JP5019116B2 - Fluorescence spectrometer - Google Patents

Description

  The present invention relates to a fluorescence spectroscopic analyzer.

  In the fluorescence correlation spectroscopy (FCS method), the autocorrelation function of the fluorescence intensity is obtained by analyzing the fluctuation of the light generated by the Brownian motion of the fluorescent molecule in the micro observation region of the microscope field of view. This is a technique for analyzing the average number of molecules, and is described in Non-Patent Document 1, for example.

  Fluorescence cross-correlation spectroscopy (FCCS method) is a technique for analyzing the relationship between two fluorescent signals by calculating the cross-correlation function between different fluorescent signals. It is used for analysis and is described in detail in Non-Patent Document 2 and Non-Patent Document 3, for example. A similar analysis method is confocal fluorescence coincidence analysis (CFCA method), which is described in detail in Non-Patent Document 4.

Here, for example, in cross-correlation spectroscopy (FCCS method), measurement is usually performed with two light sources and two light receivers. However, in this method (measurement), color separation is usually performed by a color separation filter, but crosstalk occurs in the fluorescence spectrum of the fluorescent dye, and the accuracy of the measurement result is lowered. Patent Document 1 discloses a technique for eliminating crosstalk by performing the FCCS method while alternately switching excitation light.
"Single molecule detection by fluorescence correlation spectroscopy", Kinjo, Protein Nucleic Acid Enzyme, 1999, vol. 44, N09 1431-1438 "Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution", Petra. Schwille et al, Biophysical Journal 1997, 72, 1878-1886 A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy, Petra. Schwille et al, Methods 29 (2003) 74-85 Confocal fluorescence coincidence analysis (CFCA), Winkler et al., Proc. Natl. Acad. Sci. USA 96: 1375-1378, 1999 JP 2005-283264 A

  Although the switching FCCS method according to Patent Document 1 eliminates crosstalk, the loss of fluorescence intensity data occurs in a specific delay time zone (a delay time zone in which there is no fluorescence intensity on one side). For this reason, a defect occurs in a specific delay time zone of the correlation curve obtained as a measurement result, and as a result, the reliability of the measurement result is lowered.

  An object of the present invention is to provide a fluorescence spectroscopic analyzer capable of acquiring a correlation curve without a defect while performing a switching FCCS method.

  The fluorescence spectroscopic analyzer according to the present invention includes an excitation light irradiation unit that irradiates a sample while switching a plurality of excitation lights having different wavelengths, an excitation light control unit that changes the switching of the excitation light over time, and the excitation light Correlation based on a fluorescence detection unit that detects a plurality of fluorescences having different wavelengths generated from the sample in response to irradiation, and a wavelength of excitation light irradiated on the sample and a fluorescence intensity detected by the fluorescence detection unit And an arithmetic unit for performing analysis.

  ADVANTAGE OF THE INVENTION According to this invention, the fluorescence spectroscopy analyzer which can acquire the correlation curve without a defect | deletion is implemented, implementing the switching FCCS method.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 schematically shows a fluorescence spectrometer according to an embodiment of the present invention. As shown in FIG. 1, the fluorescence spectroscopic analysis apparatus 100 includes a microscope unit 110, an excitation light control unit 130, a fluorescence detection unit 140, a signal processing unit 150, and a calculation unit 160. The microscope unit 110 generates a stage 112 for supporting the sample S, an objective lens 114 disposed below the stage 112, a dichroic mirror 116 for separating excitation light and fluorescence, and excitation light for irradiating the sample S. And an excitation light irradiation unit 120 to be activated. Excitation light generated by the excitation light irradiation unit 120 is applied to the sample S via the dichroic mirror 116 and the objective lens 114. The sample S includes a plurality of types of fluorescent dyes having different excitation wavelengths. The excitation light irradiation unit 120 irradiates the sample while switching excitation light having different wavelengths. The wavelength of the excitation light emitted from the excitation light irradiation unit 120 corresponds to the excitation wavelength of the fluorescent dye included in the sample S, respectively.

