JP2009192490A - Fluorescence analyzer and analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence analyzer and analysis method that facilitate fluorescence analysis of a measuring sample. <P>SOLUTION: This fluorescence analyzer 1A comprises an excitation light irradiation system for irradiating a sample S with an exciting light; a fluorescence detecting system for detecting the fluorescence from a fluorescence probe in a measuring area; a photon-counting section 35 for counting the number of photons, based on a detected signal, an analysis condition setting section 51 for setting the analysis condition to the number-of photons measuring data, and a measurement result analysis section 52 for performing the fluorescence analysis. The setting section 51 sets bin width for binning the measuring data, and the reference number of photons to one molecule of the fluorescence probe 1. The analysis section 52 performs binning of the measuring data, and analyses the number of fluorescence probes in the measuring area with reference to the reference number of photons. The setting section 51 sets the bin width using the variation with the bin width of the measure S/N ratio of the number of photons and the reference S/N ratio by shot-noise characteristics. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fluorescence analysis apparatus and a fluorescence analysis method for detecting fluorescence generated from a fluorescent probe in a measurement sample and analyzing the measurement sample.

Fluorescence Correlation Spectroscopy (FCS) is used to irradiate a micro area in a measurement sample of a solution containing an extremely low concentration of a fluorescent substance with an excitation light to generate the measurement area. By measuring the intensity of the fluorescent light in time series and obtaining its autocorrelation function, information on the translational diffusion movement of the fluorescent substance in the measurement sample is obtained (for example, Patent Documents 1 and 2; (See Patent Documents 1 to 3). Such fluorescence analysis by FCS can be applied to immunoassay in clinical diagnosis, for example.
US Pat. No. 6,200,188 JP 2004-347608 A Thorsten Winkler et al., "Confocal fluorescence coincidence analysis: An approach to ultrahigh-throughput screening", Proc. Natl. Acad. Sci. USA, Vol.96, pp.1375-1378, 1999 Bo Huang et al., "PhotonCounting Histogram: One-Photon Excitation", ChemPhysChem 2004, 5, pp. 1523-1531 Kerstin Korn et al., “Gene expression analysis using single molecule detection”, Nucleic Acids Research, 2003, Vol. 31, No. 16 e89

  In fluorescence analysis such as immunoassay by FCS, a molecular probe (fluorescent probe) labeled with a fluorescent substance is added to a measurement sample, and fluorescence generated from the fluorescent probe in the measurement region is detected and translated from its autocorrelation function. Get information about diffusion movements. Thereby, the amount of reaction between the target molecule in the sample to be analyzed and the fluorescent probe can be known, and information about the measurement sample can be acquired. Since this sample analysis method is a non-separation type measurement method in a homogeneous assay, it is possible to reduce the consumption cost by reducing the consumption of consumables compared to the heterogeneous assay such as ELISA method. It is. In addition, the analysis apparatus can be reduced in size and price.

  On the other hand, in the fluorescence analysis by FCS, the improvement of the measurement sensitivity is calculated | required from a viewpoint of the detection of a trace amount substance. As a configuration for improving measurement sensitivity in FCS, a configuration is used in which a flow is applied to a measurement sample by a vibration such as a flow path such as a capillary or a microchip or a piezoelectric actuator.

  In the configuration in which a flow is imparted to the measurement sample in this way, in the measurement sample, in addition to the translational diffusion movement, the movement movement by the flow can increase the passing frequency of the molecule with respect to a minute measurement region, This can improve the measurement sensitivity. However, in such a configuration, the analysis of the translational diffusion motion using the autocorrelation function is complicated due to the movement of the molecule, and particularly when the speed of the movement is increased, the fluorescence for the translational diffusion motion is increased. There is a problem that analysis becomes difficult.

  The present invention has been made to solve the above problems, and a fluorescence analysis apparatus capable of facilitating fluorescence analysis of a measurement sample by measuring fluorescence from a fluorescent probe in a measurement region. It is another object of the present invention to provide a fluorescence analysis method.

  In order to achieve such an object, the fluorescence analyzer according to the present invention includes (1) excitation light irradiation means for irradiating excitation light to a measurement region set for a measurement sample, and (2) excitation light irradiation means. Fluorescence detection means for detecting the fluorescence generated from the fluorescent probe in the measurement region of the measurement sample irradiated with the excitation light by (3), and detection by the fluorescence detection means based on the detection signal output from the fluorescence detection means A photon counting means for counting the number of photons in a time-series manner with a predetermined counting time width, and (4) setting an analysis condition for fluorescence analysis for time-series photon number measurement data acquired by the photon counting means. And (5) based on the analysis conditions set by the analysis condition setting means, the fluorescence solution is obtained for the photon number measurement data indicating the measurement result of the measurement sample including the fluorescent probe. (6) The analysis condition setting means sets the bin width for binning the photon number measurement data and the reference photon number for one fluorescent probe molecule in the bin width as the analysis conditions. The measurement result analysis means bins the photon number measurement data with the bin width set by the analysis condition setting means, and performs a fluorescence analysis on the number of fluorescent probes in the measurement region of the measurement sample with reference to the reference photon number. (7) The analysis condition setting means is the change in the measured SN ratio, which is the SN ratio of the photon number in the photon number measurement data, due to the bin width, and the SN ratio of the photon number when shot noise characteristics are assumed. The bin width is set using a change in the reference SN ratio due to the bin width.

  In addition, the fluorescence analysis method according to the present invention includes (1) an excitation light irradiation step in which a measurement region set for a measurement sample is irradiated with excitation light, and (2) a measurement in which excitation light is irradiated in the excitation light irradiation step. A fluorescence detection step for detecting fluorescence generated from the fluorescence probe in the measurement region of the sample; and (3) a predetermined count of the number of photons detected in the fluorescence detection step based on the detection signal acquired in the fluorescence detection step. (4) an analysis condition setting step for setting analysis conditions for the fluorescence analysis for the time-series photon number measurement data acquired in the photon counting step; ) Based on the analysis conditions set in the analysis condition setting step, the fluorescence analysis is performed on the photon number measurement data indicating the measurement results of the measurement sample including the fluorescent probe. (6) The analysis condition setting step sets, as analysis conditions, a bin width for binning photon number measurement data and a reference photon number for one fluorescent probe molecule in the bin width. The analysis step bins the photon number measurement data with the bin width set in the analysis condition setting step, performs the fluorescence analysis on the number of fluorescent probes in the measurement region of the measurement sample with reference to the reference photon number, (7) In the analysis condition setting step, a change in the measured SN ratio, which is the SN ratio of the photon number in the photon number measurement data, due to the bin width, and a reference SN ratio which is the SN ratio of the photon number when the shot noise characteristic is assumed The bin width is set using a change in the bin width.

  In the fluorescence analysis apparatus and the fluorescence analysis method described above, a minute measurement region is set in the measurement sample by the excitation light irradiation system and the fluorescence detection system, and fluorescence generated from the fluorescent probe in the measurement region is detected. Thus, time-series photon number measurement data is generated. The binning bin width and the reference photon number corresponding to the number of photons per molecule of the fluorescent probe are set as analysis conditions for the photon number measurement data of the measurement results, and measurement is performed based on the analysis conditions. Fluorescence analysis (photon burst analysis) is performed on the number of fluorescent probes in the region.

  In this way, by re-binning the photon number measurement data acquired in a predetermined counting time width with a set bin width, and applying the reference photon number to the binned measurement data, the photon burst analysis is performed. For example, it is possible to facilitate the fluorescence analysis of the measurement sample by measuring the fluorescence from the fluorescent probe in the measurement region, such as the analysis of the reaction between the target molecule and the fluorescent probe in the measurement sample.

  Further, in such photon burst analysis, regarding the setting of the bin width for the photon count measurement data, the bin width dependence of the reference SN ratio when the shot noise characteristic is assumed with respect to the bin width dependence of the measured SN ratio. The binning bin width is set based on these relationships. According to such a configuration, the bin width applied to the photon number measurement data in the photon burst analysis can be suitably set according to the characteristics of the measurement data actually acquired for the measurement sample. Thereby, it is possible to improve the sensitivity of the fluorescence measurement for the measurement sample and the analysis accuracy of the photon burst analysis.

  Here, it is preferable that the fluorescence analyzer includes a flow applying unit that applies a flow to the measurement sample including the fluorescent probe. Similarly, the fluorescence analysis method preferably includes a flow applying step for applying a flow to the measurement sample including the fluorescent probe. The photon burst analysis method for photon number measurement data and the method for setting the analysis conditions according to the above configuration can be used particularly effectively in a configuration in which fluorescence measurement is performed with a flow applied to a measurement sample including a fluorescent probe. it can.

