JP2009281831A - Fluorescence analyzer and analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence analyzer and an analysis method capable of executing properly fluorescence analysis of a sample. <P>SOLUTION: This analyzer 1A is constituted of: an excitation light source 22 for irradiating excitation light to a measuring domain of the sample S; a beam splitter 30 for branching fluorescence from a fluorescence probe in the measuring domain; first and second photodetectors 31, 32 for detecting branched fluorescence components respectively; a photon counting part 35 for counting in time series, the number of photons detected respectively by the photodetectors 31, 32, and acquiring first and second measurement data F<SB>A</SB>(t), F<SB>B</SB>(t); and a measuring control part 50 including an analysis data generation part 51 and a measurement result analysis part 52. The generation part 51 generates photon number analysis data n<SB>AB</SB>(t) wherein, in the case of F<SB>A</SB>×F<SB>B</SB>=0, n<SB>AB</SB>=0, and in the case of F<SB>A</SB>×F<SB>B</SB>>0, n<SB>AB</SB>=f(F<SB>A</SB>, F<SB>B</SB>) in terms of a prescribed function f, and the analysis part 52 performs fluorescence analysis relative to the analysis data. <P>COPYRIGHT: (C)2010,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 a measurement region by irradiating a minute region in a measurement sample solution containing an extremely low concentration of a fluorescent substance with excitation light, and measuring the intensity of fluorescence generated in the measurement region. Information such as translational diffusion motion of the fluorescent substance in the sample is obtained by measuring in time series and obtaining the autocorrelation function (see, for example, Patent Documents 1 to 3). Such a fluorescence analysis method can be applied to, for example, immunoassay in clinical diagnosis.
JP 2007-20565 A JP 2007-316017 A JP 2006-17628 A

  In the fluorescence analysis described above, a molecular probe (fluorescent probe) labeled with a fluorescent substance is added to the measurement sample, and the fluorescence from the fluorescent probe in the measurement region is detected. Get information. 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 fluorescence analysis using a technique such as FCS, further improvement in measurement sensitivity, efficiency, and the like is required. As one of such methods, a fluorescence analysis method has been proposed in which a plurality of fluorescent probes are bound to a target molecule so as to aggregate and the fluorescence from these fluorescent probes is detected (for example, Patent Document 1). 2). However, such a method has a problem that it is difficult to distinguish a fluorescence detection event from a target molecule to which a plurality of fluorescent probes are bound from other noise events.

  The present invention has been made to solve the above-described problems, and is capable of suitably executing a fluorescence analysis of a measurement sample by measuring fluorescence from a fluorescent probe in a measurement region. An object is to provide an apparatus and 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. Light branching means for branching the fluorescence generated from the fluorescent probe in the measurement region of the measurement sample irradiated with the excitation light into two fluorescent components, and (3) detecting the first branched first fluorescent component. 1 fluorescence detection means, (4) second fluorescence detection means for detecting the other branched second fluorescence component, and (5) the number of photons detected by the first fluorescence detection means and the second fluorescence detection means, respectively. (6) first photon counting means for obtaining first photon number measurement data F _A (t) and second photon number measurement data F _B (t) by counting in a time series with a predetermined counting time width; number of photons measured data F _{A (t)} and the second photon number measurement data Based on the _B (t), the measured data _{F A (t), n AB} (t) = 0 in the case of at least one of 0 _F B (t)
When both of the measurement data F _A (t) and F _B (t) are not 0, n _AB (t) = f (F _A (t), F _B (t)) with a predetermined function f
And (7) the photon number analysis data n _AB (t) generated by the analysis data generation means, the analysis data generation means for generating the photon number analysis data n _AB (t) And a measurement result analyzing means for performing a fluorescence analysis on the measurement result.

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 light branching step for branching fluorescence generated from a fluorescent probe in a measurement region of the sample into two fluorescent components; (3) a first fluorescence detecting step for detecting one branched first fluorescent component; ) A second fluorescence detection step for detecting the other branched second fluorescence component; and (5) time series of the number of photons detected in the first fluorescence detection step and the second fluorescence detection step in a predetermined counting time width. Counting to obtain first photon number measurement data F _A (t) and second photon number measurement data F _B (t), and (6) first photon number measurement data F _A (t ) And second photon number measurement data Based on the _B (t), the measured data _{F A (t), n AB} (t) = 0 in the case of at least one of 0 _F B (t)
When both of the measurement data F _A (t) and F _B (t) are not 0, n _AB (t) = f (F _A (t), F _B (t)) with a predetermined function f
An analysis data generation step for generating photon number analysis data n _AB (t) to be (7), and (7) for the photon number analysis data n _AB (t) generated in the analysis data generation step, And a measurement result analysis step for performing fluorescence analysis on the measurement result.

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. Further, a light branching means is provided for the fluorescence generated by the fluorescent probe in the measurement region, and the first and second fluorescence detection means are provided as the fluorescence detection system to provide the first and second photon number measurement data F _A ( t) and F _B (t) are acquired. Then, instead of directly using these measurement data for the fluorescence analysis, the analysis data n _AB (t) that becomes 0 when F _A (t) · F _B (t) = 0 is generated to perform the fluorescence analysis. Is going.

In such a configuration, by using the photon number analysis data n _AB (t) instead of the photon number measurement data F _A (t) and F _B (t), fluorescence detection by the first and second fluorescence detection means is performed. Fluorescence analysis can be executed in consideration of the simultaneity. This makes it possible to improve the accuracy of fluorescence analysis of the measurement sample, for example, by improving the accuracy of distinguishing fluorescence detection events from target molecules to which multiple fluorescent probes are bound from other noise events. Become.

  Here, with regard to excitation light and fluorescence used for fluorescence analysis on the measurement sample, the excitation light is excitation light in a single wavelength region, and the first fluorescence component and the second fluorescence component are fluorescence components in the same wavelength region. Can be used. Such a configuration is suitably used for fluorescence analysis when only one type of fluorescent probe is used as a fluorescent probe to be added to a measurement sample, and when two or more same types of fluorescent probes are bound to target molecules contained in the sample. can do.

Regarding data conversion from photon number measurement data to analysis data, specifically, for example, in the generation of analysis data, when both measurement data F _A (t) and F _B (t) are not 0, measurement data F _A (T), a function f that is a geometric mean of F _B (t), n _AB (t) = √ (F _A (t) · F _B (t))
It is preferable to generate the photon number analysis data n _AB (t) by Thus, by calculating | requiring analysis data using a geometric mean, the fluorescence analysis in consideration of the simultaneousness of fluorescence detection can be performed suitably. In this method, even when at least one of the measurement data F _A and F _B is 0, the above function √ (F _A · F _B ) of the geometric mean is n _AB = 0.

