specification, the term “signal” refers to a signal representing light from the luminescent particles unless otherwise specified.

  According to said structure, 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. In a device that performs optical analysis technology, even if the size of the light detection area, that is, the confocal volume, is larger than the setting that is normally used, the light detection of the light receiving surface larger than that of the commonly used photodetector. Without using a detector, it becomes easy to receive a light beam (a light beam emitted from within the light detection region and passed through the objective lens) that passes through the re-imaging region on the light receiving surface of the light detector. In the apparatus of the present invention, as described above, the first light beam that passes through the re-imaging region is received as a multimode optical fiber that optically couples the light beam that passes through the re-imaging region to the light receiving surface of the photodetector. By using a tapered optical fiber whose core diameter at the end is larger than the core diameter of the second end that emits light to the light receiving surface of the photodetector, the diameter of the light beam directed to the light receiving surface of the photodetector is reduced. It is possible to make the diameter smaller than the diameter of the light beam on the incident side of the optical fiber. Therefore, even if the confocal volume is enlarged and the diameter of the light beam passing through the re-imaging region is enlarged, the light beam is received by the optical fiber without missing the peripheral edge of the light beam, and is further emitted from the optical fiber. Can be easily introduced into the light-receiving surface of the photodetector without lacking the periphery. Note that tapered multimode optical fibers in which the core diameter at one end is larger than the core diameter at the other end are already used and marketed in fields such as optical communications, and are therefore relatively inexpensive. This is advantageous in that it does not significantly increase the cost of the device.

  In the above-described configuration, more specifically, in a tapered multimode optical fiber, it is preferable that the light beam having the core diameter at the first end passed through the re-imaging region at the first end. The cores at the first and second ends so that the diameter of the light beam emitted from the second end at the light receiving surface of the photodetector is smaller than the diameter of the light receiving surface of the photodetector. The diameter is selected. As a result, the light beam that has passed through the re-imaging region is received at the incident end (first end) of the optical fiber and the light receiving surface of the photodetector without being protruded, and the effect of expanding the confocal volume is achieved. Will be fully demonstrated.

  In the above-described apparatus of the present invention, the size of the light detection region, that is, the confocal volume may be changeable. As already mentioned, when the concentration of luminescent particles in the sample solution is low and the number of luminescent particles entering the light detection area per unit time is small, the light detection area is expanded to enter the light detection area per unit time. It is expected that the number of luminescent particles to be increased will increase the measurement results with sufficient accuracy in a short measurement time. Therefore, in the apparatus of the present invention, as described above, the size of the light detection region can be changed, so that measurement of a sample solution with a wider range of luminescent particle concentrations can be performed in a short measurement time, and Can be achieved with sufficient accuracy.

  Furthermore, in the above apparatus, a first end mounting fixture that achieves optical coupling to the first end of the light beam that has passed through the re-imaging region, and light reception by the photodetector from the second end. A second end mounting fixture that achieves optical coupling to a possible surface is preferably provided on the outer surface of the device, allowing the user to attach the optical fiber to the device. Generally, when an optical fiber is disposed in an arbitrary optical path, a standardized mounting fixture for optical connection such as an FC connector is used. When using such a fixture, the position of the fixture is adjusted in advance on the optical path so that optical coupling is achieved by mechanically connecting the optical fiber to the fixture. Is done. Accordingly, as described above, the first end mounting fixing portion and the second end mounting fixing portion are provided on the outer surface of the apparatus, so that the user can attach the optical fiber to the apparatus. In addition, it is possible to minimize the optical adjustment operation when the optical fiber is exchanged and attached. The user can easily select and attach an optical fiber suitable for the size of the confocal volume and the size or diameter of the light receiving surface of the photodetector.

  The present invention described above may be applied to an apparatus that executes the “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 light detection region moving unit, and a signal processing unit for individually detecting a signal representing light from each of the luminescent particles detected by the light detector while moving the position of the light detection region in the sample solution. May contain.

  In the basic configuration of the apparatus of the present invention, first, 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. Then, when 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. Then, 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. Specifically, for example, in the apparatus of the present invention described above, 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). According to such a configuration, 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. In particular, if 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. Of course, instead of directly determining the absolute number density value or concentration value, the relative number density or concentration ratio with respect to a plurality of sample solutions or a standard sample solution as a reference of the concentration or number density is calculated. It may be like this. In the present invention, 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. (If vibration or flow acts on the sample solution, the physical properties of the particles may change.) And since the structure which distribute | circulates a sample solution is not required, it can measure and analyze with a trace amount (about 1-several dozen microliters) sample solution like the case of FCS, FIDA, etc.

  Further, in the processing of the signal processing unit of the apparatus that executes the above-described scanning molecule counting method, it is determined whether or not one luminescent particle has entered the light detection region from the signal from the sequential light detection unit. This may be done based on the shape of a signal representing time-series light detected by the detector. 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.