Regarding the specific setting method of the bin width which is the analysis condition of the photon burst analysis, the fluorescence analysis apparatus is configured such that the analysis condition setting means uses the photon number measurement data for the measurement signal amount S in the photon number measurement data. A measurement noise amount N and a reference noise amount N ′ assuming a shot noise characteristic are obtained, and an evaluation for bin width setting defined by using the measurement SN ratio S / N and the reference SN ratio S / N ′. Quantity dN / S = (N−N ′) / S
It is preferable to set the bin width with reference to FIG.

Similarly, in the analysis condition setting step, the fluorescence analysis method uses the measurement noise amount N in the photon number measurement data and the reference noise when the shot noise characteristic is assumed with respect to the measurement signal amount S in the photon number measurement data. A quantity N ′ is obtained, and an evaluation quantity for bin width setting defined using the measured SN ratio S / N and the reference SN ratio S / N ′ dN / S = (N−N ′) / S
It is preferable to set the bin width with reference to FIG.

を用いることにより、光子数測定データに対するビン幅を好適に評価、設定することが可能となる。また、この場合の具体的なビン幅の設定方法の一例としては、評価量ｄＮ／Ｓのビン幅（時間幅）依存性において、評価量ｄＮ／Ｓが最大となる時間幅によってビン幅を設定する方法を用いることができる。 Thus, the evaluation amount of the following formula (1) defined by comparing the measured SN ratio S / N and the reference SN ratio S / N ′

By using, it is possible to suitably evaluate and set the bin width for the photon number measurement data. In addition, as an example of a specific bin width setting method in this case, the bin width is set according to the time width at which the evaluation amount dN / S is maximum in the bin width (time width) dependency of the evaluation amount dN / S. Can be used.

  Further, with respect to the reference noise amount N ′ and the reference S / N ratio S / N ′ obtained by the shot noise characteristics obtained in the above configuration, the fluorescence analysis apparatus is configured so that the analysis condition setting unit responds to changes in the measurement noise amount N due to the bin width. It is preferable that the reference bin width is set, and the change in the reference noise amount N ′ due to the bin width is obtained with the measurement noise amount N in the reference bin width as an initial value. Similarly, in the fluorescence analysis method, in the analysis condition setting step, the reference bin width is set with respect to the change due to the bin width of the measurement noise amount N, and the reference noise amount N is set with the measurement noise amount N in the reference bin width as an initial value. It is preferable to obtain the change due to 'bin width.

  Further, regarding a specific method for setting the reference photon number, which is another analysis condition for photon burst analysis, the fluorescence analysis apparatus is configured such that the analysis condition setting means uses a self-calculation obtained by fluorescence correlation spectroscopy from photon number measurement data. It is preferable to set the reference photon number in the bin width based on the waveform of the correlation function, the fluorescence intensity per unit time measured by the photon number measurement data, and the set bin width.

  Similarly, in the fluorescence analysis method, in the analysis condition setting step, the waveform of the autocorrelation function obtained by the fluorescence correlation spectroscopy from the photon number measurement data, the fluorescence intensity per unit time measured by the photon number measurement data, It is preferable to set the reference photon number in the bin width according to the set bin width.

  According to such a configuration, the average number of molecules of the fluorescent probe in the measurement region is obtained from the waveform of the autocorrelation function G (τ) obtained from the photon number measurement data, and the calculated average number of molecules and the measurement The reference photon number with respect to the binning bin width can be suitably set by referring to the fluorescence intensity per unit time.

  As an example of a specific setting method, the average number of molecules of the fluorescent probe in the measurement region is obtained from the waveform of the autocorrelation function, and the unit number of times is determined by the average number of molecules of the fluorescent probe and the fluorescence intensity per unit time. Fluorescence intensity per molecule per fluorescent probe is determined. Then, a method for setting the average number of photons per molecule of the fluorescent probe, which is the reference number of photons in the bin width, based on the obtained fluorescence intensity per molecule of the fluorescent probe and the set bin width. Can be used.

  According to the fluorescence analysis apparatus and the fluorescence analysis method of the present invention, the binning bin width and the reference photon number for one molecule of the fluorescent probe are analyzed for the photon number measurement data obtained by the fluorescence measurement of the measurement sample. The photon burst analysis is performed for the number of fluorescent probes in the measurement region, and the bin width dependency of the measurement SN ratio and the reference SN ratio when the shot noise characteristic is assumed in the bin width setting are set. By setting the bin width using the bin width dependency, the fluorescence analysis of the measurement sample is facilitated, and the sensitivity of the fluorescence measurement for the measurement sample and the analysis accuracy of the photon burst analysis can be improved. Become.

  Hereinafter, preferred embodiments of a fluorescence analysis apparatus and a fluorescence analysis method according to the present invention will be described in detail with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described.

  FIG. 1 is a diagram schematically showing a configuration of an embodiment of a fluorescence analysis apparatus according to the present invention. The fluorescence analyzer 1A according to the present embodiment detects fluorescence generated from a fluorescent probe in a measurement region set for the measurement sample S with respect to the fluorescent probe in the measurement sample S to detect time-series photon number measurement data. And information on the measurement sample S is acquired by performing a predetermined fluorescence analysis on the obtained photon number measurement data. Hereinafter, a fluorescent substance (including a molecule labeled with a fluorescent substance) in the measurement sample S to be measured is referred to as a “fluorescent probe”.

  An example of an analysis target by the fluorescence analysis apparatus 1A includes a target molecule (target material) T and fluorescence probes P1 and P2 shown in FIG. The schematic diagram of FIG. 2 shows an example of a homogeneous assay that acquires information about the target molecule T by using the specific binding of the fluorescent probe P to the target molecule T such as a biomolecule.

  Specifically, in FIG. 2, the fluorescence having a recognition site that specifically binds to the recognition site t1 with respect to a sample containing the target molecule T having three recognition sites t1 to t3 (FIG. 2 (a)). The labeled molecular probe P1 and the fluorescently labeled molecular probe P2 (FIG. 2B) having a recognition site that specifically binds to the recognition site t2 are mixed as a fluorescent probe, as shown in FIG. 2C. These are reacted with each other to obtain a measurement sample S to be subjected to fluorescence analysis. Information on the target molecule T can be obtained by performing fluorescence analysis (photon burst analysis) on the number of fluorescent probes as described later on such a measurement sample S. Hereinafter, the configuration of the fluorescence analysis apparatus 1A according to the present embodiment will be described together with the fluorescence analysis method.

  1A includes a sample holder 10, a reflux device 12, an objective lens 20, a dichroic mirror 21, an excitation light source 22, a reflection mirror 23, an optical filter 24, and an imaging lens 25. The pinhole 26 and the photodetector 27 are provided, and each of these elements constitutes a fluorescence microscope section in the fluorescence analysis apparatus 1A. In addition to the elements of the fluorescence microscope unit, the fluorescence analysis apparatus 1A includes a detection signal processing unit 30, a photon counting unit 35, and a measurement control unit 50.

  The sample holder 10 is sample holding means for holding a measurement sample S including a fluorescent probe to be subjected to fluorescence measurement and analysis, and its bottom surface is constituted by a container-like flow path member that functions as a slide glass. Further, in the fluorescence analysis apparatus 1 </ b> A of the present embodiment, the reflux device 12 is provided for the measurement sample S held in the flow path member 10. Further, as shown in FIG. 1, a circulation channel through which the solution-like measurement sample S flows is provided between the channel member 10 and the reflux device 12. In such a sample circulation channel, the reflux device 12 functions as a flow imparting unit that imparts a flow to the liquid sample of the sample S in a predetermined direction and a flow velocity together with the channel member 10.

  3A and 3B are diagrams showing an example of the configuration of the flow path member 10 used as the sample holder. FIG. 3A is a side view and FIG. 3B is a bottom view. The flow path member 10 of this configuration example includes a flow path body 13 that forms an upper portion thereof and a cover member 18 that forms a lower portion thereof. The flow channel body 13 is made of, for example, a glass substrate, and is opened downward between the introduction flow channel 14 and the discharge flow channel 15 penetrating between the upper surface and the lower surface thereof, and between the introduction flow channel 14 and the discharge flow channel 15. And a groove-shaped main channel 16 formed in a state.

  The cover member 18 is made of, for example, a cover glass and is provided so as to cover the groove-shaped main flow path 16 of the flow path body 13 from below. In such a configuration, the sample channel 11 in the channel member 10 is constituted by the introduction channel 14, the main channel 16, and the discharge channel 15. Of these, the main channel 16 is a measurement channel used for fluorescence measurement of the sample S. An example of the configuration of the measurement channel 16 is a channel structure having a rectangular cross-sectional shape with a width of 100 μm and a height of 25 μm. Since such a flow channel structure is very small, the measurement sample S to which the flow is applied by the flow channel member 10 and the reflux device 12 is a very small amount of liquid sample. The reflux device 12 is driven and controlled by the measurement control unit 50.