Alternatively, in the analysis data generation, when both of the measurement data F _A (t) and F _B (t) are not 0, the function f is a harmonic average of the measurement data F _A (t) and F _B (t) n _AB (t) = 2 / {1 / F _A (t) + 1 / F _B (t)}
A configuration for generating the photon number analysis data n _AB (t) may be used. As described above, by obtaining the analysis data using the harmonic average, similarly to the case of the geometric average, it is possible to suitably execute the fluorescence analysis in consideration of the synchronization of the fluorescence detection.

Specifically, for fluorescence analysis using photon number analysis data, at least one of autocorrelation analysis or photon burst analysis is performed as fluorescence analysis on photon number analysis data n _AB (t) in the measurement result analysis. It is preferable. Thus, by using the autocorrelation analysis method, the photon burst analysis method, or an analysis method combining them, the fluorescence analysis of the measurement sample can be performed with high accuracy.

In the measurement result analysis, the photon number analysis data n _AB (t) may be binned with a predetermined bin time width, and the fluorescence analysis may be performed on the binned analysis data. In this case, the accuracy of the fluorescence analysis can be improved by setting the bin time width of the analysis data according to specific measurement conditions and the like.

According to the fluorescence analysis apparatus and the analysis method of the present invention, the light branching means is provided for the fluorescence from the fluorescent probe in the measurement region set in the sample, and the first and second fluorescence detection means are provided. The first and second photon number measurement data F _A (t), F _B (t) are obtained, and the analysis data n _AB (t) is such that n _AB = 0 when at least one of the measurement data is 0 By performing the fluorescence analysis by generating the fluorescence analysis, it is possible to improve the accuracy of the fluorescence analysis of the measurement sample by executing the fluorescence analysis in consideration of the simultaneous detection of the fluorescence by the two detection means.

  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, acquires time-series photon number measurement data, and obtains measurement data obtained. The information about the measurement sample S is acquired by performing a predetermined fluorescence analysis on the sample. In the following, a fluorescent substance (including a molecule labeled with a fluorescent substance) in the 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 solution-like measurement sample S to be subjected to fluorescence analysis.

  In such a sample S, when the fluorescent probes P1 and P2 are the same type of molecular probe, or when the fluorescent substances of the fluorescent probes P1 and P2 are the same type of substance, the fluorescence generated in the sample S has a single wavelength. The region becomes fluorescent. In the following, it is assumed that a plurality of fluorescent probes of the same type bind to the target molecule T as a main target of fluorescence analysis. Hereinafter, the configuration of the analysis apparatus 1A according to the present embodiment will be described together with the fluorescence analysis method.

  A fluorescence analysis apparatus 1A shown in FIG. 1 includes a sample holder 10, an objective lens 20, a dichroic mirror 21, an excitation light source 22, an optical filter 23, a reflection mirror 24, an imaging lens 25, and a pinhole 26. The collimating lens 27, the beam splitter 30, and the photodetectors 31 and 32 are provided, and each of these elements constitutes a fluorescence microscope section in the fluorescence analyzer 1A. In addition to the elements of the fluorescence microscope unit, the fluorescence analysis apparatus 1A includes detection signal processing units 33 and 34, 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 that functions as a slide glass. An objective lens 20 is installed at a predetermined position with respect to the sample holder 10. 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.

  In the configuration shown in FIG. 1, the objective lens 20, the dichroic mirror 21, and the excitation light source 22 constitute excitation light irradiation means for irradiating the measurement region of 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 light supplied from the excitation light source 22 is reflected by the dichroic mirror 21 and is collected as a beam spot while being condensed via the objective lens 20 (excitation light irradiation). Step). As an 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, the objective lens 20, the optical filter 23, the reflection mirror 24, the imaging lens 25, the pinhole 26, the collimating lens 27, the beam splitter 30, the first photodetector 31, and the second optical detection. The device 32 constitutes fluorescence detection means. The fluorescence of the measurement sample S from the fluorescent probe is collected by the objective lens 20, passes through the dichroic mirror 21, passes through the optical filter 23, and then is reflected toward the beam splitter 30 by the reflection mirror 24. As the optical filter 23, 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. .

  The fluorescence whose optical path has been changed by the reflection mirror 24 passes through the imaging lens 25 and the pinhole 26 in order, is converted into a parallel light beam by the collimating lens 27, and then enters the beam splitter 30. 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 beam splitter 30 is a light branching unit that branches the fluorescence, and branches the fluorescence from the fluorescent probe in the measurement region of the measurement sample S irradiated with the excitation light into two fluorescent components (light branching step). In other words, the beam splitter 30 guides each photon of the fluorescence from the sample S to either the optical path to the first photodetector 31 or the optical path to the second photodetector 32, thereby Branches into fluorescent component. As such a beam splitter 30, for example, a beam splitter that equally divides fluorescence into two fluorescent components with a 50:50 branching ratio is used.

  One of the first fluorescence components branched by the beam splitter 30 (photons transmitted through the beam splitter 30) is detected by the photodetector 31 constituting the first fluorescence detection means, and a detection signal is output (first fluorescence detection). Step). The other second fluorescence component branched by the beam splitter 30 (photons reflected by the beam splitter 30) is detected by the photodetector 32 constituting the second fluorescence detection means, and a detection signal is output (first signal). 2 fluorescence detection step). As a specific example of the photodetector, a photomultiplier tube can be used. Each optical element from the optical filter 23 to the lens 27 is shared by the first and second fluorescence detecting means.

  The above-described excitation light irradiation means and fluorescence detection means constitute a fluorescence microscope using a confocal optical system that sets a minute measurement region in the sample S, irradiates excitation light, and detects fluorescence from the measurement region. . Further, the fluorescence microscope of the present embodiment is configured to include a light branching unit and two corresponding fluorescence detection units for the fluorescence from the sample S.

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. Further, when an example of the molecular concentration of the fluorescent probe contained in the sample S 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 an example of the setting of the measurement area and the like, and the specific setting differs in each case.

  Detection signals output from the first and second photodetectors 31 and 32 are input to the first and second detection signal processing units 33 and 34, respectively. The detection signal processing units 33 and 34 are configured by a signal amplification circuit such as a preamplifier and a signal processing circuit such as a wave height discriminator, for example, and perform predetermined signal processing on the detection signals from the photodetectors 31 and 32. A detection pulse signal sequence indicating a single photon detection event in each of the photodetectors 31 and 32 is generated and output.

Detection pulse signal sequences from the signal processing units 33 and 34 are input to the photon counting unit 35. The photon counting unit 35 is a counting unit that acquires measurement data used for fluorescence analysis, and determines the number of detected photons based on detection signals from the photodetectors 31 and 32 (signal trains from the processing units 33 and 34). The time-series first and second photon number measurement data F _A (t) and F _B (t) are generated (photon counting step). Thereby, the fluorescence measurement by the single photon counting with respect to the sample S is attained. Here, in the following, the amount related to the detection result in the first photodetector is indicated by the subscript “A”, and the amount related to the detection result in the second photodetector is indicated by the subscript “B”.