  Further, in the above apparatus, 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. As will be appreciated by those skilled in the art, 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. In particular, as 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.

  Furthermore, in the above apparatus, 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. As described above, when performing the scanning molecule counting method, 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. Thus, it becomes difficult to make the detected signal correspond to the presence of one luminescent 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 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 optical path of the optical system for moving the position of the light detection region may be changed by an arbitrary method. For example, 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.

  Furthermore, according to the above-described apparatus of the present invention, 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. In this regard, a novel method is provided that can fully exert its effect when the confocal volume is expanded. That is, according to another aspect of the present invention, an optical analysis method uses a light detector that passes through a re-imaging region where an image of a light detection region in a sample solution of a microscope optical system is formed. A multimode optical fiber that is optically coupled to the light receiving surface of the second optical fiber having a core diameter at the first end that receives the light passing through the re-imaging region; A tapered optical fiber larger than the end core diameter is used.

  Even in such a method, the core diameter of the first end is larger than the diameter of the light beam that has passed through the re-imaging region at the first end, and the light detector of the light beam that has exited the second end The core diameters of the first and second ends of the tapered optical fiber may be selected so that the diameter at the light receiving surface is smaller than the diameter of the light receiving surface of the photodetector. Also preferably, the method of the present invention includes the step of changing the size of the light detection region.

  When the method of the present invention is applied to the scanning molecule counting method, 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 It is characterized by including.

  Even in such a method, 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 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.

  In general, according to the present invention, in a photoanalysis technique such as a scanning molecule counting method using an optical system of a confocal microscope or a multiphoton microscope, FCS, FIDA, PCH, etc., a re-imaging region and light reception by a photodetector. By adopting a tapered optical fiber as an optical fiber to achieve the optical connection between the surfaces, re-imaging without expanding the light receiving surface of the photodetector when the confocal volume is expanded The transmission of the light beam from the region to the light receiving surface of the photodetector will be achieved substantially without a part of the light beam. Therefore, when expanding the confocal volume, it is not necessary to prepare an expensive photodetector having a particularly wide light receiving surface, and a significant increase in the cost of the apparatus can be avoided. With this configuration, when measuring a sample solution having a low concentration of luminescent particles, it is possible to achieve a reduction in measurement time by increasing the confocal volume, improvement in accuracy of measurement results, and the like at low cost.

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

FIG. 1A is a schematic diagram of the internal structure of an optical analyzer that 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. 2A is a schematic view of a part of the optical analyzer and an optical fiber, and FIG. 2B is a schematic cross-sectional view of an optical fiber mounting and fixing part in the optical analyzer. 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. 4A is a schematic diagram of a configuration of optical connection from the pinhole (re-imaging region) to the light receiving surface of the photodetector. FIG. 4B is a schematic diagram of the incident end of the optical fiber, and shows that the diameter of the incident light beam varies depending on the size of the pinhole. FIG. 4C is a schematic diagram of a tapered multimode optical fiber employed in the present invention. FIG. 5 is a flowchart showing the processing procedure of the scanning molecule counting method to which the present invention is applied. FIGS. 6A and 6B 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, respectively. 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. 6C shows the signal processing process of the detection signal in the processing procedure for detecting the presence of the luminescent particles from the measured time-series light intensity data (time change of the photon count) according to the scanning molecule counting method. It is a figure explaining an example. FIG. 7 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. In the figure, a signal labeled “noise” is ignored as it is a signal due to noise or foreign matter.

1 ... Optical analyzer (confocal microscope)
DESCRIPTION OF SYMBOLS 1a ... Outer surface of optical analyzer 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 ... Mounting fixing part 15 which connects a multimode optical fiber to the optical path from a pinhole ... Multimode optical fiber 15a, 15b ... End part 16 of multimode optical fiber ... Light Detector 16a ... Mounting fixing portion 17 for connecting the multimode optical fiber to the optical path to the light receiving surface of the photodetector ... Mirror deflector 17a ... Stage position changing device 18 ... Computer 30a, 30b ... Relay lens