  The excitation light control unit 130 controls the excitation light irradiation unit 120 so that excitation light having different wavelengths is irradiated to the sample S exclusively at different times. Therefore, the excitation light irradiation unit 120 can repeatedly irradiate the same part of the sample S with excitation light having different wavelengths at a predetermined timing. The excitation light control unit 130 further controls the excitation light irradiation unit 120 so as to change excitation light switching with time. Therefore, the excitation light control unit 130 includes an excitation light operation signal generation unit 134 and a change pattern generation unit 132. The change pattern generation unit 132 always outputs predetermined values for various parameters. The various parameters are parameters that change the cycle and duty ratio over time. The excitation light operation signal generation unit 134 generates an excitation light operation signal according to the values of various parameters input from the change pattern generation unit 132 and outputs the excitation light operation signal to the excitation light irradiation unit 120. The excitation light operation signal generation unit 134 may be configured by a general arbitrary signal generator, or may be configured by a logic circuit configuration represented by, for example, FPGA.

  The fluorescence detection unit 140 detects fluorescence generated from the sample S. From the sample S, fluorescence having different wavelengths (wavelength bands) is generated in response to irradiation with excitation light having different wavelengths. The fluorescence detection unit 140 performs detection by separating fluorescence for different wavelengths.

  The signal processing unit 150 generates fluorescence intensity data reflecting the fluorescence intensity detected by the fluorescence detection unit 140. The calculation unit 160 performs correlation analysis of fluorescence fluctuations for different wavelengths based on the wavelength of the excitation light applied to the sample S and the intensity of the fluorescence detected by the fluorescence detection unit 140. For example, the calculation unit 160 performs autocorrelation analysis, cross-correlation analysis, or confocal fluorescence coincidence analysis analysis based on comparison of output signals from the fluorescence detection units 140 corresponding to the respective fluorescences.

  FIG. 2 shows a configuration example of the excitation light irradiation unit 120. In this example, the excitation light irradiation unit 120 includes a plurality of light sources, an objective lens (see FIG. 1), and a plurality of dichroic mirrors. The plurality of light sources 122a, 122b, 122c,... Emit excitation light having different wavelengths. The dichroic mirrors 124a, 124b, 124c,... Reflect the excitation light emitted from the light sources 122a, 122b, 122c,. The light sources 122a, 122b, 122c,... Are continuously driven and continue to emit excitation light having different wavelengths. The dichroic mirrors 124a, 124b, 124c,... Transmit excitation light emitted from the light sources 122a, 122b, 122c,. The excitation light irradiation unit 120 further includes an acousto-optic element (AOTF) 126 that can control the transmission band. It is disposed between the dichroic mirror 124a and the dichroic mirror 116 (see FIG. 1). Further, the acoustooptic device 126 passes one of the light emitted from the light sources 122a, 122b, 122c,... According to the excitation light operation signal supplied from the excitation light control unit 130 (see FIG. 1). Thereby, the excitation light with which the sample S is irradiated can be selected from the excitation light having different wavelengths.

  In this configuration, the light sources 122a, 122b, 122c,... May be replaced with this single light source. In this case, a single light source emits light that includes all of the wavelength bands of light emitted by these light sources. When the light source is single, the dichroic mirrors 124a, 124b, 124c,.

  FIG. 3 shows another configuration example of the excitation light irradiation unit 120. Also in this example, the excitation light irradiation unit 120 includes a plurality of light sources 122a, 122b, 122c,... And a plurality of dichroic mirrors 124a, 124b, 124c,. The functions of the light sources 122a, 122b, 122c,... And the dichroic mirrors 124a, 124b, 124c,. The light sources 122a, 122b, 122c,... Can be controlled on and off. The excitation light irradiation unit 120 further includes a switch 128 that selects the light sources 122a, 122b, 122c,. The switch 128 selectively turns on one of the light sources 122a, 122b, 122c,... According to the pumping light operation signal supplied from the pumping light controller 130 of FIG. . Thereby, the excitation light with which the sample S is irradiated is selected from the excitation light having different wavelengths emitted from the light sources 122a, 122b, 122c,.