  In the analysis apparatus 1A, the counting time width in the photon counting unit 35 is the initial condition (minimum bin width) of the bin width of the fluorescence analysis. As the photon counting unit 35, specifically, for example, a multi-channel scaler (MCS) can be used. Further, the time resolution of the MCS that is the counting time width in the photon counting unit 35 is, for example, 200 nsec (nanoseconds) or less.

The time-series photon number measurement data F _A (t) and F _B (t) generated by the photon counting unit 35 are input to the measurement control unit 50, and the measurement control unit 50 performs fluorescence analysis on the measurement data. Done. The measurement control unit 50 according to the present embodiment includes an analysis data generation unit 51 and a measurement result analysis unit 52. 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 data generation unit 51 generates photon number analysis data n _AB (t) used for fluorescence analysis from the photon number measurement data F _A (t), F _B (t) (analysis data generation step). Specifically, the generation unit 51 sets n _AB (t) when at least one of the measurement data is 0 (F _A · F _B = 0) based on the measurement data F _A (t) and F _B (t). ) = 0
When both of the measurement data are not 0 (F _A · F _B > 0), n _AB (t) = f (F _A (t), F _B (t)) with a predetermined function f
The photon number analysis data n _AB (t) is generated.

によって解析データｎ_ＡＢ（ｔ）を生成する方法を用いることができる。なお、相乗平均の場合、その定義式で測定データの少なくとも一方が０の場合にｎ_ＡＢ＝０となる条件が満たされるため、その場合も含めて上記式（１）で解析データが定義される。 As specific photon number analysis data, preferably, the geometrical average of the measurement data F _A (t) and F _B (t) is used, and the following formula (1)

A method for generating analysis data n _AB (t) can be used. In the case of the geometric mean, since the condition that n _AB = 0 is satisfied when at least one of the measurement data is 0 in the definition formula, the analysis data is defined by the formula (1) including that case. .

The measurement result analysis unit 52 is an analysis unit that performs necessary fluorescence analysis. The analysis result n _AB (t) generated by the generation unit 51 is subjected to fluorescence analysis on the measurement result of the measurement sample S including the fluorescent probe. Perform (measurement result analysis step). Thereby, for example, necessary information about the measurement sample S such as information on the target molecule in the 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, the sample S is minutely formed by the excitation light irradiation system and the fluorescence detection system including the objective lens 20, the excitation light source 22, the photodetectors 31, 32, and the like. Set the measurement area. Further, a beam splitter 30 is provided for the fluorescence generated by the fluorescence probe in the measurement region, and first and second fluorescence detection means are installed as a fluorescence detection system, so that the first photon number measurement data F _A (t) and Second photon number measurement data F _B (t) is acquired. Then, from the measurement data F _A (t), F _B (t), the analysis data n _AB (t) that generates n _AB (t) = 0 when F _A (t) · F _B (t) = 0 is generated. And fluorescence analysis.

In such a configuration, by using photon number analysis data n _AB (t) instead of the photon number measurement data F _A (t) and F _B (t), the first and second photodetectors 31 and 32 use them. Fluorescence analysis can be executed in consideration of the simultaneous detection of fluorescence. Thereby, for example, it is possible to improve the accuracy of fluorescence analysis of the sample S, such as improving the discrimination accuracy between fluorescence detection events from target molecules to which a plurality of fluorescent probes are bound and other noise events. Become.

  Regarding the excitation light and fluorescence in the fluorescence analysis, it is possible to use a configuration in which the excitation light is excitation light in a single wavelength region, and the first and second fluorescence components are fluorescence components in the same wavelength region. As described above with reference to FIG. 2, such a configuration uses only one type of fluorescent probe as a fluorescent probe to be added to the measurement sample S, and two or more same types of fluorescent probes are present on the target molecule contained in the sample S. It can apply suitably for the fluorescence analysis in the case of combining. Moreover, in the analysis apparatus 1A of FIG. 1, the single excitation light source 22 is used corresponding to such an analysis object.

  Here, in the method using two or more types of fluorescent probes as the fluorescent probes, it is necessary for a plurality of types of fluorescent probes to react equally, and it is difficult to select a probe. In addition, for example, there may be a problem such as a mismatch of measurement regions for each type of fluorescent probe (see Patent Document 3). On the other hand, the fluorescence analysis method using one type of fluorescent probe is free from these problems and can be easily measured, and the accuracy of fluorescence analysis can be improved.

The specific details of the fluorescence analysis using the number of photons analysis data, the analyzing unit 52, to the number of photons analysis data n _AB, it is preferable that at least one of the self-correlation analysis or photon burst analysis. Thus, the fluorescence analysis of the sample S can be performed with high accuracy by using the autocorrelation analysis method, the photon burst analysis method, or the analysis method combining them. Further, the analysis unit 52 may be configured to bin the analysis data n _AB with a predetermined bin time width and perform fluorescence analysis on the binned analysis data. In this case, the accuracy of the fluorescence analysis can be improved by setting the bin time width of the analysis data according to specific measurement conditions and the like.

  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.

によって求めることができる。 First, general fluorescence analysis using the photon number measurement data F _A (t) and F _B (t) will be described. When one type of fluorescent probe is added to the sample S and measured, the autocorrelation function G (τ) based on the measurement results of the photodetectors 31 and 32 is as follows for the photon number measurement data F _A and F _B , Equation (2), Equation (3)

Can be obtained.

によって求められる。ここで、これらの相関関数Ｇ_ＡＢ（τ）、Ｇ_ＢＡ（τ）の対称性

を考慮すると、下記の式（７）で定義される関数Ｇ_Ｃ（τ）＝Ｇ_{ＣＲＯＳＳ}（τ）

を光検出器３１、３２間の相互相関関数として用いることができる。 Further, the cross-correlation function between the photodetectors 31 and 32 is expressed by the following equations (4) and (5).

Sought by. Here, the symmetry of these correlation functions G _AB (τ), G _BA (τ)

Is considered, the function G _C (τ) = G _CROSS (τ) defined by the following equation (7)

Can be used as a cross-correlation function between the photodetectors 31 and 32.

によって求められる。ここで、＜Ｎ＞は試料Ｓで共焦点光学系によって構築される測定領域内に滞在する蛍光プローブの平均分子数を示し、また、τ_Ｄは拡散時定数、ｒ_０、ｚ_０は構造パラメータ（ストラクチャパラメータ）、Ｔは三重項状態へと遷移する割合、λは三重項遷移の時定数をそれぞれ示している。これらのパラメータは、取得された相関関数に対してフィッティング計算による波形解析を行うことで求めることができる。 In general, in one-component waveform analysis in fluorescence correlation spectroscopy, the correlation function G (τ) for both autocorrelation and cross-correlation is expressed by the following equation (8).

Sought by. Here, <N> indicates the average number of molecules of the fluorescent probe staying in the measurement region constructed by the confocal optical system in the sample S, τ _D is a diffusion time constant, and r ₀ and z ₀ are structural parameters. (Structure parameter), T represents the rate of transition to the triplet state, and λ represents the time constant of the triplet transition. These parameters can be obtained by performing waveform analysis by fitting calculation on the acquired correlation function.