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

Configuration of Optical Analysis Apparatus The present invention includes an optical system and a photodetector of a confocal microscope capable of executing a scanning molecule counting method, FCS, FIDA, PCH, etc. as schematically illustrated in FIG. It is applied to an optical analysis device formed by combining With reference to FIG. 1 (A), the optical analysis apparatus 1 is comprised from the optical systems 2-17, and the computer 18 for acquiring and analyzing data while controlling the action | operation of each part of an optical system. 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. 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 beam is focused in the sample solution in the sample container or well 10 to form a region (focus region) having a high light intensity as schematically shown in FIG. In the sample solution, light-emitting particles that are the object of observation, typically molecules to which a light-emitting label such as a fluorescent dye is added are dispersed or dissolved, and when the light-emitting particles enter the focal region, light is emitted during that time. The 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 condensed by the condenser lens 12, and an image of the focal region is formed by the pinhole 13 ( Reimaging area). That is, as known to those skilled in the art, the pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, and thereby, from within the focal area as illustrated in FIG. Only emitted light passes through the pinhole 13, and light from other than the focal plane is blocked. The focal region illustrated in FIG. 1B is a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL, and is referred to as a confocal volume. In the confocal volume, the light intensity is typically a Gaussian or Lorentzian distribution with the center of the region at the apex, and the effective volume is bounded by the surface where the light intensity is 1 / e ^2. The volume of a substantially elliptic sphere. In the apparatus of the present invention, it is preferable that the size of the confocal volume, that is, the size of the light detection region can be changed. Changing the size of the confocal volume may be performed by adjusting the position of the lens 4 in the optical path of the excitation light and / or exchanging the objective lens used (selecting the numerical aperture of the objective lens). In the case of the confocal microscope, when the size of the confocal volume in the sample solution is changed, the diameter of the pinhole 13 is also changed in accordance with the change. Therefore, in the apparatus of the present invention, pinholes with various diameters are prepared, and pinholes with diameters selected appropriately may be arranged and used from among them.

  Thus, the light that has passed through the re-imaging region formed in the pinhole 13 passes through the barrier filter 14 (here, only a light component in a specific wavelength band is selected), and enters the multimode optical fiber 15. Is introduced and reaches the corresponding photodetector 16. In the photodetector 16, the sequentially arriving light is converted into a time-series electric signal and input to the computer 18, and processing for optical analysis is performed in a manner described later. As the photodetector 16, an ultrasensitive photodetector that can be used for photon counting is preferably used, so that light from one luminescent particle, for example, one or several fluorescent dye molecules can be used. The weak light can be detected. The photodetector 16 typically employs a photodiode, more preferably an APD. Thus, with the above configuration, the intensity of light from the luminescent particles is measured.

  Further, in the optical system of the above optical analyzer, particularly when the scanning molecule counting method described in detail later is executed, 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. 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. Such a mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope. Further, in order to achieve a desired movement pattern of the position of the light detection region, 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.) Although not shown, the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 up and down. As described above, according to the configuration in which the position of the light detection region is moved by changing the optical path of the optical system instead of moving the sample solution, mechanical vibrations and hydrodynamic actions are substantially caused in the sample solution. It is possible to eliminate the influence of the dynamic action on the observation object and to achieve stable measurement.

  As an additional configuration, 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. Good. The operation of the stage position changing device 17a may be controlled by the computer 18.

  When the luminescent particles emit 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. When the luminescent particles emit light by phosphorescence, the optical system of the confocal microscope is used as it is. Further, in the optical analyzer 1, as shown in the figure, 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.

  Furthermore, the apparatus of this embodiment may be preferably configured such that the multimode optical fiber 15 can be arbitrarily replaced by the user. In that case, specifically, as illustrated in FIG. 2A, the optical path of the multimode optical fiber 15 and the optical path passing through the barrier filter 14 from the pinhole 13 is formed on the outer surface 1a of the device 1. The mounting and fixing portion 14a to be achieved and the mounting and fixing portion 16a that achieves optical connection between the multimode optical fiber 15 and the light receiving surface of the photodetector 16 are exposed, so that the multimode optical fiber 15 is attached to each mounting and fixing portion. The end portions 15a and 15b can be connected by the user. As schematically illustrated in FIG. 2 (B), standard mounting fixtures for optical connection such as FC connectors, SMA connectors, ST connectors, SC connectors, etc. are used for the mounting fixtures. When the end portions 15 a and 15 b of the multimode optical fiber 15 are mechanically connected to the respective mounting and fixing devices, an optical connection is established between the optical path of the lens fixed inside the mounting and fixing device and the multimode optical fiber 15. The optical path and lens are adjusted to achieve. With this configuration, the user selects and uses the multimode optical fiber 15 to be used according to the purpose of measurement, or when the multimode optical fiber 15 is arbitrarily replaced, the optical coupling of the multimode optical fiber is performed. Therefore, the operation for this is greatly simplified.

Use of Tapered Multimode Optical Fiber As mentioned in “Summary of Invention”, optical analysis technology using optical system of confocal microscope or multiphoton microscope such as scanning molecule counting method, FCS, FIDA, PCH, etc. However, when the concentration of the luminescent particles in the sample solution is low, the number of luminescent particles entering the confocal volume (focus area) per unit time is small. Time is needed. This problem can be solved by enlarging the confocal volume more than usual and increasing the number of luminescent particles entering the confocal volume per unit time. However, if the confocal volume is enlarged, the image of the confocal volume will increase, so that the peripheral portion of the light beam in the optical path from the confocal volume to the light receiving surface of the photodetector will be It may protrude from the light receiving surface of the element, and there may be a case where the effect of expanding the confocal volume is not sufficiently exhibited.