  FIG. 4 shows a configuration example of the fluorescence detection unit 140. The fluorescence detector 140 includes a plurality of beam splitters 142a, 142b, 142c,..., A plurality of fluorescence filters 144a, 144b, 144c,..., And a plurality of light receiving elements 146a, 146b, 146c,. have. The plurality of beam splitters 142a, 142b, 142c,... Divides the fluorescence incident from the microscope unit 110. If the dichroic mirror is used instead of the plurality of beam splitters 142a, 142b, 142c,..., The light use efficiency can be increased. The plurality of fluorescent filters 144a, 144b, 144c,... Are a plurality of light receiving elements 146a, 146b, 146c that selectively transmit specific fluorescence from the fluorescence divided by the beam splitters 142a, 142b, 142c,. ,... Detect the fluorescence transmitted through the fluorescent filters 144a, 144b, 144c,. The fluorescent filters 144a, 144b, 144c,... Have different transmission bands, and selectively transmit one of a plurality of fluorescence emitted from the sample S. The light receiving elements 146a, 146b, 146c,... Receive light in a wavelength band corresponding to the transmission bands of the fluorescent filters 144a, 144b, 144c,.

  In this configuration, the plurality of light receiving elements 146a, 146b, 146c,... May be replaced with a single light receiving element. In this case, the beam splitters 142a, 142b, 142c,. Further, the fluorescent filters 144a, 144b, 144c,... May be configured such that the corresponding tendency fluorescent filters are inserted into the optical path in accordance with switching of the excitation light.

  Hereinafter, the operation of the fluorescence spectrometer of the present embodiment will be described with reference to the flowchart of FIG. In the following description, an example of switching between two excitation lights (first excitation light and second excitation light) will be described for the sake of simplicity.

  Prior to the generation of the excitation light operation signal by the excitation light controller 130, the excitation light switching timing is set in order to change the excitation light switching over time.

  Based on the set excitation light switching timing, the change pattern generation unit 132 always outputs predetermined values for parameters whose period and duty ratio change with time. The excitation light operation signal generation unit 134 generates an excitation light operation signal according to the parameter value input from the change pattern generation unit 132 and outputs the excitation light operation signal to the excitation light irradiation unit 120.

  The excitation light operation signal is a binary signal of “0” and “1” as shown in FIG. When the excitation light irradiation unit 120 has the configuration shown in FIG. 2, the acoustooptic device 126 selectively transmits the first excitation light when the excitation light operation signal is “0”, and the excitation light operation signal is “1”. Sometimes the second excitation light is selectively transmitted. When the excitation light irradiation unit 120 has the configuration shown in FIG. 3, the switch 128 selectively turns on the first light source 122 a that emits the first excitation light when the excitation light operation signal is “0”. When the operation signal is “1”, the second light source 122b that emits the second excitation light is selectively turned on. As a result, as shown in FIG. 7, the excitation light applied to the sample S is alternately switched between the first excitation light and the second excitation light.

  Further, for example, as shown in FIG. 6, the excitation light operation signal is obtained by repeating block 1 and block 2 having different periods. Thereby, the excitation light irradiation unit 120 switches between the first excitation light and the second excitation light while changing the switching cycle of the first excitation light and the second excitation light for each block. Here, the excitation light operation signal is a repetition of two blocks, but it may be a repetition of three or more blocks having different periods. That is, the period of the excitation light operation signal is different for each block having a constant time interval. As a result, the excitation light control unit 130 changes the excitation light switching cycle for each block at a constant time interval.

  Fluorescence is generated from the sample S in response to the excitation light irradiation. The first light receiving element 146 a detects the first fluorescence generated from the sample S in response to the irradiation of the first excitation light, and outputs the first fluorescence detection signal shown in FIG. 7 to the signal processing unit 150. The second light receiving element 146b detects the second fluorescence generated from the sample S in response to the irradiation of the second excitation light, and outputs the second fluorescence detection signal shown in FIG.

  The signal processing unit 150 is supplied with the first fluorescence detection signal and the second fluorescence detection signal from the first light receiving element 146a and the second light receiving element 146b of the fluorescence detection unit 140, respectively. The signal processing unit 150 converts the supplied first fluorescence detection signal and second fluorescence detection signal into a fluorescence intensity signal for each predetermined time, and combines the fluorescence intensity signal and the excitation light control signal in an optimal form for calculation. Data is generated and output to the calculation unit 160.

  The calculation unit 160 performs autocorrelation analysis, cross-correlation analysis, or confocal fluorescence coincidence analysis based on the calculation data supplied from the signal processing unit 150.