The analysis method using the equations (7) and (8) is a conventional method, but the analysis apparatus 1A shown in FIG. 1 that uses the analysis data n _AB without directly using the measurement data also uses such an analysis method. Analyzing parameters such as the average number of molecules, translational diffusion time constant, triplet, etc. is suitable for grasping the initial state of the fluorescent probe. If the transition probability to the triplet state is T = 0 in the equation (8), G (τ = 0) = 1 / <N> for the y-intercept of G (τ) when the time delay τ = 0.
The relationship is obtained. That is, the average number of molecules <N> in the measurement region corresponding to the concentration of the fluorescent probe in the sample S is obtained from the y intercept of the correlation function G (τ).

Here, since the cross-correlation function G _C (τ) shown in the equation (7) is obtained using one kind of fluorescent probe, the waveform analysis can be performed by the equation (8). Such a fluorescence analysis method has an advantage that noise of the photodetector can be canceled by cross-correlation. However, in the cross-correlation using two photodetectors as described above, the S / N ratio is reduced to 1 / √2 times as compared with the case of one photodetector.

Next, fluorescence analysis using photon number analysis data n _AB (t) will be described. Here, in the analysis apparatus 1A of FIG. 1, when only one fluorescent probe is present in the measurement region of the sample S, fluorescence is not detected simultaneously by the photodetectors 31 and 32. When fluorescence is detected simultaneously by the photodetectors 31 and 32, it is assumed that a plurality of fluorescent probes are always present in the measurement region.

In this case, analysis data n _AB (t) by the geometric mean shown in the above formula (1).
n _AB (t) = √ (F _A (t) · F _B (t))
By using, it is possible to eliminate a detection event by one fluorescent probe from the viewpoint of synchronization of photon detection. Further, in this analysis data, for the evaluation of simultaneity, the analysis data is obtained without being excluded even when one or both of the measurement data F _A and F _B are 0 and n _AB = 0. Further, the above analysis data based on the geometric mean can be particularly preferably applied to fluorescence analysis when a beam splitter having a branching ratio of 1: 1 is used as the light branching means.

となる。なお、相乗平均による解析データは実数データであるため、この式（９）の相関関数を求める演算も実数演算となる。このような演算を含む蛍光解析は、測定制御部５０でソフトウェア的に解析演算を行う構成において好適に実現することできる。 When this analysis data n _AB is used as time-series photon number data, an autocorrelation function G _new (τ) taking into account the coincidence of fluorescence detection is obtained, the following equation (9)

It becomes. Since the analysis data based on the geometric mean is real number data, the calculation for obtaining the correlation function of Equation (9) is also a real number calculation. The fluorescence analysis including such calculation can be suitably realized in a configuration in which the measurement control unit 50 performs analysis calculation in software.

In the autocorrelation function of Equation (9), when the bin time width of the photon number data is as small as 1 μsec, for example, there is a high probability that n _AB = 0, and thus a correlation waveform cannot be obtained. On the other hand, when the bin time width of the analysis data n _AB is increased by binning, the probability of n _AB = 0 gradually decreases and a correlation waveform appears. That is, the above correlation waveform G _new (τ) indicates that when an arbitrary bin time width is set for the photon number data acquired by the two photodetectors 31 and 32, the photons are simultaneously within the bin width. This indicates the degree of detection.

In the autocorrelation function G (τ), as described above, the y-intercept corresponds to the reciprocal 1 / <N> of the average number of molecules of the fluorescent probe. Therefore, when there are many fluorescent probes in the measurement region of the sample S, the probability that they are simultaneously observed increases and the y-intercept of the correlation waveform decreases. On the other hand, when there are few fluorescent probes, the probability of simultaneous observation is reduced and the y-intercept is increased. The correlation function G _new (τ) taking into account the synchronism of fluorescence detection varies depending on the above characteristics with respect to a general correlation function G _C (τ).

As for the translational diffusion movement of the fluorescent probe, the same movement is observed in any correlation function. For this reason, also in the waveform analysis of the correlation function G _new (τ) in Expression (9), the parameters of the diffusion time constant τ _D , the transition ratio T, and the time constant λ obtained for the cross correlation function G _C (τ) Can be applied with the same parameter values.

In the derivation of the correlation function G _new (τ), the bin time width of the analysis data n _AB is appropriately set with respect to the concentration of the fluorescent probe, thereby staying in the measurement region due to the limitation due to the simultaneous detection of the fluorescence. It is possible to estimate the apparent number of molecules small. This is because the frequency of simultaneously detecting fluorescence from a plurality of fluorescent probes in the measurement region with two photodetectors varies depending on the size of the bin time width. Regarding the bin time width, it is preferable to set and change the bin time width by performing binning as necessary for the photon number data acquired using the counting time width in the photon counting unit 35 as the initial bin width.

The above-mentioned property of the fluorescence analysis using the analysis data n _AB is particularly effective for the fluorescence analysis in the case where a plurality of fluorescent probes are bound to the target molecule so as to aggregate. In this case, increasing the number of fluorescent probes added to the sample S increases the fluorescence detection events due to the target molecules to which a plurality of fluorescent probes are bound. In addition, since the translational diffusion movement is changed by the binding of a plurality of fluorescent probes to the target molecule, the ability to detect the target molecule is improved in the fluorescence analysis by FCS. As for free fluorescent probes, even when multiple probes stay in the measurement area, the stay timing is shifted and the simultaneity is reduced, so the event due to free fluorescent probes is effectively excluded by setting the bin time width. Is possible.

によって求められる。ここで、式（１０）、式（１１）において、添え字ｉは標的分子に対する蛍光プローブの結合状態が異なる場合の各蛍光成分を示している。また、αは蛍光の発光効率に関する係数である。また、式（１１）の相関波形成分では、三重項に関する波形部分を省略している。 In the fluorescence analysis by FCS in the case where a plurality of fluorescent probes are bound to the target molecule, the correlation function G (τ) is expressed by the following equations (10) and (11).

Sought by. Here, in the expressions (10) and (11), the subscript i indicates each fluorescent component when the binding state of the fluorescent probe to the target molecule is different. Α is a coefficient relating to the luminous efficiency of fluorescence. Further, in the correlation waveform component of Expression (11), the waveform portion related to the triplet is omitted.