  More specifically, as briefly mentioned in connection with FIG. 1, in the confocal microscope or multiphoton microscope optical system, as shown schematically in FIG. The light from the volume CV is imaged again after passing through the objective lens 8, passes through the pinhole 13, and is then introduced into one end of the optical fiber 15 by the lenses 30 a, 30 b, etc. 15 is transmitted from the other end of 15 to the light receiving surface of the photodetector 16. Then, light re-imaging area from within the confocal volume CV, that is, light passing outside the opening area of the pinhole 13 other than the image of the focal area of the objective lens is blocked at the outer edge portion. It becomes. In other words, the confocal volume, that is, the light detection area is defined by the size of the diameter δ μm of the pinhole 13, and when the confocal volume CV is enlarged, the light detector passes through the re-imaging area. The width of the light beam toward the 16 light receiving surfaces is also enlarged. [Strictly speaking, the region where the light detected in the case of the confocal microscope is emitted, that is, the diameter of the confocal volume CV of the detected light is determined by the aperture δ μm of the pinhole 13. In the case of measurement using excitation light, the confocal volume CV of the excitation light and the confocal volume CV of the detection light are substantially such that an image of the focal region of the excitation light is formed in the pinhole 13. The optical system is adjusted so as to match as much as possible. Therefore, in the confocal microscope using the excitation light, when the confocal volume CV is enlarged, the focal area of the excitation light and the diameter δ μm of the pinhole 13 are enlarged. On the other hand, in a confocal microscope that does not use excitation light, the confocal volume CV is expanded simply by increasing the diameter δ μm of the pinhole 13. In the case of a multiphoton microscope that does not require the pinhole 13, the confocal volume CV is expanded by expanding the focal region of the excitation light. In any case, when the confocal volume CV is enlarged, the diameter of the light beam from the re-imaging area toward the light receiving surface of the photodetector 16 is also enlarged. ]

  In the above configuration, when the diameter of the light beam passing through the pinhole 13 or the re-imaging region is increased due to the expansion of the confocal volume CV, the light beam passes through the lenses 30a and 30b and travels toward the incident end 15a of the optical fiber. In some cases, the diameter of the optical fiber increases and becomes larger than the diameter of the light receiving surface of the incident end 15a of the optical fiber. For example, in a typical device example, the diameter of the pinhole 13 is set to about 50 μm, and an optical fiber having a core diameter of about 100 μm is used. In that case, even if the diameter of the pinhole 13 is increased to 100 μm, the light beam that has passed through the pinhole 13 is transmitted into the optical fiber without protruding from the light receiving surface (FIGS. ii)). However, when the diameter of the pinhole 13 is enlarged to exceed 100 μm, for example, when the diameter is set to 200 μm, the light beam that has passed through the pinhole 13 protrudes from the light receiving surface of the optical fiber at the peripheral portion thereof. (FIG. 4 (B) (iii)). In other words, the size of the confocal volume CV is limited to the diameter of the light receiving surface of the optical fiber. Therefore, even if the diameter of the pinhole 13 is enlarged to some extent (for example, even if the diameter is increased to 200 μm), the light beam that has passed through the pinhole 13 is transmitted into the optical fiber without protruding from the light receiving surface. In order to do so, it is necessary to use an optical fiber having a larger core diameter at the incident end (FIGS. 4B and 4Iv).

  However, in the optical analyzer using the optical system of the confocal microscope or the multiphoton microscope described above, normally, multimode optical fibers having the same core diameter at both ends are used (for example, Fiber Guide Industries, Inc.). (SFS100TO110N from Fiberguide Industries (New Jersey USA) etc.) And the diameter of the light beam emitted from the optical fiber with a large core diameter is also increased, so it is possible to cope with the enlargement of the confocal volume CV and / or the pinhole 13 When a multimode optical fiber having a large core diameter is used, in the optical connection from the pinhole 13 to the incident end of the optical fiber, the light beam is transmitted into the optical fiber without protruding from the light receiving surface. In the optical connection from the emission end of the optical fiber to the light receiving surface of the photodetector, FIG. As with the situation described in connection with), it is possible for light rays to protrude from the light-receiving surface of the photodetector, in this regard, in an optical analyzer using a confocal microscope or a multiphoton microscope optical system. As a photodetector, typically, an APD standard product having a light receiving surface diameter of about 180 μm is used (for example, SPCM- of Perkin Optoelectronics (Fremont CA, USA)). AQRH-13 etc.) The standard specification of the light receiving part of such APD is usually standardized so that, for example, a multi-mode optical fiber with a core diameter of 100 μm suitable for an FC connector can be connected. When a multi-mode optical fiber is attached to the light receiving part of such an APD, the optical connection from the light emitting end of the optical fiber to the light receiving surface of the photodetector is such that the light beam passes from the light receiving surface of the photodetector. (See Fig. 2 (B).) Therefore, if a multimode optical fiber with a core diameter of more than 100 µm is connected to the light receiving portion of the APD, the light beam will be detected by a photodetector. One method for solving this problem is to use an APD whose light receiving surface is larger than the standard product, but an APD having a light receiving surface larger than the standard product is very It is expensive and increases the cost of the optical analyzer.