  The objective lens 20 is installed at a predetermined position with respect to the sample holder 10 of the flow path member that holds the measurement sample S. As the objective lens 20, for example, a water immersion (immersion) objective lens can be suitably used. Further, with respect to such an objective lens 20, the sample holder 10 is configured such that its shape corresponds to the working distance of the objective lens 20. As shown in FIG. 1, in the configuration using a flow path member as the sample holder 10, the objective lens 20 is disposed at a predetermined position with respect to the sample flow path 11 in the flow path member 10.

  In the configuration shown in FIG. 1, the objective lens 20, the dichroic mirror 21, and the excitation light source 22 constitute excitation light irradiating means for irradiating the measurement region set for the measurement sample S with excitation light. As the excitation light source 22, for example, a laser light source can be suitably used. The excitation laser beam supplied from the excitation light source 22 is reflected by the dichroic mirror 21 and condensed through the objective lens 20 and condensed and irradiated as a beam spot onto the measurement sample S in the sample holder 10 ( Excitation light irradiation step). Here, as a specific example of the excitation light source 22, a laser light source that supplies laser light with a wavelength of 473 nm, 532 nm, or 635 nm can be used.

  Further, in the configuration shown in FIG. 1, in the measurement region of the measurement sample S irradiated with the excitation light by the objective lens 20, the reflection mirror 23, the optical filter 24, the imaging lens 25, the pinhole 26, and the photodetector 27. Fluorescence detection means for detecting the fluorescence generated from the fluorescent probe is provided. The fluorescence from the fluorescent probe of the measurement sample S is collected by the objective lens 20, passes through the dichroic mirror 21, and then is reflected toward the photodetector 27 by the reflection mirror 23. The fluorescence whose optical path has been changed by the reflection mirror 23 passes through the optical filter 24 and is incident on the photodetector 27 while being imaged by the imaging lens 25.

  As the optical filter 24, for example, a bandpass filter selected according to the fluorescence spectrum of the fluorescent probe to be measured for removing extraneous light components such as scattered light and stray light from the sample S can be used. . A pinhole 26 is provided between the imaging lens 25 and the photodetector 27. The pinhole 26 is installed at a position that becomes confocal with respect to the fluorescence condensed by the imaging lens 25, removes out-of-focus light, and measures a measurement area of a beam spot formed in the sample S. Only the fluorescence from is passed.

  The fluorescence that has passed through the pinhole 26 is detected by the photodetector 27, and a detection signal that is an electrical signal indicating the detection result is output (fluorescence detection step). With the above excitation light irradiation means and fluorescence detection means, a minute measurement region is set in the measurement sample S, the measurement region of the sample S is irradiated with excitation light, and the fluorescence from the fluorescent probe in the measurement region is detected. A fluorescence microscope using a confocal optical system is configured. As a specific example of the photodetector 27, a photomultiplier tube can be used.

Here, the measurement region set in the measurement sample S by the fluorescence microscope having the above-described configuration is a very small region of about 1 fl (femtoliter), for example. As an example of the molecular concentration of the fluorescent probe contained in the sample S, if the concentration is 1 nM, the average number of molecules of the fluorescent probe in the measurement region is 6 · 10 ²³ × 1 · 10 ⁻⁹ × 1 · 10 ^{−. 15} = 0.6. In the homogeneous assay illustrated in FIG. 2, the concentration of the target molecule is, for example, about 1 pM. However, these numerical values are examples of settings for the measurement region and the like, and the specific settings are different in each case.

  The detection signal output from the light detector 27 of the fluorescence detection means is input to the detection signal processing unit 30. The detection signal processing unit 30 is configured by a signal amplification circuit such as a preamplifier and a signal processing circuit such as a discriminator (wave height discriminator), and performs predetermined signal processing on the analog detection signal from the photodetector 27. To generate and output a detection pulse signal sequence indicating individual single photon detection events in the photodetector 27.

  The detection pulse signal sequence output from the detection signal processing unit 30 is input to the photon counting unit 35. The photon counting unit 35 determines the number of photons detected by the photodetector 27 of the fluorescence detection means based on the detection signal from the photodetector 27 (detection pulse signal sequence signal-processed by the detection signal processing unit 30). Photon counting means for generating time-series photon number measurement data by counting in time series with a counting time width (time resolution) (photon counting step). With such a configuration, fluorescence measurement by single photon counting on the measurement sample S can be performed.

  Here, in the fluorescence analysis apparatus 1A, the counting time width in the photon counting unit 35 is an initial condition (minimum bin width) of the bin width in the fluorescence analysis. Further, the photon number measurement data counted by the photon counting unit 35 includes a noise event, a background event, and the like by the photodetector 27 in addition to a photon detection event due to fluorescence from the measurement sample S.

  Specifically, for example, a multi-channel scaler (MCS) can be used as the photon counting unit 35. In addition, the MCS counting time resolution that is the counting time width in the photon counting unit 35 is, for example, about 200 nsec (nanoseconds).

  The time-series photon number measurement data generated by the photon counting unit 35 is input to the measurement control unit 50, and the measurement control unit 50 performs fluorescence analysis on the measurement data. In the present embodiment, the measurement control unit 50 includes an analysis condition setting unit 51 and a measurement result analysis unit 52, and the fluorescent probe in the measurement region of the measurement sample S with respect to the photon number measurement data is measured. Information on the measurement sample S is acquired by performing fluorescence analysis (photon burst analysis) on the number. Such a measurement control part 50 can be comprised by the computer for control which the software for fluorescence analysis operates, for example.

  The analysis condition setting unit 51 is a setting for setting the analysis conditions for the fluorescence analysis to be performed on the time-series photon number measurement data acquired by the photon counting unit 35 with a predetermined counting time width (initial bin width). Means (analysis condition setting step). In addition, the measurement result analysis unit 52 performs an analysis for performing photon burst analysis as fluorescence analysis on the photon number measurement data indicating the measurement result of the measurement sample S including the fluorescent probe based on the analysis conditions set by the analysis condition setting unit 51. Means (measurement result analysis step).

  The analysis condition setting unit 51 sets a bin width (time width of each bin) for binning the photon number measurement data and a reference photon number for one molecule of the fluorescent probe in the bin width as analysis conditions for the measurement data. . Here, the bin time width for the photon number measurement data is preferably set to an integral multiple of the initial bin width in the photon counting unit 35. In this case, binning of the photon number measurement data can be easily performed by adding a predetermined number of pieces of count data continuous in time. In addition, the bin width of the binning needs to be set to a value suitable for accurately executing the photon burst analysis corresponding to the fluorescence analysis executed by the analysis unit 52.

  Specifically, the setting unit 51 changes the measurement SN ratio, which is the SN ratio of the photon number in the photon number measurement data, according to the bin width and the reference SN that is the SN ratio of the photon number when the shot noise characteristic is assumed. The binning bin width is set using the ratio change with bin width. The setting unit 51 obtains the reference photon number for one molecule of the fluorescent probe in the bin width for the set bin width. That is, for the measurement data after binning with the set bin width, the number of photons corresponding to one fluorescent probe molecule is obtained for the count data of each bin, and is used as the reference number of photons in the photon burst analysis.

  Based on the analysis conditions set as described above, the measurement result analysis unit 52 bins the photon number measurement data with the set bin width to generate measurement data for fluorescence analysis, and the measurement data after the binning is generated. The photon burst analysis is performed on the number of fluorescent probes in the measurement region of the measurement sample S with reference to the reference number of photons. Thereby, for example, information about the measurement sample S such as information on target molecules in the measurement sample S can be acquired.

  The effects of the fluorescence analysis apparatus and the fluorescence analysis method according to the above embodiment will be described.

  In the fluorescence analysis apparatus 1A and the fluorescence analysis method shown in FIG. 1, a minute measurement region in the measurement sample S by the excitation light irradiation system and the fluorescence detection system including the objective lens 20, the excitation light source 22, the photodetector 27, and the like. Set. In addition, fluorescence generated from a fluorescent probe in the measurement region (for example, a fluorescent substance labeled with the fluorescent probe) is detected by the photodetector 27, and the photon counting unit 35 determines the number of photons in time series from the detection signal. Generate measurement data.