となる。ここで、添え字ｉ＝１はフリーの蛍光プローブによる蛍光成分を示し、ｉ＝２は標的分子に蛍光プローブが１個結合した場合の蛍光成分を示し、ｉ＝３は標的分子に蛍光プローブが２個結合した場合の蛍光成分を示している。また、この場合の蛍光プローブの総分子数＜Ｎ＞は、下記の式（１３）

によって求められる。 When only one type of molecular probe is used as the fluorescent probe, and the measurement data acquired by the analysis apparatus 1A of FIG. 1 is analyzed by the cross-correlation function G _C (τ) of Expression (7), the coefficient α _i is a positive integer. The average number of molecules <N _i > is determined accordingly. For example, when only up to two fluorescent probes bind to the target molecule, the correlation function G (τ) of equation (10) is expressed by the following equation (12):

It becomes. Here, the subscript i = 1 indicates the fluorescent component by the free fluorescent probe, i = 2 indicates the fluorescent component when one fluorescent probe is bound to the target molecule, and i = 3 indicates that the fluorescent probe is present on the target molecule. The fluorescence component when two are bonded is shown. In this case, the total number of molecules <N> of the fluorescent probe is expressed by the following formula (13).

Sought by.

Using these equations (12) and (13), the coefficient of each correlation waveform component g _i (τ) is obtained for the cross-correlation function G _C (τ) of equation (7), so that Information about the state, the amount of the target molecule, and the like can be acquired. However, in such a fluorescence analysis, if the amount of target molecules is small and there are many free fluorescent probes, the correlation waveform will be represented mainly by the fluorescent component of the free fluorescent probes, and information about the target molecules will be displayed. It becomes difficult to obtain with sufficient accuracy.

In such a case, information on the target molecule is suitably obtained by using the correlation function G _new (τ) based on the analysis data n _AB instead of the correlation function G _C (τ) based on the measurement data F _A and F _B. be able to. That is, in the correlation function G _new (τ) taking into account the synchronism of fluorescence detection, the number of free fluorescent probes and only one fluorescent probe bound to the target molecule is estimated to be low. <N ₃ > can be quantified with high accuracy.

Further, the photon number analysis data n _AB (t) itself shown in the equation (1) corresponds to the fluorescence fluctuation when a plurality of fluorescent probes stay in the measurement region of the sample S at the same time. For this reason, in addition to the autocorrelation analysis described above, the photon burst analysis (analysis of the number of detected photons in the photon number data) is also performed on the analysis data to obtain information on the target molecule. It is possible to obtain. The photon burst analysis of the analysis data will be specifically described later.

Here, in the homogeneous assay using the specific binding of the fluorescent probe P to the target molecule T (see FIG. 2), specifically, for example, for the sample S containing the target molecule, (1) the fluorescent probe before the reaction The fluorescence analysis is performed in three steps: (2) a reaction step between the target molecule and the fluorescent probe, and (3) a fluorescence evaluation step after the reaction. In this case, in the evaluation step (1) before the reaction, the cross-correlation function G _C (τ) of Equation (7) is obtained, and the waveform analysis is performed by applying Equation (8) to determine the necessary parameter values. . Further, analysis is performed using the analysis data of Equation (1) and the correlation function G _new (τ) of Equation (9) to obtain the initial value of the state before the reaction, and fluorescence that is important in evaluating simultaneity. Set the bin time width for analysis.

  In the evaluation step (3) after the reaction, the analysis is performed according to the equations (1) and (9) by applying the previously obtained bin time width for fluorescence analysis, and further the equation ( 10) The amount of the target molecule in the sample S can be estimated by performing a waveform analysis using Equation (11). Alternatively, instead of waveform analysis, photon burst analysis may be performed on the analysis data of Equation (1). The bin time width setting method and the like will be specifically described later.

によって求められるが、時間遅れτが例えば１００ｎｓｅｃ〜１ｓｅｃの範囲全体で一定のビン幅１００ｎｓｅｃの分解能を適用すると演算量が膨大となり、また、時間遅れτが大きい領域で求められる波形のＳＮ比が劣化する場合がある。 In calculating the autocorrelation function G (τ), a multiple tau method may be used in which the correlation function is obtained by changing the bin width according to the time delay τ without performing the calculation with a constant bin time width. That is, the general autocorrelation function is expressed by the following equation (14).

However, if a resolution with a constant bin width of 100 nsec is applied over the entire time delay τ range of 100 nsec to 1 sec, for example, the calculation amount becomes enormous, and the S / N ratio of the waveform required in the region where the time delay τ is large is deteriorated. There is a case.

  On the other hand, according to the multiple tau method in which the bin width is changed according to the time delay τ so that the bin time width becomes large in the region where the time delay τ is large, the calculation amount of the correlation waveform is reduced and the time delay τ SN ratio in a large area can be improved. Specifically, for example, when performing analysis calculation on time-series measurement data having an initial bin width of 100 ns, the calculation is performed with the initial bin width of 100 ns at τ = 1 μs, and the bin width is 1 μs at τ = 1 ms. It is possible to use a method of performing calculation using the photon number data binned as described above.

  In general, when calculating the correlation function, set the minimum bin width to be applied in the region where the time delay τ is small, and bin the bin width with an integer multiple of the minimum bin width in the region where the time delay τ is large. Can be used. Such a calculation method can be suitably realized in a configuration in which the measurement control unit 50 performs fluorescence analysis in software. In other words, the software-analyzed configuration has a high degree of freedom for analysis calculations, and since data processing can be executed after fluorescence measurement, various fluorescence analyzes are performed, for example, the calculation is repeated many times under different analysis conditions. It is possible.

Next, the fluorescence analysis using the analysis data n _AB and the correlation function G _new (τ) will be further described along with specific examples thereof. In the following, the counting time width in the photon counting unit 35, which is the initial bin width of the measurement data F _A and F _B in the fluorescence analysis, is 100 ns. In the calculation of the correlation function, the above-described multiple tau method is basically used.

FIG. 3 is a graph showing a correlation function obtained for the photon number measurement data. In FIG. 3, the graph (a) shows the autocorrelation function G _A (τ) calculated by the equation (2) from the measurement data F _A at the photodetector 31. Further, the graph (b) shows the cross-correlation function G _C (τ) calculated by the equation (7) from the measurement data F _A and F _B at the photodetectors 31 and 32. These correlation functions are all calculated by a multiple tau method with a minimum bin width of 100 ns. FIG. 3 shows a correlation function obtained from measurement data obtained by repeating the fluorescence measurement for 3 seconds 10 times with the analysis apparatus 1A of FIG.

In the graph (a) of the autocorrelation function in FIG. 3, in the fast time region up to about τ = 1 μs, the waveform is superimposed with the after-pulse noise of the photodetector, and is effective only for the correlation waveform after 1 μs. Can be analyzed. Further, in the graph (b) of the cross-correlation function, the transition process to the triplet state is sufficiently observed, and the state of the fluorescent probe can be accurately known by analyzing the correlation waveform. The average number of detected photons in the fluorescence measurement is 35.3 kcps for the first photodetector 31 and 32.9 kcps for the second photodetector 32, and the fluorescence branching ratio by the beam splitter 30 is about 52:48. . Further, when the waveform fitting is performed with the equation (8) for the correlation waveform of the graph (b), the value of each parameter is <N>: 21.4.
τ _D : 0.256 ms
T: 0.123
λ: 0.396 μs
r ₀ / z ₀ : 0.2
In this case, the average number of molecules is <N> = 21.4.