  Therefore, in the present invention, as an optical fiber for optical connection from the pinhole 13 to the light receiving surface of the photodetector, a core at one end as schematically illustrated in FIG. A tapered multi-mode optical fiber designed so as not to cause a loss of light propagation despite the fact that the diameter is larger than the core diameter at the other end and the core diameter gradually changes is employed. In its use, the end 15a having the larger core diameter is connected to the mounting fixing portion 14a on the pinhole 13 side, and the end 15b having the smaller core diameter is connected to the mounting fixing portion 16a on the photodetector 16 side (see FIG. 2 (A)). Then, even when the diameter of the pinhole 13 is larger than the normal diameter (for example, 100 μm), the light beam from the pinhole 13 protrudes from the light receiving surface into the optical fiber at the end of the optical fiber on the pinhole 13 side. Thus, even at the end of the optical fiber on the side of the photodetector 16, the emitted light beam is incident on the light receiving surface of the photodetector without protruding therefrom. As a result, the restriction on the expansion of the confocal volume CV can be relaxed, and when the concentration of the luminescent particles in the sample solution is low, the confocal volume CV can be appropriately expanded to be good in a short time. Measurement results can be obtained with high accuracy. The tapered multi-mode optical fiber as described above is already used and marketed in the field of optical communications (for example, SFT200TO100Y of Fiberguide Industries (New Jersey USA)) and is relatively inexpensive. Therefore, a significant increase in the cost of the optical analyzer is avoided.

Scanning Molecule Counting The configuration of the present invention is applied to an apparatus that executes a scanning molecule counting method, an optical analysis technique such as FCS, FIDA, PCH, as already described. In this specification, a case where the present invention is applied to the scanning molecule counting method will be described. The principle and specific operation of the “scanning molecule counting method” will be outlined below.

1. Principle of scanning molecule counting method 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. However, in the spectroscopic analysis techniques such as FCS and FIDA, in principle, 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 If 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.

  Therefore, the applicant of the present application disclosed in Japanese Patent Application No. 2010-044714 and PCT / JP2011 / 53481 even when the concentration of the luminescent particles is lower than the level required by the above-described spectroscopic analysis techniques such as FCS and FIDA. We proposed a "scanning molecule counting method" based on a novel principle that enables the detection of the properties of luminescent particles.

  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 drawing) 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. Thus, 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. Thus, the luminescent particles are individually detected, and information on the characteristics of the luminescent particles can be acquired. In the principle of the scanning molecule counting method, statistical calculation processing such as calculation of fluctuations in fluorescence intensity is not performed, and luminescent particles are detected one by one. Therefore, FCS, FIDA, etc. have sufficient accuracy. 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.

2. Processing Operation Process of Scanning Molecule Counting Method In the scanning molecule counting method using the optical analyzer 1 illustrated in FIG. 1A, specifically, (1) a sample solution preparation process including luminescent particles, (2) The light intensity measurement process of the sample solution and (3) the measured light intensity analysis process are executed. FIG. 5 shows a processing process in the present embodiment expressed in the form of a flowchart.

(1) Preparation of sample solution 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. For example, 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. In addition, 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.

(2) Measurement of light intensity of sample solution In the process of measuring light intensity by the scanning molecule counting method of this embodiment, 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 performing scanning within the solution (FIG. 5-Step 100). 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 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). 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. Typically, 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 μsec is measured, and the time-series light intensity data is time-series photon count data. It's okay.

  Regarding 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. 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 (the excitation light intensity in the light detection region decreases outward with the center of the region as the apex), and significant light intensity changes corresponding to individual light emitting particles (light emission) It is difficult to specify the signal representing the light from the particles. Therefore, preferably, as shown in FIG. 6B, the particles cross the light detection region CV in a substantially straight line, whereby light corresponding to each particle in the time-series light intensity data is obtained. As illustrated in the uppermost part of FIG. 6C, the intensity change profile has a substantially bell shape substantially the same as the excitation light intensity distribution, and the correspondence between the individual luminescent particles and the light intensity can be easily specified. As described above, the moving speed of the position of the light detection region is set faster than the average moving speed (diffusion moving speed) due to the Brownian motion of the particles.