  The analysis condition setting unit 51 sets the binning bin width and the reference photon number corresponding to the number of measurement photons per molecule of the fluorescent probe as analysis conditions for the photon number measurement data of the measurement result, The result analysis unit 52 performs photon burst analysis, which is fluorescence analysis for the number of fluorescent probes in the measurement region, based on the analysis conditions.

  In this way, the photon number measurement data acquired for the measurement sample S with a predetermined counting time width (initial bin width) is binned again with the set bin width, and the reference photon number is set in the measurement data after the binning. By applying the photon burst analysis by applying, for example, analysis of the reaction between the target molecule and the fluorescent probe in the measurement sample S, the fluorescence of the measurement sample S is measured by measuring the fluorescence from the fluorescent probe in the measurement region. It becomes possible to facilitate fluorescence analysis.

  Further, in such a fluorescence analysis of the measurement sample S, the reference S / N ratio when the shot noise characteristic is assumed with respect to the bin width dependency of the measured S / N ratio with respect to the bin width setting for the photon number measurement data. The bin width dependency is obtained, and the binning bin width is set from the relationship. According to such a configuration, the bin width applied to the photon number measurement data in the photon burst analysis can be suitably set according to the characteristics of the measurement data actually acquired for the measurement sample S. Thereby, it is possible to improve the sensitivity of the fluorescence measurement for the measurement sample S and the analysis accuracy of the photon burst analysis.

  Further, in the fluorescence analysis apparatus 1A of FIG. 1, time-series photon number measurement data acquired by the photon counting unit 35 is input to a measurement control unit 50 including an analysis condition setting unit 51 and a measurement result analysis unit 52. The measurement control unit 50 is configured to execute fluorescence analysis on measurement data by operating a fluorescence analysis program or the like corresponding to the above configuration. Thereby, the fluorescence analysis including the setting and change of the bin width for the photon number measurement data can be suitably executed according to the content of the measurement data.

  Further, in the fluorescence analysis apparatus 1A according to the present embodiment, the reflux device 12 is provided as a flow imparting unit that imparts a flow to the measurement sample S with respect to the measurement sample S held in the flow path member of the sample holder 10. . The photon burst analysis method for the photon number measurement data having the above-described configuration and the method for setting the analysis conditions are such that fluorescence measurement is performed in a state in which a flow is applied to the measurement sample S including the fluorescent probe by the flow applying means such as the reflux device 12. In the structure to perform, it can use especially effectively.

  That is, in the configuration in which a flow is applied to the measurement sample S, the measurement sensitivity of fluorescence measurement is improved, but there is a problem that it is difficult to analyze the translational diffusion motion by the autocorrelation function using the fluorescence correlation spectroscopy. On the other hand, by setting the analysis conditions with the above-described configuration and applying the photon burst analysis method, it is possible to suitably perform analysis on such fluorescence measurement data. As for the flow applying means for applying a flow to the sample S, the flow is simply applied to the measurement sample S by using the flow path member 10 having the sample flow path 11 and the reflux device 12 as described above. can do.

  The fluorescence analysis apparatus 1A shown in FIG. 1 and the fluorescence analysis method executed in the analysis apparatus 1A will be described more specifically.

  First, the application of the flow to the measurement sample S by the flow path member 10 and the reflux device 12 will be described. As described above, in the configuration in which a flow is applied to the measurement sample S, molecules move in the sample S due to the flow in addition to the translational diffusion motion. Thereby, the passage frequency of molecules with respect to a minute measurement region set in the measurement sample S can be increased, and the measurement sensitivity with respect to the sample S can be improved.

FIG. 4 is a graph showing an autocorrelation function obtained by applying fluorescence correlation spectroscopy to photon number measurement data. This graph shows a fluorescence measurement for 60 seconds with respect to a measurement sample S having a fluorescent probe concentration of 4.5 × 10 ⁻¹¹ M, and analysis of the photon number measurement data of the measurement result by fluorescence correlation spectroscopy. The autocorrelation function G (τ) obtained by performing is shown.

  In the graph of FIG. 4, the horizontal axis represents time τ (sec) corresponding to the time delay parameter when calculating the autocorrelation function, and the vertical axis represents the normalized autocorrelation function. Graph A1 shows the autocorrelation function when the flow is not applied to the sample S (no flow), and graph A2 shows the autocorrelation function when the flow is applied (with flow).

  The graph A1 shows the translational diffusion movement of the fluorescent probe in the sample S in a state where no flow is applied to the sample S. Further, in the graph A2 to which the flow is applied, a regular flow is generated in the measurement sample S at a moving speed exceeding the translational diffusion motion, and this is observed in the autocorrelation function. In addition, by controlling the flow velocity of the sample S, a waveform that is intermediate between the graphs A1 and A2 can be obtained. In this case, the effects of both the translational diffusion motion and the movement motion due to the flow of the sample S are expressed in the autocorrelation function. Observed.

  As can be seen from these graphs A1 and A2, in the graph A2 with the flow, the moving speed of the fluorescent probe is about one digit larger than the graph A1 without the flow. Thereby, as described above, the passing frequency of molecules with respect to the measurement region in the sample S can be increased, and the measurement sensitivity can be improved.

  As for the excited state of the fluorescent dye molecule of the fluorescent probe, when the moving speed of the fluorescent probe is improved as described above, the passing time of the measurement region is shortened, so that the excitation time by the excitation light irradiation to the fluorescent probe is reduced. Shorter. For this reason, even if the transition to the triplet state is frequently confirmed under the no-flow condition, the frequency is reduced by the presence of the flow, and the fluorescence intensity per molecule can be suitably measured. Become.

  As described above, when the passing frequency of the fluorescent probe with respect to the measurement region in the sample S is increased by applying the flow using the reflux device 12, the concentration of the fluorescent probe in the sample S does not change. The average number of staying probes does not vary, and the speed of entering and exiting the fluorescent probe with respect to the measurement region changes. On the other hand, in the fluorescence analysis apparatus 1A having the above configuration, the binning bin width of the photon number measurement data can be set according to the moving speed of the fluorescence probe, and the photon burst analysis can be performed with high accuracy. In such a configuration, it is possible to perform fluorescence analysis by setting optimum analysis conditions according to measurement conditions such as whether or not flow is applied to the sample S, and flow rate when flow is applied. is there.

によって求めることができる。また、自己相関関数の算出においては、上記の式（２）において、自己相関関数を求める際の時間遅れτを測定データＦ（ｔ）の時間分解能の整数倍、図１に示した構成では光子計数部３５での計測時間幅（初期ビン幅）の整数倍に設定すれば、単純に時系列の光子数測定データＦ（ｔ）でのビン間の積によって、自己相関関数Ｇ（τ）を求めることができる。 Here, the autocorrelation function G (τ) obtained by fluorescence correlation spectroscopy will be briefly described. The autocorrelation function G (τ) is expressed by the following equation (2) using time series photon number measurement data as F (t)

Can be obtained. In calculating the autocorrelation function, in the above equation (2), the time delay τ when obtaining the autocorrelation function is an integral multiple of the time resolution of the measurement data F (t). In the configuration shown in FIG. If it is set to an integral multiple of the measurement time width (initial bin width) in the counting unit 35, the autocorrelation function G (τ) is simply calculated by the product between bins in the time-series photon number measurement data F (t). Can be sought.

  Further, since the autocorrelation function G (τ = 0) in the case of the time delay τ = 0 is the product of its own bin, the influence of noise of the photodetector 27 cannot be ignored. Therefore, when obtaining this G (0), it is preferable to estimate it from the waveform of G (τ) in the range of τ> 0. Further, in the estimation of G (0), there is a possibility that problems such as superposition of fixed noise due to the after-pulse of the photodetector 27 may occur in the vicinity of τ = 0, so the entire waveform of G (τ) is handled. It is preferable to perform waveform analysis.

によって求められる。この式（３）によれば、時間遅れτ＝０のときに、Ｇ（τ）のｙ切片について
Ｇ（τ＝０）＝１／ｎ
の関係が得られる。 In general, in one-component waveform analysis in fluorescence correlation spectroscopy, the autocorrelation function G (τ) is expressed by the following equation (3):

Sought by. According to this equation (3), G (τ = 0) = 1 / n for the y-intercept of G (τ) when time delay τ = 0.
The relationship is obtained.

Here, in the above formula (3), n represents the average number of molecules of the fluorescent probe in the measurement region of the measurement sample S, τ _D is the diffusion time constant, and r ₀ and z ₀ are structural parameters (structure parameters). ) Respectively. These variables can be obtained by performing waveform analysis by fitting calculation on the autocorrelation function G (τ) calculated by the equation (2). At this time, the average molecular number n indicating the concentration of the fluorescent probe in the measurement sample S is obtained from the y intercept of G (τ) described above. The average molecular number n thus obtained can be referred to in setting the reference photon number used for photon burst analysis, as will be described later.

