JP2009058405A - Optical analysis apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical analysis apparatus that irradiates a sample with a plurality of irradiation lights and measures and analyzes a plurality of radiation lights, and can acquire measurement data without crosstalk without increasing a signal processing burden of the measurement data. <P>SOLUTION: This optical analysis apparatus includes light irradiating means for selectively irradiating the sample with the plurality of irradiation lights, a plurality of light detecting means for selectively detecting each of the plurality of radiation lights, and measurement controlling means for controlling measurement of the radiation light. When the measurement controlling means selects one of the plurality of irradiation lights to be emitted to the sample, photodetection permitting means permits the detection of the radiation light of only the light detecting means for detecting the radiation light to be emitted from the sample correspondingly to the selected irradiation light. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention irradiates a sample with light (illumination light, excitation light or other irradiation light), and emits light (fluorescence, phosphorescence, scattered light, chemiluminescence, bioluminescence or other emitted light) from the sample. ), And more specifically, radiated light from the sample emitted in response to the irradiated light by irradiating the sample with irradiated light of a plurality of wavelength bands or wavelength characteristics The present invention relates to an optical analysis device that measures and analyzes the above.

  In the field of spectroscopic analysis technology, as described above, a sample to be measured or analyzed is often irradiated with at least two types of irradiation lights having different wavelength bands or wavelength characteristics, and each of these irradiation lights is irradiated. Correspondingly, radiated light having different wavelength bands or wavelength characteristics emitted from the sample is separately measured and analyzed. For example, in fluorescence cross-correlation spectroscopy (FCCS, Fluorescence Cross-Correlation Spectroscopy-see Non-Patent Document 1) and confocal fluorescence coincidence analysis (CFCA, Confocal fluorescence coincidence analysis-see Non-Patent Document 2), excitation and fluorescence wavelength characteristics Intensity of fluorescence from each of a plurality of types of fluorescent molecules emitted by irradiating a sample such as a biomolecule having a plurality of different types of fluorescent labels with excitation light that excites each of the plurality of types of fluorescent molecules. Are measured in time series with separate optical receivers, and the cross-correlation function (FCCS) of the fluorescence intensities from the multiple types of fluorescent molecules or the frequency with which the fluctuations in the fluorescence intensities of the multiple types of fluorescent molecules coincide (CFCA) is calculated. The cross-correlation function or frequency value thus obtained indicates whether or not a plurality of types of fluorescent molecules in the sample are in Brownian motion integrally. It is possible to obtain various information related to the dissociation state, structural change, or intermolecular interaction. Such information is difficult to obtain easily by a method of irradiating one sample with a single irradiation light and measuring a single radiated light from the sample.

  In the optical analysis method of irradiating a sample with irradiation light having a plurality of wavelength bands or wavelength characteristics as described above and measuring radiation light having a plurality of wavelength bands or wavelength characteristics from the sample, a certain wavelength band is used. The measurement value of the radiated light emitted from the sample in response to the irradiated light is acquired in a state completely separated from the radiated light emitted from the sample in response to the irradiated light in the other wavelength bands, that is, It is required that the measured value of the photoreceiver that receives the radiated light emitted from the sample in response to the irradiated light be in a state where there is no contribution from the radiated light emitted from the sample in response to the other irradiated light. There is a case. For example, in the case of the above-mentioned examples of FCCS and CFCA, if the measurement value of the fluorescence intensity from one fluorescent molecule includes the contribution of the fluorescence intensity from another fluorescent molecule, the fluorescence from a plurality of types of fluorescent molecules It becomes impossible to accurately calculate the cross-correlation function of the intensity (the value of the correlation function is apparently increased). Therefore, in the apparatus, in order to separately measure the fluorescence intensity of each fluorescent molecule, a light receiver that receives only light in each emission wavelength band of the plurality of fluorescent molecules is prepared, and the plurality of fluorescent molecules The selection is made so that the emission wavelength of each fluorescent molecule does not overlap with the detection wavelength band of the receiver for another fluorescent molecule, so that one receiver only measures the fluorescence from one type of fluorescent molecule An attempt is made to achieve a state to do.