Specifically, the time Δτ required for the light-emitting particles having the diffusion coefficient D to pass through the photodetection region (confocal volume) having the radius Wo by Brownian motion is expressed by a relational expression (2Wo) ² = 6D · Δτ (1)
From
Δτ = ( ² Wo) ² / 6D (2)
Therefore, the speed (diffusion movement speed) Vdif at which the luminous particles move by Brownian motion is approximately
Vdif = 2Wo / Δτ = 3D / Wo (3)
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 luminescent particles is expected to be about D = 2.0 × 10 ⁻¹⁰ m ² / s, assuming that Wo is about 0.62 μm, Vdif is 1.0 × 10 ^{Since −3} m / s, the moving speed of the position of the light detection region may be set to 15 mm / s, which is approximately 10 times or more. In addition, when the diffusion coefficient of the luminescent particles is unknown, 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.

  As already described, the lower the concentration of 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, in order to shorten the measurement time, the confocal volume (light detection region) is expanded and the optical path from the pinhole 13 to the light detector 16 is increased. In addition, a tapered multimode optical fiber is attached to the apparatus 1 as the optical fiber 15 so as not to protrude from the incident end of the optical fiber and the light receiving surface of the photodetector 16. At this time, as already described, the end 15a having the larger core diameter is connected to the mounting / fixing portion 14a on the pinhole 13 side, and the end 15b having the smaller core diameter is connected to the mounting / fixing portion 16a on the photodetector 16 side.

(3) Analysis of light intensity When time-series light intensity data of the luminescent particles in the sample solution is obtained by the above processing, the light intensity is obtained by the computer 18 according to a program stored in the storage device. Analysis such as detection of a signal corresponding to light from the luminescent particles in the data and calculation of the concentration of the luminescent particles is performed.

(I) Detection of signal corresponding to luminescent particles In the time-series light intensity data, 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. 6 ( 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. Then, 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. Also, the light intensity distribution in the light detection area is Gaussian distribution:
I = A · exp (−2t ² / a ² ) (4)
Can be assumed that the intensity A and the width a calculated by fitting the expression (4) to a significant light intensity profile (a profile that can be clearly determined not to be background) are within a predetermined range. In some cases, it may be determined that the light intensity profile corresponds to the passage of one particle through the light detection region, and one luminescent 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 luminescent particles from time-series light intensity data, first, time-series light intensity data (FIG. 5A, uppermost “detection result (unprocessed)”) Then, a smoothing (smoothing) process is performed (FIG. 5-step 110, upper part “smoothing” in FIG. 6C). The light emitted by the luminescent particles is probabilistically emitted, and data values may be lost in a very short time. Therefore, such a data value loss can be ignored by the smoothing process. The smoothing process may be performed by, for example, a moving average method. It should be noted that the parameters for executing the smoothing process, such as the number of data points averaged at a time in the moving average method and the number of moving averages, are the moving speed (scanning of the position of the light detection region at the time of acquiring the light intensity data). Speed) and BIN TIME may be set as appropriate.

  Next, in the 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 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.

  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 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 region is specified, a bell-shaped function fitting is performed on the smoothed time-series light intensity data in the pulse existence region (see FIG. 6C, “bottom-shaped function fitting”). )), Parameters such as the peak peak (maximum value) intensity Ipeak, the pulse width (full width at half maximum) Wpeak, and the correlation coefficient (of the least squares method) in the fitting (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 luminescent particle passes through the light detection region, That is, it is determined whether or not the peak intensity, pulse width, and correlation coefficient of the pulse are within predetermined ranges (step 150). Thus, as shown in the left of FIG. 7, 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. Thus, one luminescent particle is detected. On the other hand, as shown in the right of FIG. 7, 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 addition, 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.

(Ii) Determination of Luminescent Particle Concentration Further, 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. A solution having a known particle concentration (control solution) was detected by measuring the light intensity, detecting the luminescent particles, and counting as described above under the same conditions as the measurement of the sample solution to be examined. It may be determined from the number of luminescent particles and the concentration of luminescent particles in the control solution. Specifically, for example, for a control solution having a concentration C of luminescent particles, if the number of detected luminescent particles in the control solution is N, the total volume Vt of the region through which the photodetection region has passed is
Vt = N / C (5)
Given by. In addition, as a 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. When Vt is given, the concentration c of the luminescent particles in the sample solution whose counting result of the luminescent particles is n is
c = n / Vt (6)
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. Further, in the optical analyzer of the present embodiment, 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.

  Thus, according to the present invention described above, in the scanning molecule counting method in which the luminescent particles are individually detected by scanning in the sample solution by the light detection region, counting of the luminescent particles in the sample solution, determination of the concentration, etc. Is possible.

  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.