によって定義される。ここで、δＦ（ｔ）は蛍光ゆらぎを示すものであり、

によって定義される。 The autocorrelation function G (τ) will be described in detail. The autocorrelation function G (τ) is expressed by the following equation (4) with respect to the photon number measurement data F (t).

Defined by Here, δF (t) indicates fluorescence fluctuation,

Defined by

すなわち、この式（６）に示すように、上記仮定のもとでは、自己相関関数Ｇ（τ）の定義式（４）は、式（２）のように表すことができる。また、自己相関関数の波形解析に用いられる式（３）は、定義式（４）に対してレーザ光強度分布式、拡散方程式などを用いてパラメータを導入して式を解くことで得られるものである。 Using this δF (t) and assuming that the sum of products of the fluorescence fluctuation δF (t) and the average fluorescence intensity <F> converges to 0, the right side of the above equation (2) is transformed as follows: it can.

That is, as shown in the equation (6), the definition equation (4) of the autocorrelation function G (τ) can be expressed as the equation (2) under the above assumption. The equation (3) used for the autocorrelation function waveform analysis is obtained by solving the equation by introducing parameters to the definition equation (4) using a laser light intensity distribution equation, a diffusion equation, and the like. It is.

As for the autocorrelation function equation (3), if the process of transitioning to the triplet state is included, the equation becomes more complicated, and parameters such as multi-component systems and flow systems increase. It becomes difficult to determine accurately. On the other hand, in the one-component analysis, when measuring the same sample S, the diffusion time constant τ _D can be fixed. In addition, since the structural parameter indicates whether the measurement region can be constructed with good reproducibility, it is also effective in confirming that the confocal optical system and the fluorescent probe are in the standard state.

  As for the flow applying means for applying a flow to the measurement sample S including the fluorescent probe, in the embodiment shown in FIG. 1, an example of the circulation flow path of the liquid sample configured using the flow path member 10 and the reflux device 12 is illustrated. However, specifically, the flow providing means having various configurations other than such a configuration may be used.

  Next, a bin width setting method, which is an analysis condition for photon burst analysis on photon number measurement data, will be described.

を参照して、ビン幅を設定することが好ましい。 For a specific method of setting the binning bin width, which is an analysis condition when performing photon burst analysis on the photon number measurement data, the analysis condition setting unit 51 compares the measurement signal amount S in the photon number measurement data with respect to the measurement signal amount S. Then, the measurement noise amount N in the photon number measurement data and the reference noise amount N ′ when the shot noise characteristic is assumed are obtained and defined using the measurement SN ratio S / N and the reference SN ratio S / N ′. Evaluation amount dN / S for setting the bin width of the formula (1)

It is preferable to set the bin width with reference to FIG.

  Thus, by using the evaluation quantity dN / S obtained by comparing the measured SN ratio S / N and the reference SN ratio S / N ′, the bin width for the photon number measurement data is preferably evaluated and set. It becomes possible to do. In addition, as an example of a specific bin width setting method in this case, the bin width is set according to the time width at which the evaluation amount dN / S is maximum in the bin width (time width) dependency of the evaluation amount dN / S. It is preferable to do.

  The above-described evaluation amount dN / S and setting of the bin width using the evaluation amount will be specifically described. FIG. 5 is a graph showing the bin width dependence of the S / N ratio in the photon number measurement data. In the graph of FIG. 5, the horizontal axis indicates the bin time width (sec) when binning the photon number measurement data, and the vertical axis indicates the S / N ratio S / N obtained for each bin width. . Graph B1 shows the measured SN ratio when there is no flow, and graph B2 shows the measured SN ratio when there is a flow.

  Here, in the calculation of the S / N ratio in the photon number measurement data, for the signal amount S, the average value per bin time width of the photon number recorded for a predetermined measurement time (for example, 60 seconds) by the photon counting unit 35 is measured. The signal amount is S. As for the noise amount N, the degree of variation in the number of photons with respect to the bin time width, that is, the standard deviation of the number of measured photons is used as the measurement noise amount N. From these measurement signal amount S and measurement noise amount N, the measurement SN ratio S / N can be obtained.

In the measured SN ratio graphs B1 and B2 in FIG. 5, when the bin width is as small as about 1 μsec, the SN ratio is hardly affected by the movement speed of the fluorescent probe, and shows the SN ratio of the luminescence phenomenon itself. At this time, the number of photons in each bin within the measurement time shows a Poisson distribution similar to that of the shot noise characteristic. When the bin time width is w _t , the measurement signal amount S, which is the average value of the number of photons, It increases in proportion to w _t . Further, the measurement noise amount N, which is the standard deviation of the number of photons, is proportional to the square root √w _{t of the} time width from the characteristics of the Poisson distribution.

Accordingly, in the dependence of the measured SN ratio on the bin width, the measured SN ratio S / N is proportional to the square root √w _t of the bin time width w _{t in} the region where the bin time width is small and exhibits the same characteristics as the shot noise characteristics. Further, in such a region where the shot noise characteristic is established, the S / N ratio is improved by one digit when the bin width is increased by two digits.

On the other hand, when the bin width of the photon number measurement data is increased, the number of photons counted in each bin and the S / N ratio thereof are determined by the movement speed of the fluorescent probe in the measurement sample S and the fluorescence with respect to the measurement region. Under the influence of the probe coming and going, it is observed as fluorescence fluctuation in the photon count measurement data. At this time, the bin width dependency of the measured SN ratio deviates from the shot noise characteristic due to the fluorescence fluctuation. Therefore, by evaluating the degree of deviation of the S / N ratio from the shot noise characteristic due to the fluorescence fluctuation, it is possible to evaluate the bin time width w _t suitable for acquiring information about the movement of the fluorescent probe.

  As a specific method, for example, in addition to the noise amount N actually measured with the photon number measurement data with respect to the measurement signal amount S, the noise amount when the shot noise characteristic is assumed is set as the reference noise amount N ′. Asking. Then, by evaluating how far the SN ratio S / N due to the measurement noise amount N deviates from the SN ratio S / N ′ due to the reference noise amount N ′, the bin time to be applied in the fluorescence analysis of the photon number measurement data The width can be set suitably. In this case, in the evaluation of the deviation of the SN ratio from the shot noise characteristic, the evaluation amount dN / S of the above-described formula (1) can be suitably used.

  Here, in calculating the reference noise amount N ′ with respect to the measurement noise amount N, a reference bin width is set with respect to a change due to the bin width of the measurement noise amount N obtained from the photon number measurement data, and the reference bin width is set. It is preferable to obtain the change due to the bin width of the reference noise amount N ′ due to the shot noise characteristics, with the measurement noise amount N at 1 being the initial value. FIG. 6 is a graph showing the bin width dependence of the measured S / N ratio and the reference S / N ratio obtained by such a method.

In this analysis example, the minimum bin width (for example, the initial count when the photon counting unit 35 counts the number of photons is considered in consideration of the fact that the measured S / N ratio shows the same characteristics as the shot noise characteristics in a region where the bin time width is small. Bin width) is the reference bin width. Then, the measurement noise amount N, which is the standard deviation of the number of photons obtained from the photon number measurement data at the minimum bin width, is used as an initial value, and the reference noise is utilized by using the fact that the noise amount is proportional to √w _t in the shot noise characteristics. The bin width dependency of the quantity N ′ is obtained.

In FIG. 6, with respect to the noise amount thus obtained, the SN ratio S / N graph C1 calculated by the measurement signal amount S and the measurement noise amount N and the SN ratio calculated by the reference noise amount N ′. The graph C0 of S / N ′ is shown. It can be seen from these graphs C0 and C1 that the measured SN ratio S / N deviates from the reference SN ratio S / N ′ based on the shot noise characteristics as the bin time width w _t increases.

  By using the measured SN ratio S / N and the reference SN ratio S / N ′ shown in FIG. 6, the above-described evaluation amount dN / S = (N−N ′) / S for setting the bin width can be obtained. it can. FIG. 7 is a graph showing the bin width dependency of the evaluation amount dN / S for setting the bin width. In the graph of FIG. 7, the horizontal axis indicates the bin time width (sec) when binning the photon number measurement data, and the vertical axis indicates the evaluation amount dN / S obtained for each bin width. . The graph D1 indicates dN / S when there is no flow, and the graph D2 indicates dN / S when there is a flow.