  In the switching FCCS, when a plurality of excitation lights having different wavelengths are switched and measured at a constant period, fluorescence intensity data corresponding to the fluorescent dye is lost while the fluorescent dye is not excited. When the correlation calculation is performed based on such fluorescence intensity data that is partially missing, a missing section occurs in the correlation curve obtained by the correlation calculation. For example, it is assumed that measurement is always performed while switching between the first excitation light and the second excitation light in the cycle T1 of the block 1 in FIG. In this case, when correlation analysis is performed based on the fluorescence intensity data acquired by measurement, a deficient section is generated in the correlation curve as shown in the upper part of FIG. In addition, it is assumed that the measurement is always performed while switching between the first excitation light and the second excitation light in the cycle T2 of the block 2 in FIG. In this case, when the correlation analysis is performed based on the fluorescence intensity data acquired by the measurement, a deficient section is generated in the correlation curve as shown in the middle part of FIG. In FIG. 8, the discontinuous section of the correlation curve with the period T2 coincides with the continuous section of the correlation curve with the period T1. However, in practice, the discontinuous section coincides with a very small part of the continuous section of the correlation curve with the period T1.

  Therefore, in the present embodiment, measurement is performed while changing the excitation light with time. Specifically, the correlation analysis is performed while switching between the first excitation light and the second excitation light while changing the period for each block at a constant time interval. As a result, as shown in the lower part of FIG. 8, a correlation curve having no missing section due to missing fluorescence intensity data is obtained.

  Hereinafter, a mechanism in which data loss occurs in the switching FCCS that switches a plurality of excitation lights at a constant period will be described with reference to FIGS. In the description that follows, an example in which two excitation lights (first excitation light and second excitation light) are switched will be described to simplify the explanation. The data loss occurrence mechanism is the same in the switching FCCS that switches between three or more excitation lights.

  In FCCS, a product-sum operation is performed on two pieces of fluorescence intensity data (first fluorescence intensity data and second fluorescence intensity data) for each delay time. In the switching FCCS, only the product-sum operation is performed when two pump lights are emitted with a delay time between them. However, for each delay time that is a multiple of the pump light switching period, there is a part where the product-sum operation data does not exist. Arise. In addition, since the data just before the excitation light switching is invalidated, the delay time range in which product-sum operation data does not occur is further expanded. In the graph with the delay time on the horizontal axis, data loss occurs in this delay time range.

  12 to 16 show combinations of fluorescence intensity data of product-sum operation at various delay times and results of product-sum operation execution. In the figure, s indicates that there is effective fluorescence intensity data, o indicates that an effective calculation result is obtained by a product-sum operation between effective fluorescence intensity data, and x indicates effective fluorescence intensity data. It indicates that there is no or no valid operation result.

  FIGS. 17 and 18 show combinations of fluorescence intensity data of product-sum operations at various delay times and results of the product-sum operation. The product-sum operation shown in FIGS. 12 to 16 is equivalent to performing a product-sum operation in a vertical column while shifting the first fluorescence intensity data to the right one by one as shown in FIGS. 17 and 18.

FIG. 19 shows a list of product-sum operation results and a graph of product-sum operation data shown in FIGS. 17 and 18. The product-sum operation data number s is 0 when τ = ₀ , 2 when τ = τ ₀ , and 4 when τ = 2τ ₀ , and increases as the delay time increases, and reaches a maximum of 16 when τ = 8τ ₀ . Thereafter, it decreases to 14 as τ = 9τ ₀ and 12 as τ = 10τ ₀ , and decreases as the delay time increases, and becomes ₀ again as τ = 16τ ₀ . Thereafter, tau = the 17τ ₀ 1, and 2, τ = 18τ _0, incremented as the delay time increases, the τ = 24τ ₀ in 8. Then, τ = 25τ ₀ in 7, and τ = 26τ ₀ At 6, decremented as the delay time increases, becomes zero at τ = 32τ _0, then always zero.

  FIG. 20 and FIG. 21 show the product-sum operation at various delay times in the fluorescence intensity data (data2) acquired at a sampling period that is 1/2 of the sampling period of the fluorescence intensity data (data1) shown in FIGS. The combination of the fluorescence intensity data and the result of the product-sum operation are shown.