  As sample solutions, solutions were prepared in which the fluorescent dye ATTO633 was dissolved in a phosphate buffer (containing 0.1% Pluronic F-127) so that the concentration would be 10 pM. Next, 30 μL of the prepared sample solution was dispensed into the wells of the microplate. In addition, 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 1 mW of 633 nm laser light, and the detection wavelength band was a wavelength band of 660-710 nm using a bandpass filter. In light measurement, time-series light intensity data (photon count data) was obtained for each of the sample solutions according to the embodiment described in “(2) Measurement of light intensity of sample solution”. . The light detection area in the sample solution was moved at a moving speed of 15 mm / sec. In addition, BIN TIME was set to 10 μs, and the measurement time was set to 2 seconds. Furthermore, in order to verify the effect of expansion of the confocal volume and the use of the tapered multimode optical fiber in the measurement of such light, the measurement was performed by setting the pinhole diameter to 50 μm, 100 μm, and 200 μm, respectively. A taper type optical fiber having a normal type optical fiber (SFS100TO110N) having a core diameter of 100 μm at both ends, a pinhole aperture set to 200 μm, an end having a core diameter of 200 μm, and an end having a core diameter of 100 μm It performed about each when (SFT200TO100Y) was used. APD (SPCM-AQRH-13) was used as the photodetector.

After measuring the light intensity, according to the processing procedure described in “(3) (i) Detection of signal corresponding to luminescent particles” above, the time-series photon count data acquired for the sample solution under each condition The signals representing the light from the luminescent particles detected in were counted. In the smoothing of the data by the moving average method in step 110, nine data points were averaged at a time, and the moving average process was repeated five times. In the fitting of step 140, a Gaussian function was fitted to the time series data by the least square method, and the peak intensity (in the Gaussian function), the peak width (full width at half maximum), and the correlation coefficient were determined. . Further, in the determination process in step 150, the following conditions:
20 μsec <pulse width <400 μsec peak intensity> 1.0 [pc / 10 μs] (A)
Correlation coefficient> 0.95
Only a pulse signal satisfying the above condition is determined to be a signal corresponding to the luminescent particle, while a pulse signal not satisfying the above condition is ignored as noise, and the number of signals determined to be a signal corresponding to the luminescent particle is expressed as “ Counted as “number of pulses”.

The number of pulses detected under the above measurement conditions was as follows.
Pinhole diameter [μm] Optical fiber Pulse number ratio a) 50 Normal type 203 1
b) 100 normal type 739 1.91
c) 200 Normal type 1833 3.01
d) 200 taper type 3224 3.99
The “ratio” is the ratio of the square root of the number of pulses in each case to the square root of the number of pulses of a.

In the scanning molecule counting method described above, the number of pulses, that is, the number of detected luminescent particles is proportional to the cross-sectional area (scanning cross-sectional area) of the plane perpendicular to the scanning direction of the confocal volume, and the pinhole diameter is N When it is doubled, the scanning cross-sectional area is N ² times. Therefore, the square root of the number of pulses is proportional to the pinhole diameter. Thus, with reference to the above results a) to c), when a normal type optical fiber is used, the pinhole diameter of b) is 100 μm, that is, twice that of a), based on the case of a). In this case, the ratio of the square root of the number of pulses was almost doubled. However, when the pinhole diameter of c) was 200 μm, that is, four times that of a), the ratio of the square root of the number of pulses was about Tripled. This means that even if the pinhole diameter is enlarged, a part of the light beam that has passed through the pinhole protrudes from the optical fiber, and the effect of expanding the confocal volume corresponding to the expansion of the pinhole diameter is reflected in the result. Suggests not. On the other hand, when a tapered optical fiber is used, the ratio of the square root of the number of pulses is approximately 4 when the pinhole diameter of d) is 200 μm, that is, four times that of a) with reference to the case of a). Doubled. This is because even if the pinhole diameter is increased by using a tapered optical fiber, the light beam that has passed through the pinhole reaches the photodetector without substantially protruding from the optical fiber, and the confocal volume is increased. Suggests that this is reflected in the results.

  Thus, according to the present invention, it has been clarified that the effect of expanding the confocal volume can be sufficiently exhibited by using the tapered optical fiber when expanding the confocal volume.

FCS was performed under the same measurement conditions as in Example 1. However, the concentration of the fluorescent dye in the sample solution was 1 nM. The detected translational diffusion times were as follows:
Pinhole diameter [μm] Optical fiber Translational diffusion time [μs] Ratio a) 50 Normal type 140.7 1
b) 100 normal type 565.9 2.006
c) 200 Normal type 1597.9 3.370
d) 200 taper type 2611.0 4.308
The “ratio” is the ratio of the square root of the translational diffusion time in each case to the square root of the translational diffusion time of a.