For the same measurement data F _A and F _{B as in} FIG. 3, the analysis data n _AB of equation (1) is obtained, and the correlation function G _new (τ) taking into account the simultaneity of equation (9) is calculated. 4 shows. FIG. 4 shows a correlation function calculated by a multiple tau method by binning with a predetermined time width larger than the initial bin width of 100 ns as a minimum bin width. In FIG. 4, graphs A1, A2, A3, and A4 show correlation functions when the minimum bin width is 6.4 μs, 25.6 μs, 102.4 μs, and 409.6 μs, respectively. Here, for example, photon number data with a bin width of 6.4 μs is obtained by binning processing in which 64 photon number data with an initial bin width of 100 ns are added. The reason that the minimum bin width is set to a time width larger than the initial bin width is that the count value of most bins becomes 0 in consideration of simultaneity at the minimum bin width of 100 ns.

From the graphs A1 to A4 in FIG. 4, by increasing the minimum bin width when obtaining the correlation function G _new (τ), the y-intercept of the correlation function corresponding to 1 / <N> decreases, and within the measurement region It can be seen that the average number of molecules <N> of the fluorescent probe in consideration of the simultaneity is increased. By applying the above parameter values obtained in the graph (b) in FIG. 3 to parameters other than the number of molecules <N> for the correlation function in FIG. 4 and performing analysis by waveform fitting, graphs A1 to A4 For each, the average number of molecules <N> at each minimum bin width is determined.

FIG. 5 is a graph showing the bin width dependence of the number of molecules obtained from the correlation function G _new (τ). In FIG. 5, the horizontal axis represents the minimum bin time width (sec) in the calculation of the correlation function by the multiple tau method, and the vertical axis represents the average number of molecules <N> obtained from the y-intercept of the correlation function at each minimum bin width. Is shown. Here, in the photon number analysis data n _AB , the simultaneity is evaluated on the assumption that a bin with a count value of 0 exists in the time-series measurement data. Therefore, the time domain the frequency count value is increasing fluorescence amount per bin time width by binning process becomes 0 is low, in the region bin width is not less than 0.3ms in FIG. 5 for example, obtains the analysis data n _AB Thus, the effect of evaluating simultaneity is not sufficiently obtained.

On the other hand, for example, in the correlation function G _new (τ) when the bin width is 6.4 μs, the average number of molecules <N that was 21.4 in the correlation function G _C (τ) in the graph (b) of FIG. > Has decreased to 6.1. This shows that the apparent number of molecules in the measurement region of the sample S was estimated to be small by considering the simultaneous detection of fluorescence with the analysis data n _AB . As described above, such analysis conditions reduce the influence of background events caused by a plurality of free fluorescent probes in a fluorescence analysis when a plurality of fluorescent probes are aggregated to a target molecule. It is particularly effective in that it is done.

FIG. 6 is a graph showing another example of the correlation function obtained for the photon number measurement data. The graph of FIG. 6 shows the cross-correlation function G _C (τ) obtained by performing the fluorescence measurement under the condition where the concentration of the fluorescent probe is lower than that in the case of FIG. In this graph, the average number of detected photons in the fluorescence measurement is 2.3 kcps in the first photodetector 31 and 2.2 kcps in the second photodetector 32, and the fluorescence branching ratio by the beam splitter 30 is about 51:49. It has become. When the waveform fitting is performed with the equation (8) for the correlation waveform of the graph of FIG. 6, the value of each parameter is <N>: 1.66.
τ _D : 0.256 ms
T: 0.247
λ: 2.28 μs
r ₀ / z ₀ : 0.2
In this case, the average number of molecules is <N> = 1.66.

FIG. 7 is a graph showing the bin width dependence of the number of molecules obtained from the correlation function G _new (τ) for the same measurement data as FIG. In this graph, for example, in the correlation function G _new (τ) when the bin width is 6.4 μs, the average number of molecules <N> that was 1.66 in the correlation function G _C (τ) in FIG. 6 is 0. Reduced to 27. As can be seen from the graphs of FIGS. 5 and 7, the bin time width when binning the photon number measurement data and analysis data in fluorescence analysis refers to the bin width dependence of the number of molecules obtained from the correlation function. It is preferable to set the bin time width. In addition, the bin width dependency of the number of molecules varies depending on conditions such as the concentration of the fluorescent probe, but if the conditions are the same, the reproducibility is considered high.

となり、Ｆ_Ａ・Ｆ_Ｂ＞０の場合に所定の関数ｆで

となるように求められる。また、Ｆ_Ａ・Ｆ_Ｂ＞０の場合の解析データの具体的な算出方法としては、上記に例示した相乗平均による解析データｎ_ＡＢ

を用いることができる。これにより、蛍光検出の同時性が考慮された蛍光解析を好適に実行することができる。また、この方法では、Ｆ_Ａ・Ｆ_Ｂ＝０の場合においても、相乗平均の上記関数でｎ_ＡＢ＝０となる。 Next, data conversion from the photon number measurement data F _A and F _B to the analysis data n _AB will be described. Analytical data considering simultaneity is generally when F _A · F _B = 0

When F _A · F _B > 0, the predetermined function f

It is requested to become. In addition, as a specific calculation method of analysis data in the case of F _A · F _B > 0, analysis data n _AB based on the geometric average exemplified above is used.

Can be used. Thereby, the fluorescence analysis in consideration of the synchronization of fluorescence detection can be suitably executed. In this method, even when F _A · F _B = 0, n _AB = 0 in the above-mentioned geometric mean function.

を用いることができる。なお、この方法では、相加平均の上記関数ではＦ_Ａ、Ｆ_Ｂの一方のみが０の場合にｎ_ＡＢ＝０とならない。このため、Ｆ_Ａ・Ｆ_Ｂ＝０の場合については、別にｎ_ＡＢ＝０と定義する。 As another method for calculating analysis data when F _A · F _B > 0, analysis data n _{AB obtained} by arithmetic mean is used.

Can be used. In this method, in the above arithmetic mean function, when only one of F _A and F _B is 0, n _AB = 0 is not satisfied. For this reason, in the case of F _A · F _B = 0, it is separately defined as n _AB = 0.

を用いることができる。これにより、相乗平均の場合と同様に、蛍光検出の同時性が考慮された蛍光解析を好適に実行することができる。また、この方法でも、Ｆ_Ａ・Ｆ_Ｂ＝０の場合については、別にｎ_ＡＢ＝０と定義する。 Alternatively, as another calculation method of analysis data in the case of F _A · F _B > 0, analysis data n _AB by harmonic average is used.

Can be used. As a result, as in the case of geometric averaging, it is possible to suitably execute fluorescence analysis in consideration of the synchronization of fluorescence detection. Also in this method, when F _A · F _B = 0, it is separately defined as n _AB = 0.