  By referring to the bin width dependency of the evaluation amount dN / S, a suitable bin time width can be set when performing photon burst analysis on the photon number measurement data. That is, in the change in dN / S due to the bin width, the value of the evaluation amount dN / S described above is small in the region where the measured SN ratio shows the same characteristics as the shot noise characteristics. On the other hand, in the region where the influence of the fluorescence fluctuation due to the movement of the fluorescent probe in the measurement sample S is large, the value of dN / S becomes large. In addition, an increase in the value of dN / S in this way indicates that the measurement sensitivity to the fluorescent probe is high in the photon number measurement data to which the bin width is applied.

In setting the binning bin width in the fluorescence analysis of the photon number measurement data, the characteristic of the evaluation quantity dN / S is taken into consideration, and the time when the evaluation quantity dN / S = (N−N ′) / S is maximized. It is preferable to set the bin width according to the width. In such a setting method, in the example shown in FIG. 7, when there is no flow, the binning bin width for the photon number measurement data is set to w _t = 0.73 msec with reference to the graph D1. When there is a flow, the bin width is set to w _t = 0.10 msec with reference to the graph D2.

  Alternatively, the specific setting method of the bin width using the evaluation amount dN / S is not limited to the method of setting the binning bin width by the time width in which the dN / S is maximized as described above. Various configurations can be used. As such a configuration, for example, a range having a dN / S value of a predetermined ratio or more, for example, 70% or more is set from the peak position where the dN / S is maximum, and the bin width is set by the time width selected within the range. A configuration or the like to be set can be used.

  Next, a method for setting the reference photon number, which is an analysis condition for photon burst analysis on the photon number measurement data, will be described.

  Regarding the reference photon number for one fluorescent probe molecule, which is an analysis condition for performing photon burst analysis on the photon number measurement data, the analysis condition setting unit 51 sets the photon number measurement data and the bin width set as the analysis condition. From this, it is preferable to obtain the average number of photons per molecule of the fluorescent probe and use the obtained average number of photons as the reference number of photons.

  In calculating the average photon number from the photon number measurement data, the autocorrelation function obtained by applying fluorescence correlation spectroscopy to the photon number measurement data is used, and the average photon number is obtained from the measurement data by fluorescence correlation spectroscopy. Based on the autocorrelation function waveform, the fluorescence intensity per unit time measured in the measurement data, and the set bin width, the average number of photons per molecule that is the reference photon number in the bin width is set. It is preferable to do. In this case, the average number of molecules of the fluorescent probe in the measurement region is obtained from the waveform of the autocorrelation function G (τ) obtained from the photon number measurement data, and the calculated average number of molecules per unit time measured. The reference photon number with respect to the bin width can be suitably set with reference to the fluorescence intensity.

  As an example of such a setting method, the average number of molecules of the fluorescent probe in the measurement region is obtained from the waveform of the autocorrelation function G (τ), the average number of molecules of the fluorescent probe, and the fluorescence intensity per unit time. The fluorescence intensity per molecule of the fluorescent probe per unit time is obtained. Then, a method for setting the average number of photons per molecule of the fluorescent probe, which is the reference number of photons in the bin width, based on the obtained fluorescence intensity per molecule of the fluorescent probe and the set bin width. Can be used.

  A specific example of setting the reference photon number will be described. First, the autocorrelation function G (τ) shown in FIG. 4 is obtained from the photon number measurement data. Here, as described above, if the average number of molecules of the fluorescent probe in the measurement region of the measurement sample S is n, G (τ = 0) = 1 / n holds. Therefore, by performing waveform analysis on the autocorrelation function G (τ) and obtaining its y intercept, the average number of molecules n of the fluorescent probe can be obtained. In addition, since this average number of molecules does not change depending on the flow of the measurement sample S, regardless of actual fluorescence measurement conditions, for example, if the average number of molecules n is obtained from photon number measurement data without flow, good.

  Next, the average fluorescence intensity per unit time is obtained from the photon number measurement data obtained under actual fluorescence measurement conditions. Then, from the obtained average number of molecules n and the average fluorescence intensity, a fluorescence intensity (FPM: Fluorescence Intensity Per Molecule) per molecule is calculated, and further, this FPM value, the set bin time width, Thus, the average number of photons per molecule that is the reference number of photons in the bin width is obtained.

  For example, in the measurement example shown in FIG. 4, the average fluorescence intensity in the measurement without flow (graph A1) is 2.73 kcps, and the fluorescence probe in the measurement region obtained by applying fluorescence correlation spectroscopy is used. The average number of molecules was n = 0.15. Further, assuming that the actual fluorescence measurement condition is a flow (graph A2), the fluorescence intensity under that condition was 5.24 kcps. At this time, considering that the background is 300 cps, the fluorescence intensity per molecule of the fluorescent probe is obtained as FPM = 32.9 kcps.

  Further, with respect to the photon number measurement data under the fluorescence measurement conditions, if the binning bin width of the measurement data is set to 0.1 msec with reference to the graph D2 in FIG. The average number of photons is 3.29 counts. That is, when the bin width is set to 0.1 msec in the photon number measurement data, when one fluorescent probe molecule enters the measurement region in the measurement sample S, an average photon count value of 3.29 counts is obtained.

  Therefore, the number of fluorescent probes in the measurement region is analyzed by analyzing the number of photons in each bin in the data obtained by binning the photon number measurement data using the average photon number of 3.29 counts as the reference photon number. It is possible to perform a photon burst analysis for. For example, in the above example, in the photon number measurement data binned with a bin width of 0.1 msec, if the number of photons in the bin (the number of events counted) is 3 to 4 counts, one fluorescent probe is present in the measurement region. It can be estimated that it exists. If the number of photons in the bin is 6 to 7 counts, it can be estimated that two fluorescent probes are present in the measurement region. In addition, when there are three or more fluorescent probes, estimation can be performed by the same method.

  For example, when the fluorescence analysis is performed with the molecular system shown in FIG. 2 as the measurement target, the target molecule T can be quantitatively measured by performing the photon burst analysis by the above method. Further, even when two different fluorescent probes are involved in the target molecule, each is labeled with the same fluorescent dye, and the fluorescence analysis is performed by the above method using the same amount of the fluorescent probes as the target molecule. Quantification is possible.

  Such a method can be similarly applied when three or more different fluorescent probes are involved. Thus, when there are many fluorescent probes, the identification accuracy of the target molecule by photon burst analysis becomes high. For example, as described above, when the average number of fluorescent probes in the measurement region is as small as 0.15, two or more free fluorescent probes that have not reacted with the target molecule accidentally enter the measurement region. The probability of coming is low. Therefore, by performing photon burst analysis on the fluorescent probe and identifying the counting events of two or more fluorescent probes, it is possible to reliably acquire information on the target molecule. The above method can also be applied when a plurality of types of fluorescent probes labeled with different fluorescent dyes are involved.

  Specifically, various methods may be used for analyzing the photon number measurement data using the reference photon number set for one molecule of the fluorescent probe. As an example of such a method, for example, a method can be used in which a threshold for the number of photons is set for the measurement data based on the reference number of photons, and an event by the target molecule is identified by applying the threshold. Alternatively, the number of photons in each bin in the photon number measurement data is normalized by the reference photon number, and the number of fluorescent probes in the measurement region and its time change are evaluated with reference to the normalized photon number data obtained. The method can be used.

  Next, the fluorescence analysis method executed in the fluorescence analysis apparatus 1A shown in FIG. 1 will be further described along with specific examples thereof. 8 to 15 are flowcharts showing an example of the fluorescence analysis method according to the present invention.

  FIG. 8 is a flowchart illustrating an example of a method for acquiring photon number measurement data. The measurement method shown in FIG. 8 is used, for example, when acquiring measurement data for setting analysis conditions. In this method, first, a fluorescently labeled molecular probe used for fluorescence measurement is placed in the sample holder 10 as a measurement sample S (step S101). Then, the measurement sample S is measured under the condition of no flow in the fluorescence analyzer 1A shown in FIG. 1, and the photon counting unit 35 acquires the photon number measurement data (S102). The acquired measurement data is input to the measurement control unit 50 and stored in a memory or the like of the data storage unit (S103).

  Next, it is confirmed whether or not the fluorescence measurement is performed under the condition with the flow (S104). When it is necessary to perform measurement, a flow is imparted to the sample S by the reflux device 12 (S105), and fluorescence measurement (S106) and measurement data acquisition and storage (S107) are performed with the flow present. In this fluorescence analysis method, it is preferable that the excitation light intensity is the same in all measurements with respect to the intensity of the excitation light supplied from the excitation light source 22 at the time of fluorescence measurement. In addition, with respect to the detection of fluorescence by the photodetector 27, it is preferable to perform the measurement under conditions that are not more than the maximum count value so that the photodetector 27 is not saturated.