  However, realizing the requirement to remove the contribution from the radiation emitted from the sample in response to the other illumination light from the measured value of the radiation emitted from the sample corresponding to the one illumination light as described above. , Often difficult. In the case of the above-mentioned FCCS and CFCA, the range of types of fluorescent molecules that can be selected as a fluorescent probe is limited in consideration of artifacts with respect to samples (particularly biomolecules). It is usually difficult to select the fluorescent molecules to be used so that each of the bands does not overlap with the detection wavelength band of the receiver for another fluorescent molecule, so the fluorescence of one fluorescent molecule is different. May be detected by a light receiver for the fluorescent molecules. That is, in a state in which a sample is irradiated with a plurality of irradiation lights at the same time, from the measured value of one radiated light emitted from the sample corresponding to one irradiation light, the other emitted from the sample corresponding to the other irradiation light. It is difficult to achieve a state where the contribution of synchrotron radiation is removed (the contribution of another synchrotron radiation included in the measurement of one synchrotron radiation is called “crosstalk”).

In this regard, in order to avoid the above-described crosstalk in FCCS or CFCA, Patent Documents 1 and 2 simultaneously irradiate a sample with a plurality of excitation lights for exciting a plurality of fluorescent molecules. Rather than irradiating the sample one by one with a plurality of excitation light one by one, that is, in a state where the excitation light irradiated to the sample becomes one at a time, the fluorescence from the sample measured at one time is one. Devices and methods have been proposed that are configured to be from a variety of fluorescent molecules. According to such an apparatus or method, it is possible to know the time during which fluorescence corresponding to each excitation light is measured by grasping the period during which each excitation light is irradiated, so that measurement data without crosstalk is acquired. (In this case, the measured value of the fluorescence intensity of each fluorescent molecule becomes intermittent, but the timing of switching the excitation light is changed with time, that is, the fluorescence molecule It is possible to calculate the cross-correlation function of the fluorescence intensity from multiple fluorescent molecules significantly by setting it so that it is sufficiently faster than the speed at which the supported molecules pass through the fluorescence observation region by Brownian motion. Become.).
JP 2006-93370 A JP 2006-93371 A "Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution", Petra. Schwilleet al, Biophysical Journal 1997, 72, 1878-1886 Confocalfluorescence coincidence analysis (CFCA), Winkler et al., Proc. Natl. Acad. Sci. USA 96: 1375-1378, 1999

  In the case of the optical measurement method in the fluorescence spectroscopic analyzer proposed in Patent Documents 1 and 2, the emission wavelength of the fluorescent molecule that emits light when the sample is irradiated with excitation light in a certain wavelength band When the band also overlaps the detection wavelength band of a photoreceiver for receiving the fluorescence of another fluorescent molecule, crosstalk occurs due to the fluorescence of the fluorescent molecule excited at that time in the measured value of the photoreceiver (See FIG. 2A). Therefore, in the case of the apparatus of the same document, after obtaining the measurement data of each light receiver, when calculating the cross-correlation function or the like, data other than the period corresponding to the irradiation time of the corresponding excitation light is corrected to zero value. By such signal processing or arithmetic processing, the influence of crosstalk in the measurement data of each light receiver is eliminated. That is, in such a configuration, when analyzing measurement data, it is necessary to execute signal processing or arithmetic processing for removing crosstalk.

  In this regard, the amount of synchrotron radiation measurement data measured in time series in the optical analysis as described above is often enormous depending on the time resolution, and the number of types of fluorescent molecules observed is increased. For this reason, the enormous amount of time-series measurement data is further increased accordingly, so that the amount of data to be subjected to signal processing or calculation processing is considerably large, and signal analysis may take a long time. In addition, as described in Patent Documents 1 and 2, when the corresponding radiated light is measured by selectively switching the irradiation light (excitation light) irradiated to the sample, as described above, the measurement is performed. Signal processing to remove the contribution of crosstalk from the measurement data, since it is performed faster and more frequently than the change in synchrotron radiation to be done (in the case of FCCS or CFCA, the speed of the molecular Brownian motion) The amount of calculation processing is also large. Therefore, in the case of the methods in Patent Documents 1 and 2, the signal processing or calculation processing burden for the analysis or analysis of the measurement data is further increased, and much time is required for the signal analysis. is there.