を用いても良い。ただし、この積による解析データは、他の相乗平均、相加平均、調和平均による解析データとは性質が異なっている。 Alternatively, as another method of calculating analysis data when F _A · F _B > 0, analysis data n _AB based on the product of measurement data is used.

May be used. However, the analysis data based on this product is different from the analysis data based on other geometric averages, arithmetic averages, and harmonic averages.

In general, in the calculation of the analysis data n _AB (t), when F _A · F _B > 0, a function f (F _A , F _B that is one of geometric average, arithmetic average, harmonic average, or product is obtained. it is preferable to obtain the analysis data _{n AB} at), in particular, the geometric mean, arithmetic mean or harmonic mean is either a function _{f (F} a,, it is preferable to obtain the analysis data _{n AB} in _{F B).}

  FIG. 8 is a graph showing the bin width dependence of the number of molecules obtained from the correlation function. 8, graph (a) corresponds to the graph when the concentration of the fluorescent probe shown in FIG. 5 is high, and graph (b) corresponds to the graph when the concentration shown in FIG. 7 is low. In graph (a), graphs B1, B2, B3, and B4 indicate the number of molecules <N> when the geometrical average, arithmetic average, harmonic average, and product are used for calculation of the analysis data, respectively. The same applies to the graphs C1, C2, C3, and C4 in the graph (b).

  FIG. 9 is a graph showing a correlation function obtained for the photon number analysis data under the condition where the concentration of the fluorescent probe shown in the graph (a) of FIG. 8 is high. In FIG. 9, graph (a) shows the correlation function when the bin width is set to 6.4 μs, and graphs D1, D2, D3, and D4 represent the geometric mean, the arithmetic mean, and the harmonic mean, respectively, for calculation of analysis data. The correlation function when using the product is shown. Further, the graph (b) in FIG. 9 shows the correlation function when the bin width is set to 102.4 μs, and the graphs E1, E2, E3, and E4 respectively represent the geometric mean, the arithmetic mean, and the harmonic for calculating the analysis data. A correlation function in the case of using an average and a product is shown.

  As shown in these graphs, when the analysis data based on the geometric mean, the arithmetic mean, and the harmonic mean are used, all show the same tendency regarding the bin width dependency of the number of molecules <N>. On the other hand, when the analysis data based on the product is used, since the average intensity and the fluctuation fluctuate in the same manner in the autocorrelation calculation, the y-intercept of the correlation waveform and the number of molecules <N> obtained thereby are almost constant values. The result is shown. In any of the above cases, the correlation function (see FIG. 9) can be analyzed by waveform fitting by applying the parameter values obtained from the cross correlation function to parameters other than the number of molecules <N>. Is possible.

  In the example shown in the graph (a) of FIG. 8, as described above, the same tendency is shown for synergy, additive, and harmonic average. In this measurement example, the average photon detection numbers in the photodetectors 31 and 32 are 35.3 kcps and 32.9 kcps, and the average count value is 0.22 in the bin width of 6.4 μs, for example. For this reason, most of the count values in each bin are either 0, 1, or 2, and there is no significant difference in synergistic, additive, and harmonic averages in detection bias between the photodetectors. Among these synergistic, additive, and harmonic averages, the fluorescence fluctuation is not overestimated, and therefore, the geometrical average and harmonic average are considered to be particularly suitable for calculation of analysis data.

Next, photon burst analysis of the photon number analysis data n _AB (t) will be described. Photon burst analysis is an analysis method that obtains information about the number of fluorescent probes in the measurement region by analyzing the number of detected photons in each bin and its temporal change with respect to time-series photon number data. is there.

FIG. 10 is a graph showing an example of the distribution of count values (number of detected photons per bin) in time-series photon number data. The count value distribution shown in FIG. 10 is based on the measurement results when the concentration of the fluorescent probe shown in FIGS. 3 to 5 is high. The graph (a) in FIG. 10 shows the count value distribution when the bin width is set to 307.2 μs by binning in the photon number data obtained by adding the measurement data F _A and F _B at the photodetectors 31 and 32. Yes. Here, in the measurement example of FIG. 10, when the average number of detected photons in the measurement data F _A and F _B is added, it becomes 68.2 kcps, and the number of molecules is <N> = 21.4. The fluorescence intensity of is about 3.2 kcps. The bin width is selected so that the count value corresponding to one molecule of the fluorescent probe is about 1, considering the fluorescence intensity. For this reason, in the graph (a), the peak count value is approximately 21.

On the other hand, the graph (b) in FIG. 10 shows the count value distribution when the analysis data n _AB by the geometric mean is obtained with the bin width of 6.4 μs and the bin width is set to 307.2 μs by binning. In this case, in terms of the average of the measurement data F _A and F _B , a count value of 0.5 corresponds to one fluorescent probe molecule. In this graph (b), it is easy to identify a phenomenon in which the count value is 5 or more and light is emitted across two or more bins. That is, in the example of FIG. 10, for example, in the case of a reaction system in which about 10 fluorescent probes are aggregated and bound to a target molecule, the measurement data graph (a) is buried in an event by a free fluorescent probe and is difficult to identify. However, it is possible to identify them in the graph (b) of the analysis data in which the synchronism of fluorescence detection is considered.

FIG. 11 is a graph showing another example of the count value distribution in the photon number data. The count value distribution shown in FIG. 11 is based on the measurement result when the concentration of the fluorescent probe shown in FIGS. 6 and 7 is low. Graphs (a) and (b) in FIG. 11 show count value distributions obtained in the same manner as the graphs (a) and (b) in FIG. 10, respectively. Here, in the measurement example of FIG. 11, when the average photon detection number in the measurement data F _A and F _B is added, it becomes 4.5 kcps, and the number of molecules is <N> = 1.66. Has a fluorescence intensity of about 2.6 kcps. At this time, in the bin width, the count value corresponding to one fluorescent probe molecule is about 0.8.

  In the graph (b) of FIG. 11, the frequency of the count value 1 is 142, the frequency of the count value 2 is 1, and the count values of all other events are 0. In such a measurement result, it is possible to perform a photon burst analysis with the bin width of 307.2 μs as described above, for example, with a count value of 0.4 as a reference.

In a reaction system in which a plurality of fluorescent probes are bound to a target molecule, the probability that the analysis data _nAB is greater than 0 is very high. For this reason, by using analysis data such as a geometric mean, it is possible to perform fluorescence analysis in a form in which background events due to a free fluorescent probe or the like are reduced. However, since the photon burst analysis is a relative evaluation, it is preferable to make a measurement curve in advance for a measurement sample with a known concentration, create a calibration curve, and perform the analysis with reference to the calibration curve. In addition, regarding the calculation method of analysis data in consideration of simultaneity, there was no significant difference in photon burst analysis even when using synergistic, arithmetic, or harmonic average. Further, since the product tends to have a large variance, it is considered that a geometric mean or the like is suitable as a method for calculating analysis data.