  Subsequently, the analysis condition setting unit 51 of the measurement control unit 50 sets analysis conditions necessary for performing photon burst analysis on the measurement data using the acquired photon number measurement data (S108). The setting of the analysis conditions here is performed using the measurement data when only the fluorescence measurement without a flow is performed. When fluorescence measurement is performed with and without a flow, analysis conditions are set using measurement data of both. If setting of analysis conditions is unnecessary, this step S108 does not need to be performed.

  FIG. 9 is a flowchart showing an example of the analysis condition setting method performed in step S108 of FIG. In this method, first, the number-of-molecules analysis is performed on the photon number measurement data acquired for setting the analysis conditions, and the average number of molecules n of the fluorescent probe in the measurement region of the measurement sample S is obtained (S121). ). Next, the bin width is analyzed, and the binning bin width used for the fluorescence analysis is set (S122).

  Further, the fluorescence intensity (FPM) per molecule of the fluorescent probe is calculated from the average number of molecules n obtained in step S121 and the average fluorescence intensity in the measurement data (S123), and this FPM value and in step S122. Based on the set bin width, the average number of photons per molecule corresponding to the reference photon number in the bin time width is obtained. Thereby, the bin width and the reference photon number of the analysis conditions of the photon burst analysis for the photon number measurement data are set (S124), the set analysis conditions are stored (S125), and the setting of the analysis conditions is finished.

  The molecular number analysis (fluorescence probe concentration analysis) in step S121 shown in the analysis condition setting method in FIG. 9 can be executed, for example, according to the procedure shown in FIG.

  In the analysis example of FIG. 10, first, photon number measurement data acquired under no-flow conditions is read from the data storage unit (S131), and fluorescence correlation spectroscopy is applied to the time-series measurement data to obtain an autocorrelation function G ( τ) is derived (S132). Then, waveform analysis of the autocorrelation function is performed by single component fitting calculation or the like (S133), and G (τ = 0) of the y-intercept is calculated (S134). As described above, the average number of molecules n of the fluorescent probe in the measurement region of the measurement sample S can be derived from the reciprocal of the value of G (0) (S135).

  Further, the bin width analysis (analysis on the time resolution of the measurement data) in step S122 shown in the analysis condition setting method of FIG. 9 can be executed by the procedure shown in FIG. 11, for example.

  In the analysis example of FIG. 11, first, photon number measurement data acquired under no flow or with flow conditions is read according to the fluorescence measurement conditions actually applied in sample analysis (S141), and bin width analysis is performed. Processing conditions for processing are read (S142). As processing conditions in this case, for example, when bin width analysis is performed using the evaluation amount dN / S shown in FIG. 7, a bin time width range (for example, from the initial bin width to about 1 second is executed). ), And the analysis interval and the number of analysis points. Such processing conditions may be prepared as, for example, a bin time width table for executing analysis.

  Next, data processing for the measured S / N ratio is performed on the photon number measurement data (S143). This measurement data process is a process for deriving a change due to the bin width of the measured S / N ratio S / N shown in the graph C1 of FIG. FIG. 12 is a flowchart illustrating an example of measurement data processing.

  In the measurement data processing shown in FIG. 12, first, the bin width (for example, the initial bin width of the photon number measurement data that is the minimum bin width) from which the measurement SN ratio is derived is set (S151). For the bin width, binning is performed on time-series measurement data as necessary (S152). Further, an average value of the number of measurement photons that becomes the measurement signal amount S and a standard deviation that becomes the measurement noise amount N are calculated from the obtained data (S153), and the measurement SN ratio S in the bin width is calculated from these values. / N is derived (S154).

  When the measurement SN ratio S / N is derived, it is confirmed whether the SN ratio derivation has been completed for all the bin widths necessary for the measurement data processing (S155). If not, the bin width is changed ( S156), the process for deriving the measured S / N ratio S / N is repeated. If the derivation of all S / N ratios has been completed, the measurement data processing ends. Specifically, for example, when analysis is performed with a bin width of 1 μs for a minimum bin width of 200 ns, the photon number measurement data is binned by integrating five consecutive bins, and measurement is performed from the obtained binning measurement data. An S / N ratio is determined.

  Returning to FIG. 11, when the measurement data processing is completed, data processing for the reference signal-to-noise ratio is further performed on the photon number measurement data (S144). This reference data process is a process for deriving a change due to the bin width of the reference S / N ratio S / N 'shown in the graph C0 of FIG. FIG. 13 is a flowchart illustrating an example of the reference data processing.

  In the reference data processing shown in FIG. 13, first, a reference bin width (for example, the minimum bin width) for determining the initial value of the reference SN ratio is set for the photon number measurement data, and the measurement signal amount S in the reference bin width is set. An average value of the number of photons to be measured and a standard deviation to be a measurement noise amount N are calculated (S161), and an SN ratio S with a reference bin width as an initial value of the reference SN ratio S / N ′ is calculated from these values. / N is derived (S162). Further, a bin width for subsequently deriving the reference SN ratio is set (S163), and the reference SN ratio S / N ′ based on the shot noise characteristic is derived based on the initial value for the bin width (S164).

  When the reference SN ratio S / N ′ is derived, it is confirmed whether or not the SN ratio derivation has been completed for all bin widths necessary for the reference data processing (S165). If not, the bin width is changed. (S166), the process for deriving the reference SN ratio S / N ′ is repeated. If the derivation of all S / N ratios has been completed, the reference data processing is terminated. Specifically, for example, when the analysis is performed with a bin width of 1 μs with respect to the reference bin width of 200 ns, the bin width is five times, so that the reference SN ratio is obtained by adding the square root of 5 to the initial value.

  Returning to FIG. 11, when the measurement data processing and the reference data processing are completed, a difference calculation is performed using the obtained measurement SN ratio S / N and reference SN ratio S / N ′, and bin width setting is performed. The evaluation amount dN / S = (N−N ′) / S is calculated (S145). Based on the change in the evaluation amount dN / S due to the bin width (see FIG. 7), the bin width used for the photon burst analysis is determined (S146).

  FIG. 14 is a flowchart showing another example of a method for acquiring photon number measurement data. The measurement method shown in FIG. 14 is used, for example, when acquiring measurement data for performing a fluorescence analysis on a measurement sample S including a target molecule and a fluorescent probe. In this method, first, an analysis target sample including a target molecule to be analyzed is placed in the sample holder 10 (S201), and a fluorescent probe is mixed and reacted with the target molecule to prepare a measurement sample S (S202). .

  Next, it is confirmed whether or not the fluorescence measurement is performed under the condition with the flow (S203). When the measurement is performed under the condition that the flow is applied to the sample S, the flow is applied to the sample S by the reflux device 12 (S204), and the fluorescence measurement is performed in this state to obtain the photon number measurement data (S205). . On the other hand, when the measurement is performed under the condition of no flow, the fluorescence measurement is performed without operating the reflux device 12 to obtain the photon number measurement data (S206).

  The acquired photon number measurement data is input to the measurement control unit 50 and stored in the data storage unit (S207). Furthermore, when it is necessary to continue to perform the fluorescence analysis on the obtained photon number measurement data, the analysis condition previously set by the analysis condition setting unit 51 in the measurement result analysis unit 52 of the measurement control unit 50 Fluorescence analysis is performed based on (S208).

  FIG. 15 is a flowchart illustrating an example of the measurement result analysis method performed in step S208 of FIG. In this method, first, photon number measurement data to be analyzed is read (S221), and analysis conditions set for photon burst analysis are read (S222). Next, the bin width set as the analysis condition by the analysis condition setting unit 51 is referred to, binning processing is performed on the photon number measurement data with the bin width (S223), and the reference photon is added to the measurement data after binning. The photon burst analysis is performed by applying the number (S224).

  This photon burst analysis will be described as an example in which an analysis is performed by setting a threshold for event identification based on the reference photon number. For example, a threshold is applied to the count value in each bin of the photon number data after binning. Then, the case where the number of measured photons exceeds the threshold is identified as one event that matches the burst condition, and the number of events is counted.

  Subsequently, it is confirmed whether or not the measurement data is for calibration (S225). If it is measurement data for calibration, a calibration curve (a calibration curve used in fluorescence analysis) is created from the result of photon burst analysis (S226). Here, in the fluorescence measurement for calibration, a sample having a known value such as the concentration of the target molecule to be analyzed is a measurement sample S, and fluorescence measurement and analysis are performed on a plurality of samples S having different concentrations. Then, a calibration curve serving as a calibration curve for estimating the amount of the target molecule is created by associating the concentration value of the target molecule with the number of events obtained by the photon burst analysis.