  Thus, one object of the present invention is an optical analyzer that measures and analyzes radiation emitted from a sample having a plurality of wavelength bands or wavelength characteristics by irradiating the sample with irradiation light having a plurality of wavelength bands or wavelength characteristics. An object of the present invention is to propose an apparatus capable of acquiring measurement values or measurement data without crosstalk as described above without increasing the signal processing or calculation processing burden of the measurement data.

  Another object of the present invention is an apparatus as described above, in which a measurement value of the intensity of radiated light from which the contribution of crosstalk has been eliminated is obtained, and the measurement value is almost directly subjected to analysis or analysis calculation processing. It is to propose a device that can be used.

  Still another object of the present invention is to propose an apparatus as described above, which can be advantageously used in fluorescence cross-correlation spectroscopy or confocal coincidence analysis.

  According to the present invention, in order to achieve the above-described problems, the crosstalk (that is, included in the measurement value of one radiated light) in the output or the measurement value of each of the light receiver or the light detection means for detecting the radiated light. There is provided an optical analysis device that achieves a state in which there is no contribution of another radiated light that does not need to be removed from the output value or measurement value of the light detection means.

  An optical analyzer for irradiating a sample with irradiation light having a plurality of wavelength bands or wavelength characteristics according to the present invention and measuring and analyzing emitted light having a plurality of wavelength bands or wavelength characteristics emitted from the sample corresponding to the irradiation light (Hereinafter referred to as “multiple light irradiation-multiple light detection type optical analyzer”) includes a light irradiation means for selectively irradiating a sample with a plurality of irradiation lights, and radiation of a plurality of wavelength bands or wavelength characteristics. A plurality of light detection means for selectively detecting each of the light, and a measurement control means for selecting the irradiation light to be irradiated on the sample and executing the measurement of the emitted light. And the measurement control means in the apparatus of the present invention selectively permits the detection of the emitted light by each of the irradiation light selection means for selecting the irradiation light emitted to the sample and the plurality of light detection means. A light detection permission means, and when the irradiation light selection means selects one of the plurality of irradiation lights to be emitted from the sample, the light detection permission means includes the plurality of irradiation lights. The detection of the radiated light of the light detection means for detecting the radiated light to be emitted from the sample corresponding to the selected irradiation light is permitted. The light source in the light irradiation means is typically a laser, but may be any one used in the field of other optical analysis techniques such as a mercury lamp and a xenon lamp. In the light detection means, the light receiving element that actually receives the light may be any one used in the field of optical analysis technology such as a photodiode or CCD.

  In the conventional multiple light irradiation-multiple light detection type optical analyzer, as already described, depending on the wavelength characteristic of the radiation light of the sample, another radiation light is used as the light detection means for detecting one radiation light. If crosstalk occurs in which the contributions of both are mixed and the accuracy of the result of the desired optical analysis deteriorates, the crosstalk component is removed from the measurement data during signal analysis or analysis using the measurement data. This is necessary, and this increases the burden of signal processing or arithmetic processing. Therefore, in the apparatus of the present invention, as can be understood from the above configuration, a light detection permission unit that selectively permits detection of the emitted light by each of the plurality of light detection units is provided, and a plurality of light detection permission units are provided. Only the light detection means for detecting the radiated light to be emitted from the sample corresponding to the selected radiated light among the radiated light is configured to allow detection of the radiated light. According to such a configuration, while a certain irradiation light is irradiated on the sample, only the light detection means for detecting the wavelength band of the emitted light emitted from the sample corresponding to the irradiation light is allowed to detect the emitted light. Accordingly, the detection of the radiated light by other light detection means is prohibited. Therefore, the output value of each light detection means does not include the crosstalk of the radiated light that should be emitted corresponding to the irradiation light different from the corresponding irradiation light, and the output value of each light detection means is Thus, the measurement data can be used for subsequent signal analysis or analysis without executing a signal processing process for removing crosstalk. In order to execute multiple light irradiation-multiple light detection type light detection, that is, detection of a plurality of radiated lights by a plurality of irradiation lights, in the analysis apparatus of the present invention, there are a plurality of irradiation light selecting means. Select the irradiation light to irradiate the sample one by one in sequence, and the light detection permission means emits from the sample corresponding to the selected irradiation light in synchronization with the selection of the irradiation light of the irradiation light selection means. It may be adapted to allow detection of the emitted light of the light detection means for detecting the emitted light to be performed.