FIG. 22 shows a list of product-sum operation results and a graph of product-sum operation data (data2) shown in FIGS. 20 and 21, together with a graph of product-sum operation data (data2) shown in FIG. The product-sum operation data number s is 0 when τ = ₀ , 5 when τ = τ ₀ , and 10 when τ = 2τ ₀ , and increases as the delay time increases, and becomes 20 at the maximum when τ = 4τ ₀ . Then, τ = 5τ ₀ At 15, a 10, τ = 6τ _0, decremented as the delay time increases, the tau = 8? 0 again at _0. Thereafter, tau = the 9τ ₀ 4, and τ = 10τ ₀ At 8, incremented as the delay time increases, the τ = 12τ ₀ at 16. Then, τ = 13τ ₀ At 12, the τ = 14τ ₀ At 8, decremented as the delay time increases, becomes zero at τ = 16τ _0. Similarly, the increase and decrease are repeated as the delay time increases.

  In both data1 and data2, data loss occurs. In the calculation of the cross correlation, the data to be calculated are two data corresponding to the delay time. When one of the two data is in the invalid section (the section in which the data just before the excitation light switching is invalid), the calculated value is ignored as the target of the cross-correlation calculation. As a result, data loss occurs.

In Figure 22, as can be seen by comparing the data1 and data2, although the data2 in tau = 8? ₀ there is data missing, there is no missing data in the data1. Therefore, the data loss of data2 can be compensated by using data1. However, in this example, since a half sampling period of data2 are sampling period of data1, there is data missing in both data1 and data2 in τ = 16τ _0. Therefore, the missing data in the τ = 16τ ₀ can not be compensated even by combining the data1 and data2.

However, for example, if the sampling period of the data2 to 1/3 of the sampling period of data1, the data data2 in data loss occurs τ = 16τ ₀ to data1 exists. Therefore, it is possible to compensate for the loss of data in the τ = 16τ ₀ of data1 with data2.

  Examples of conditions that can compensate for the deficiency include (1) that the sampling frequency of one fluorescence intensity data is not an integral multiple of the sampling frequency of the other fluorescence intensity data, and (2) that the deficient portions of the data do not overlap. The ratio of the sampling frequency of fluorescence intensity data is reasonably large. Regarding (2), it can be said that the mutual delay time is an appropriate ratio that can compensate for the invalid section of the data.

  That is, if the period of switching the excitation light in each block is adjusted so that data loss does not occur in both data1 and data2 in the same delay time zone, the data loss of data1 and data2 can be compensated for each other. . Thereby, it is possible to obtain a correlation curve having no missing section while performing the switching FCCS method.

[Modification of excitation light operation signal]
FIG. 9 shows another excitation light operation signal that can be substituted for the excitation light operation signal shown in FIG. For example, as shown in FIG. 9, the excitation light operation signal is obtained by repeating a block whose period changes with time. Thereby, the excitation light irradiation unit 120 switches between the first excitation light and the second excitation light while changing the cycle of each switching of the first excitation light and the second excitation light in each block. In the example of FIG. 9, the cycle gradually decreases, but the method of changing the cycle is not limited to this, and it is sufficient that the cycle changes within a block having a constant time interval. Further, the method of changing the cycle is the same for each block, but is not limited to this, and may be different for each block. That is, the period of the excitation light operation signal changes with time in each block at a constant time interval. As a result, the excitation light control unit 130 changes the excitation light switching cycle with time in each block at regular time intervals. Also in this case, the data loss can be compensated for each other by adjusting the change of the excitation light switching period in each block so that the two fluorescence intensity data are not lost in the same delay time zone. . Thereby, it is possible to obtain a correlation curve having no missing section while performing the switching FCCS method.

  FIG. 10 shows another excitation light operation signal that can be substituted for the excitation light operation signal shown in FIG. For example, as shown in FIG. 10, the excitation light operation signal is obtained by repeating block 1, block 2, and block 3 having different duty ratios. Thereby, the excitation light irradiation unit 120 switches between the first excitation light and the second excitation light while changing the ratio of the irradiation time of the first excitation light and the second excitation light for each block. Here, the excitation light operation signal is a repetition of three blocks, but may be a repetition of two or four or more blocks having different duty ratios. That is, the duty ratio of the excitation light operation signal is different for each block at a constant time interval. As a result, the excitation light control unit 130 changes the duty ratio for switching excitation light for each block at a constant time interval. In this case as well, data loss can be compensated for by adjusting the excitation light switching duty ratio of each block so that both the two fluorescence intensity data are not lost in the same delay time zone. Thereby, it is possible to obtain a correlation curve having no missing section while performing the switching FCCS method.