  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. Further, the above-described fluorescence analysis method can be applied to fluorescence measurement under various conditions other than the case where only one type of fluorescent probe is added to the measurement sample. Further, since the above-described fluorescence analysis apparatus and analysis method can suppress the influence of a free fluorescent probe, it is considered to be effective as a fluorescence analysis method for a reaction system having a weak binding force in an intermolecular interaction, for example. It may also be applicable to combinations of antigen-antibody reactions that could not be applied to methods.

  INDUSTRIAL APPLICABILITY The present invention can be used as a fluorescence analysis apparatus and a fluorescence analysis method that can suitably execute fluorescence analysis of a measurement sample by measuring fluorescence from a fluorescence 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 graph which shows the correlation function obtained with respect to photon number measurement data. It is a graph which shows the correlation function obtained with respect to the photon number analysis data in consideration of simultaneity. It is a graph which shows the bin width dependence of the number of molecules calculated | required from a correlation function. It is a graph which shows the correlation function obtained with respect to photon number measurement data. It is a graph which shows the bin width dependence of the number of molecules calculated | required from a correlation function. It is a graph which shows the bin width dependence of the number of molecules calculated | required from a correlation function. It is a graph which shows the correlation function obtained with respect to the photon number analysis data in consideration of simultaneity. It is a graph which shows distribution of the count value in photon number data. It is a graph which shows distribution of the count value in photon number data.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1A ... Fluorescence analyzer, S ... Measurement sample, 10 ... Sample holder, 20 ... Objective lens, 21 ... Dichroic mirror, 22 ... Excitation light source, 23 ... Optical filter, 24 ... Reflection mirror, 25 ... Imaging lens, 26 ... Pin Hall, 27 ... collimating lens,
DESCRIPTION OF SYMBOLS 30 ... Beam splitter, 31 ... 1st photodetector, 32 ... 2nd photodetector, 33 ... 1st detection signal processing part, 34 ... 2nd detection signal processing part, 35 ... Photon counting part, 50 ... Measurement control part 51 ... Analysis data generation unit, 52 ... Measurement result analysis unit.

Claims (13)

Excitation light irradiation means for irradiating excitation light to the measurement region set for the measurement sample;
A light branching means for branching 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 into two fluorescent components;
First fluorescence detecting means for detecting one branched first fluorescence component;
Second fluorescence detection means for detecting the other branched second fluorescence component;
The number of photons detected by the first fluorescence detection means and the second fluorescence detection means is counted in a time-series manner with a predetermined counting time width, and the first photon number measurement data F _A (t) and the second photon are counted. Photon counting means for obtaining the number measurement data F _B (t);
On the basis of the first photon number measurement data _F A (t) and the second photon number measurement data _F B (t), the measured data _F A _(t), when at least one of _F B (t) is 0 n _AB (t) = 0
When both of the measurement data F _A (t) and F _B (t) are not 0, n _AB (t) = f (F _A (t), F _B (t)) with a predetermined function f
Analysis data generating means for generating photon number analysis data n _AB (t) to be
Measurement result analysis means for performing fluorescence analysis on the measurement result of the measurement sample including the fluorescent probe with respect to the photon number analysis data n _AB (t) generated by the analysis data generation means, Fluorescence analyzer. The analysis data generation means n is a function f that is a geometric mean of the measurement data F _A (t) and F _B (t) when both of the measurement data F _A (t) and F _B (t) are not zero. _AB (t) = √ (F _A (t) · F _B (t))
The fluorescence analysis apparatus according to claim 1, wherein the photon number analysis data n _AB (t) is generated by: The analysis data generating means is a function f that is a harmonic average of the measurement data F _A (t) and F _B (t) when both of the measurement data F _A (t) and F _B (t) are not 0. _AB (t) = 2 / {1 / F _A (t) + 1 / F _B (t)}
The fluorescence analysis apparatus according to claim 1, wherein the photon number analysis data n _AB (t) is generated by: The measurement result analysis means performs at least one of autocorrelation analysis or photon burst analysis as the fluorescence analysis on the photon number analysis data n _AB (t). The fluorescence analyzer according to one item. The measurement result analysis means bins the photon number analysis data n _AB (t) with a predetermined bin time width, and performs the fluorescence analysis on the binned analysis data. The fluorescence analysis device according to any one of the above.   The excitation light is excitation light in a single wavelength region, and the first fluorescent component and the second fluorescent component are fluorescent components in the same wavelength region. Fluorescence analyzer. An excitation light irradiation step for irradiating the measurement region set for the measurement sample with excitation light;
A light branching step for branching 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 into two fluorescent components;
A first fluorescence detection step of detecting one branched first fluorescence component;
A second fluorescence detection step of detecting the other branched second fluorescence component;
The number of photons detected in the first fluorescence detection step and the second fluorescence detection step is counted in time series with a predetermined counting time width, and the first photon number measurement data F _A (t) and the second photon are counted. A photon counting step to obtain the number measurement data F _B (t);
On the basis of the first photon number measurement data _F A (t) and the second photon number measurement data _F B (t), the measured data _F A _(t), when at least one of _F B (t) is 0 n _AB (t) = 0
When both of the measurement data F _A (t) and F _B (t) are not 0, n _AB (t) = f (F _A (t), F _B (t)) with a predetermined function f
An analysis data generation step for generating photon number analysis data n _AB (t) to be
A measurement result analysis step of performing fluorescence analysis on the measurement result of the measurement sample including the fluorescent probe with respect to the photon number analysis data n _AB (t) generated in the analysis data generation step, Fluorescence analysis method. In the analysis data generation step, when both of the measurement data F _A (t) and F _B (t) are not 0, the function f is a geometric mean of the measurement data F _A (t) and F _B (t) n _AB (t) = √ (F _A (t) · F _B (t))
The fluorescence analysis method according to claim 7, wherein the photon number analysis data n _AB (t) is generated by: In the analysis data generation step, when both of the measurement data F _A (t) and F _B (t) are not 0, the function f is a harmonic average of the measurement data F _A (t) and F _B (t) n _AB (t) = 2 / {1 / F _A (t) + 1 / F _B (t)}
The fluorescence analysis method according to claim 7, wherein the photon number analysis data n _AB (t) is generated by: The said measurement result analysis step WHEREIN: At least one of an autocorrelation analysis or a photon burst analysis is performed as said fluorescence analysis with _{respect to} said photon number analysis data _nAB (t). The fluorescence analysis method according to one item. The photon number analysis data n _AB (t) is binned with a predetermined bin time width in the measurement result analysis step, and the fluorescence analysis is performed on the binned analysis data. The fluorescence analysis method according to any one of the above.   The excitation light is excitation light in a single wavelength region, and the first fluorescence component and the second fluorescence component are fluorescence components in the same wavelength region. Fluorescence analysis method.   Only one type of fluorescent probe is used as the fluorescent probe to be added to the measurement sample, and the method is applied to the fluorescence analysis when two or more of the same type of fluorescent probe bind to a target molecule contained in the measurement sample. The fluorescence analysis method according to any one of claims 7 to 12, wherein:

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