  In addition, when the measurement data is for the sample S to be analyzed because the concentration of the target molecule is unknown, the sample is determined based on the prepared calibration curve and the number of events obtained by the photon burst analysis. Necessary numerical values such as the reaction amount of the fluorescent probe in S or the concentration of the target molecule are calculated (S227). In creating the calibration curve described above, the threshold value in the photon burst analysis may be made variable, and the analysis condition threshold value may be adjusted and set so that an optimum calibration curve is obtained.

  The fluorescence analysis apparatus and fluorescence analysis method according to the present invention are not limited to the above-described embodiments and configuration examples, and various modifications are possible. For example, FIG. 1 shows an example of the configuration of an apparatus such as a fluorescence microscope used for fluorescence measurement. Specifically, various configurations may be used. In addition, regarding the application of the flow to the sample S, the above embodiment does not limit the direction of the flow, and the above configuration can be applied to any direction.

  Further, the sample S to be measured is not limited to the sample in which the target molecule and the fluorescent probe shown in FIG. 2 are mixed, and various materials including fluorescent substances can be used as long as photon burst analysis is generally applicable. The above configuration can be applied to the measurement sample. In addition, since the fluorescence analysis apparatus having the above configuration can realize a low running cost, it requires a large amount of processing in the clinical diagnosis field, the food inspection field, and further drug discovery, etc., where many tests need to be performed at a low cost. It can be applied to various fields such as the screening field.

  INDUSTRIAL APPLICABILITY The present invention can be used as a fluorescence analysis apparatus and a fluorescence analysis method that can facilitate fluorescence analysis of a measurement sample by measuring fluorescence from a fluorescent probe in a measurement region.

It is a figure which shows the structure of one Embodiment of a fluorescence analyzer. It is a figure which shows an example of the analysis object by a fluorescence analyzer. It is a figure which shows an example of a structure of the flow-path member used as a sample holder. It is a graph which shows the autocorrelation function obtained with respect to photon number measurement data. It is a graph which shows the bin width dependence of SN ratio in photon number measurement data. It is a graph which shows the bin width dependence of measurement SN ratio and reference | standard SN ratio. It is a graph which shows the bin width dependence of evaluation amount dN / S for bin width setting. It is a flowchart which shows an example of the acquisition method of photon number measurement data. It is a flowchart which shows an example of the analysis condition setting method. It is a flowchart which shows an example of a molecular number analysis. It is a flowchart which shows an example of a bin width analysis. It is a flowchart which shows an example of a measurement data process. It is a flowchart which shows an example of a reference | standard data process. It is a flowchart which shows the other example of the acquisition method of photon number measurement data. It is a flowchart which shows an example of the measurement result analysis method.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1A ... Fluorescence analysis apparatus, S ... Measurement sample (liquid sample), 10 ... Sample holder (channel member), 11 ... Sample channel, 12 ... Reflux device, 13 ... Channel body, 14 ... Introduction channel, 15 ... Discharge flow path, 16 ... main flow path (measurement flow path), 18 ... cover member, 20 ... objective lens, 21 ... dichroic mirror, 22 ... excitation light source, 23 ... reflection mirror, 24 ... optical filter, 25 ... imaging lens, 26: Pinhole, 27: Photodetector, 30: Detection signal processing unit, 35: Photon counting unit, 50: Measurement control unit, 51: Analysis condition setting unit, 52: Measurement result analysis unit.

Claims (12)

Excitation light irradiation means for irradiating excitation light to the measurement region set for the measurement sample;
Fluorescence detection means for detecting fluorescence generated from a fluorescent probe in the measurement region of the measurement sample irradiated with the excitation light by the excitation light irradiation means;
Based on a detection signal output from the fluorescence detection means, a photon counting means for counting the number of photons detected by the fluorescence detection means in a time series with a predetermined counting time width;
Analysis condition setting means for setting the analysis conditions for fluorescence analysis with respect to time-series photon number measurement data acquired by the photon counting means;
Based on the analysis condition set by the analysis condition setting means, comprising a measurement result analysis means for performing fluorescence analysis on the photon number measurement data indicating the measurement result for the measurement sample including the fluorescent probe,
The analysis condition setting means sets, as the analysis condition, a bin width for binning the photon number measurement data, and a reference photon number for one fluorescent probe molecule in the bin width,
The measurement result analyzing unit bins the photon number measurement data with the bin width set by the analysis condition setting unit, and refers to the reference photon number, and the fluorescent probe in the measurement region of the measurement sample Fluorescence analysis of the number of
The analysis condition setting means includes a change in the measured SN ratio, which is an SN ratio of the number of photons in the photon number measurement data, according to a bin width, and a reference SN ratio that is an SN ratio of the number of photons when a shot noise characteristic is assumed. The fluorescence analyzer characterized by setting the said bin width using the change by a bin width. The analysis condition setting means obtains a measurement noise amount N in the photon number measurement data and a reference noise amount N ′ assuming a shot noise characteristic with respect to the measurement signal amount S in the photon number measurement data. , An evaluation amount for bin width setting defined using the measured SN ratio S / N and the reference SN ratio S / N ′ dN / S = (N−N ′) / S
The fluorescence analyzer according to claim 1, wherein the bin width is set with reference to FIG.   The fluorescence analysis apparatus according to claim 2, wherein the analysis condition setting means sets the bin width according to a time width in which the evaluation amount dN / S is maximized.   The analysis condition setting means sets a reference bin width with respect to a change in the measurement noise amount N due to the bin width, and uses the measurement noise amount N in the reference bin width as an initial value, depending on the bin width of the reference noise amount N ′. 4. The fluorescence analyzer according to claim 2, wherein a change is obtained.   The analysis condition setting means is based on the autocorrelation function waveform obtained from the photon number measurement data by fluorescence correlation spectroscopy, the fluorescence intensity per unit time measured by the photon number measurement data, and the bin width. The fluorescence analysis apparatus according to claim 1, wherein the reference photon number in the bin width is set.   The fluorescence analysis apparatus according to claim 1, further comprising a flow applying unit that applies a flow to the measurement sample including the fluorescent probe. An excitation light irradiation step for irradiating the measurement region set for the measurement sample with excitation light;
A fluorescence detection step of detecting fluorescence generated from a fluorescent probe in the measurement region of the measurement sample irradiated with the excitation light in the excitation light irradiation step;
Based on the detection signal acquired in the fluorescence detection step, a photon counting step for counting the number of photons detected in the fluorescence detection step in a time series with a predetermined counting time width,
An analysis condition setting step for setting the analysis conditions for fluorescence analysis for time-series photon number measurement data acquired in the photon counting step;
Based on the analysis condition set in the analysis condition setting step, comprising a measurement result analysis step for performing fluorescence analysis on the photon number measurement data indicating the measurement result for the measurement sample including the fluorescent probe,
The analysis condition setting step sets, as the analysis condition, a bin width for binning the photon number measurement data, and a reference photon number for one fluorescent probe molecule in the bin width,
The measurement result analyzing step bins the photon number measurement data with the bin width set in the analysis condition setting step, refers to the reference photon number, and the fluorescent probe in the measurement region of the measurement sample Fluorescence analysis of the number of
In the analysis condition setting step, the change in the measured SN ratio, which is the SN ratio of the photon number in the photon number measurement data, due to the bin width, and the reference SN ratio which is the SN ratio of the photon number when the shot noise characteristic is assumed A fluorescence analysis method, wherein the bin width is set using a change due to a bin width. In the analysis condition setting step, a measurement noise amount N in the photon number measurement data and a reference noise amount N ′ assuming a shot noise characteristic are obtained with respect to the measurement signal amount S in the photon number measurement data. , An evaluation amount for bin width setting defined using the measured SN ratio S / N and the reference SN ratio S / N ′ dN / S = (N−N ′) / S
The fluorescence analysis method according to claim 7, wherein the bin width is set with reference to FIG.   The fluorescence analysis method according to claim 8, wherein, in the analysis condition setting step, the bin width is set according to a time width in which the evaluation amount dN / S is maximized.   In the analysis condition setting step, a reference bin width is set for a change in the measurement noise amount N due to a bin width, and the measurement noise amount N in the reference bin width is set as an initial value, depending on the bin width of the reference noise amount N ′. The fluorescence analysis method according to claim 8 or 9, wherein a change is obtained.   In the analysis condition setting step, the autocorrelation function waveform obtained from the photon number measurement data by fluorescence correlation spectroscopy, the fluorescence intensity per unit time measured by the photon number measurement data, and the bin width The fluorescence analysis method according to claim 7, wherein the reference photon number in the bin width is set.   The fluorescence analysis method according to any one of claims 7 to 11, further comprising a flow applying step of applying a flow to the measurement sample including the fluorescent probe.

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