  FIG. 11 shows still another excitation light operation signal that can be substituted for the excitation light operation signal shown in FIG. For example, as shown in FIG. 11, the excitation light operation signal is a signal obtained by repeating blocks in which the duty ratio changes with time. Thereby, the excitation light irradiation unit 120 changes the ratio of the irradiation time of the first excitation light and the second excitation light at each time of switching between the first excitation light and the second excitation light in each block, The second excitation light is switched. In the example of FIG. 11, the duty ratio changes in one direction (so that “1” decreases), but the method of changing the duty ratio is not limited to this, and the duty ratio changes at a constant time interval. It is sufficient that the duty ratio is changed in the block. Further, the manner of changing the duty ratio is the same for each block, but is not limited to this, and may be different for each block. In other words, the duty ratio of the excitation light operation signal changes with time in each block at a constant time interval. Thus, the excitation light control unit 130 changes the excitation light switching duty ratio with time in each block at a constant time interval. In this case as well, data loss can be compensated for by adjusting the change in the duty ratio of the excitation light switching in each block so that no loss occurs in the same delay time zone in both of the two fluorescence intensity data. it can. Thereby, it is possible to obtain a correlation curve having no missing section while performing the switching FCCS method.

  Furthermore, the excitation light operation signal may be a combination of FIGS. 6 and 10 in which a plurality of blocks having different periods and duty ratios are repeated. In this case, the excitation light irradiation unit 120 changes the first excitation light and the second excitation light switching period and the ratio of the irradiation time of the first excitation light and the second excitation light for each block. The second excitation light is switched. That is, the excitation light control unit 130 changes the excitation light switching cycle and the duty ratio for each block at a constant time interval. Further, as a combination of FIG. 9 and FIG. 11, a block in which the cycle and the duty ratio change with time may be repeated. In this case, in each block, the excitation light irradiation unit 120 has a cycle of each switching of the first excitation light and the second excitation light, and a ratio of irradiation times of the first excitation light and the second excitation light at each switching. The first excitation light and the second excitation light are switched while changing. That is, the excitation light control unit 130 changes the excitation light switching cycle and the duty ratio with the passage of time in each block at a constant time interval.

  The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. Also good.

1 schematically shows a fluorescence spectrometer according to an embodiment of the present invention. 2 illustrates a configuration example of the excitation light irradiation unit illustrated in FIG. 1. The example of another structure of the excitation light irradiation part shown in FIG. 1 is shown. 2 illustrates a configuration example of the fluorescence detection unit illustrated in FIG. 1. The flowchart of operation | movement of the fluorescence-spectroscopy analyzer shown in FIG. 1 is shown. The excitation light operation signal is shown. 2 is a time chart of signals in the fluorescence spectroscopic analyzer shown in FIG. The correlation curve by period T1, the correlation curve by period T2, and the correlation curve by switching of period T1 and period T2 are shown. 7 shows another excitation light operation signal that can be substituted for the excitation light operation signal shown in FIG. 6. 7 shows another excitation light operation signal that can be substituted for the excitation light operation signal shown in FIG. 7 shows still another excitation light operation signal that can be substituted for the excitation light operation signal shown in FIG. 6. The combination of the fluorescence intensity data of the product-sum operation at the delay times of τ = 0, τ = τ ₀ and τ = 2τ ₀ and the result of the product-sum operation are shown. The combination of the fluorescence intensity data of the product-sum operation and the results of the product-sum operation are shown for the delay times of τ = 3τ ₀ , τ = 7τ ₀ and τ = 8τ ₀ . The combination of the fluorescence intensity data of the product-sum operation and the result of the product-sum operation are shown in the delay times of τ = 9τ ₀ , τ = 13τ ₀ and τ = 14τ ₀ . The combination of the fluorescence intensity data of the product-sum operation at the delay times of τ = 15τ ₀ , τ = 16τ ₀ and τ = 17τ ₀ and the result of the product-sum operation are shown. The combination of the fluorescence intensity data of the product-sum operation in the delay time of τ = 18τ ₀ and τ = 19τ ₀ and the result of the product-sum operation are shown. τ = 0, τ = τ ₀ , τ = 2τ ₀ , τ = 3τ ₀ , τ = 6τ ₀ , τ = 7τ ₀ , τ = 8τ ₀ Combination of fluorescence intensity data and product-sum operation in delay time of τ = 8τ ₀ The results of the implementation are shown. τ = 9τ ₀ and τ = 10τ ₀ and τ = 15τ ₀ and τ = 16τ ₀ and τ = 17τ ₀ and τ = 24τ ₀ and τ = 32τ ₀ The result of the calculation is shown. The list of the product-sum operation results and the graph of the product-sum operation data shown in FIGS. 17 and 18 are shown. In the fluorescence intensity data acquired at a sampling period ½ of the sampling period of the fluorescence intensity data shown in FIGS. 17 and 18, τ = 0, τ = τ ₀ , τ = 2τ ₀ , τ = 3τ _0, and τ = The combination of the fluorescence intensity data of the product-sum operation and the result of the product-sum operation are shown for the delay times of 4τ ₀ and τ = 5τ ₀ and τ = 6τ ₀ . In the fluorescence intensity data acquired at a sampling period that is 1/2 of the sampling period of the fluorescence intensity data shown in FIGS. 17 and 18, τ = 7τ ₀ and τ = 8τ ₀ and τ = 9τ ₀ and τ = 10τ ₀ and τ = indicates the result of a combination product-sum calculation performed in the fluorescence intensity data of the product sum operation in 14Tau ₀ and τ = 15τ ₀ and the delay time of τ = 16τ _0. The list of the product-sum operation results and the graph of the product-sum operation data shown in FIGS. 20 and 21 are shown together with the product-sum operation data graph shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Fluorescence spectroscopy analyzer, 110 ... Microscope part, 112 ... Stage, 114 ... Objective lens, 116 ... Dichroic mirror, 120 ... Excitation light irradiation part, 122a, 122b, 122c ... Light source, 124a, 124b, 124c ... Dichroic mirror, DESCRIPTION OF SYMBOLS 126 ... Acousto-optic element, 128 ... Switch, 130 ... Excitation light control part, 132 ... Change pattern generation part, 134 ... Excitation light operation signal generation part, 140 ... Fluorescence detection part, 142a, 142b, 142c ... Beam splitter, 144a , 144b, 144c ... fluorescent filter, 146a, 146b, 146c ... light receiving element, 150 ... signal processing unit, 160 ... calculation unit.