In the above FCS, the translational diffusion time τ is given by τ = (Wo) ² / D from the confocal volume diameter Wo and the diffusion constant D of the particle, so that the square root of the translational diffusion time is It is proportional to the diameter of the focal volume, that is, the pinhole diameter. Thus, with reference to the above results a) to c), when a normal type optical fiber is used, the pinhole diameter of b) is 100 μm, that is, twice that of a), based on the case of a). In this case, the ratio of the square root of the translational diffusion time was almost doubled. However, when the pinhole diameter of c) was 200 μm, that is, four times that of a), the ratio of the square root of the translational diffusion time was It was about 3.4 times. This means that even if the pinhole diameter is enlarged, a part of the light beam that has passed through the pinhole protrudes from the optical fiber, and the effect of expanding the confocal volume corresponding to the expansion of the pinhole diameter is reflected in the result. Suggests not. On the other hand, when a tapered optical fiber is used, the ratio of the square root of the translational diffusion time is about when the pinhole diameter of d) is 200 μm, that is, four times that of a), based on the case of a). Four times. This is because even if the pinhole diameter is increased by using a tapered optical fiber, the light beam that has passed through the pinhole reaches the photodetector without substantially protruding from the optical fiber, and the confocal volume is increased. Suggests that this is reflected in the results.

  Thus, as will be understood from the results of the above embodiments, according to the present invention described above, in the optical analysis technique using the optical system of the confocal microscope or the multiphoton microscope, from the pinhole or the re-imaging region. By achieving the optical connection to the light receiving surface of the photodetector with a tapered optical fiber, it is possible to reflect the effect of expanding the confocal volume, the pinhole, or the re-imaging region in the result. As a result, when the concentration of luminescent particles in the sample solution is low, execution of measurement using an enlarged confocal volume can be performed with good accuracy and without using an expensive photodetector with a large light-receiving surface. It will be achieved by time.

Claims (13)

An optical analyzer that detects and analyzes light from luminescent particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope or a multiphoton microscope,
A multimode optical fiber that optically couples a light beam passing through a re-imaging region where an image of the photodetection region in the sample solution of the optical system forms an image to a light receiving surface of a photodetector; A tapered optical fiber in which a core diameter at a first end for receiving a light beam passing through the re-imaging region is larger than a core diameter at a second end for emitting the light beam to the light receiving surface of the photodetector. A device characterized by being.   2. The apparatus of claim 1, wherein the core diameter of the first end is larger than the diameter of the light beam that has passed through the re-imaging region at the first end, and the light beam emitted from the second end. The core diameters of the first and second ends of the tapered optical fiber are selected so that the diameter at the light receiving surface of the photodetector is smaller than the diameter of the light receivable surface of the photodetector. A device characterized by.   3. The apparatus of claim 1 or 2, wherein the first end mounting fixture for achieving optical coupling to the first end of the light beam that has passed through the re-imaging region, and the second end. The second end mounting fixture that achieves optical coupling from the optical detector to the light receptive surface of the photodetector, provided on the outer surface of the device, by which the user attaches the optical fiber to the device A device characterized in that   4. The apparatus according to claim 1, wherein the size of the light detection region is changeable. The apparatus according to any one of claims 1 to 4, further comprising:
A light detection region moving unit that 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;
A signal processing unit for individually detecting a signal representing light from each of the luminescent particles detected by the photodetector while moving the position of the light detection region in the sample solution. Features device.   6. The apparatus according to claim 5, wherein the signal processing unit counts the number of signals representing light from the individually detected light emitting particles and detects the light emission detected during movement of the position of the light detection region. An apparatus characterized by counting the number of particles.   7. The apparatus according to claim 5, wherein the light detection area moving unit moves the position of the light detection area at a speed faster than a diffusion movement speed of the luminescent particles. An optical analysis method for detecting and analyzing light from luminous particles dispersed and moving randomly in a sample solution using an optical system of a confocal microscope or a multiphoton microscope,
A light beam passing through a re-imaging region where an image of the light detection region in the sample solution of the optical system is formed is a multimode optical fiber that optically couples to a light receiving surface of a photodetector, and the reconnection is performed. A tapered optical fiber having a core diameter at a first end that receives light passing through an image region is larger than a core diameter at a second end that emits light to the light receiving surface of the photodetector. how to.   9. The method of claim 8, wherein the core diameter of the first end is larger than the diameter of the light beam that has passed through the re-imaging region at the first end, and the light beam emitted from the second end. The core diameters of the first and second ends of the tapered optical fiber are selected so that the diameter at the light receiving surface of the photodetector is smaller than the diameter of the light receivable surface of the photodetector. A method characterized by.   10. A method according to claim 8 or 9, comprising the step of changing the size of the photodetection region. A method according to any of claims 8 to 10, comprising
Changing the optical detection path of the optical system in the sample solution by changing the optical path of the optical system;
Detecting light from the light detection region by the light detector while moving the position of the light detection region in the sample solution;
Individually detecting signals representing light from individual luminescent particles from the detected light.   12. The method of claim 11, further comprising counting the number of signals representing light from the individually detected luminescent particles to determine the number of luminescent particles detected during movement of the position of the light detection region. A method comprising the step of counting.   13. The method according to claim 11, wherein in the process of moving the position of the light detection region, the position of the light detection region is moved at a speed faster than the diffusion movement speed of the luminescent particles. A method characterized by that.

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