Claims (11)

An excitation light irradiation unit that irradiates the sample while switching a plurality of excitation lights having different wavelengths;
An excitation light control unit that changes the excitation light switching over time;
A fluorescence detection unit for detecting a plurality of fluorescence having different wavelengths generated from the sample in response to irradiation of the excitation light;
A fluorescence spectroscopic analyzer having a calculation unit that performs a correlation analysis based on the wavelength of excitation light applied to the sample and the intensity of fluorescence detected by the fluorescence detection unit.   The fluorescence spectroscopy analyzer according to claim 1, wherein the excitation light control unit changes a switching period of the excitation light for each block having a constant time interval.   The fluorescence spectroscopic analysis apparatus according to claim 1, wherein the excitation light control unit changes a duty ratio for switching the excitation light for each block having a constant time interval.   The fluorescence spectroscopic analyzer according to claim 1, wherein the excitation light control unit changes a duty ratio of switching of the excitation light with time in each block at a constant time interval.   The fluorescence spectroscopic analysis apparatus according to claim 1, wherein the calculation unit has a fixed complementary processing condition when performing correlation analysis.   The excitation optical unit includes a single light source that emits light in a wavelength band that includes the plurality of excitation lights, and a selection unit that selects excitation light to be applied to the sample from the light emitted from the light source. The fluorescence spectroscopic analyzer according to claim 1.   The excitation optical unit includes a plurality of light sources that respectively emit the plurality of excitation lights, and a selection unit that selects excitation light to be irradiated on the sample from a plurality of excitation lights emitted by the plurality of light sources. Item 2. The fluorescence spectroscopic analyzer according to Item 1.   The fluorescence spectroscopic apparatus according to claim 6, wherein the selection unit includes an acousto-optic element capable of controlling a transmission band.   The fluorescence spectrometer according to claim 7, wherein the selection unit includes a light source switching unit that selects a light source to emit light.   The fluorescence spectroscopic analysis apparatus according to claim 1, wherein the fluorescence detection unit includes a plurality of light receiving elements having sensitivity in different wavelength bands.   The fluorescence spectroscopic apparatus according to claim 1, wherein the fluorescence detection unit includes a single light receiving element having a light receiving band including a plurality of fluorescence wavelength bands.

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