  In the above-described embodiment of the present invention, as a configuration for selectively irradiating the sample with any one of a plurality of irradiation lights, the light irradiation means includes a plurality of light sources, and each of the light sources is a specific light source. When generating irradiation light of a wavelength, the irradiation light selection means selectively irradiates one of the light sources so that irradiation light from any one of the plurality of light sources can be given to the sample. The light may be generated. As another configuration, the irradiation light selection unit includes an optical element that selectively transmits light in the wavelength band of any of the plurality of irradiation lights, for example, an acousto-optic element (AOTF), The transmission wavelength band of the optical element may be selectively changed.

  Further, in the above-described embodiment of the present invention, as a configuration for selectively permitting or prohibiting detection of radiated light from the sample in each light detection permission means, the light detection permission means includes the sample from the sample. Including shutter means that allows radiation light to enter the light detection means by selectively opening the optical path of the radiation light to the light detection means, and controlling the operation of the shutter means to emit light to the light detection means May be transmitted or blocked. As such shutter means, an acousto-optic element (AOTF), an optical filter whose light transmittance is controlled using liquid crystal, or the like may be used. Further, as another aspect of the configuration for selectively permitting or prohibiting the detection of the emitted light, the light detection permission means is for detecting the emitted light that should be emitted from the sample in response to the selected irradiation light. The light detection means is set in a state in which radiation light can be selectively detected, and the light detection means is in a state in which radiation light cannot be detected during a period in which irradiation light that does not correspond to each light detection means is irradiated. The operation may be controlled. Here, the specific configuration that brings the light detection means into a state where radiation light can be detected or cannot be detected may be appropriately configured according to the light receiver employed in the light detection means. Should be understood. What is important is that each light detection means does not generate an output corresponding to the radiated light even if the radiated light reaches the light receiver during a period in which the corresponding irradiated light is not irradiated on the sample. That is.

  By the way, the above-described configuration of the apparatus of the present invention can be advantageously used for fluorescence analysis using a plurality of samples containing fluorescent molecules having different emission wavelength characteristics from each other, particularly in fluorescence spectroscopic analysis. Therefore, in the above configuration, when the sample includes a plurality of fluorescent molecules having different emission wavelength characteristics, excitation for selectively exciting each of the plurality of fluorescent lights with one of the plurality of fluorescent molecules. Each of the plurality of emitted light that is light and is to be detected may be any of the fluorescence emitted from the plurality of fluorescent molecules.

  The configuration of the apparatus of the present invention described above can be advantageously applied particularly to an optical analyzer capable of executing FCCS or CFCA as described in the background art section. In these FCCS or CFCA measurement systems, an optical system of a confocal microscope is used. In the configuration of the present invention, light detection is performed from the light irradiation means to the position of the sample and from the position of the sample. The optical system up to the means may constitute the optical system of the confocal microscope.

  In general, according to the characteristic configuration of the apparatus of the present invention, the crosstalk as described above does not exist in the outputs of the plurality of light detection means, and therefore, at the output stage of the light detection means, there is a certain level. Requirement to obtain measured values of radiation emitted from a sample corresponding to irradiation light in one wavelength band in a state completely separated from radiation emitted from a sample corresponding to irradiation light in another wavelength band It becomes possible to satisfy. Therefore, compared with the method described in Patent Document 1 or 2, the signal processing or calculation processing burden is reduced, and the time required for signal analysis is reduced (only for the crosstalk removal processing on the measurement data). (There is no need to increase the specifications (calculation speed, storage capacity) of the computer or arithmetic processing unit of the signal analyzer.) In addition, since crosstalk on the measurement data is not mixed, the measurement data is subjected to analysis based on FCCS or CFCA as it is (after normal processing such as normal noise removal and background subtraction). Since it can be used, the configuration of a program for signal processing or arithmetic processing can be simplified.

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

  The present invention will now be described in detail with reference to a few preferred embodiments with reference to the accompanying drawings. In the following embodiments of the present invention, a fluorescence analyzer having an optical system of a confocal fluorescence microscope will be described. However, the concept of the present invention is another type of multiple light irradiation-multiple light detection type. It should be understood that such a case may be applied to the optical analysis apparatus of the present invention, and such a case also belongs to the scope of the present invention. Further, in the following description, a case where a fluorescently labeled sample is used as the sample will be described. Therefore, the irradiation light is described as excitation light, and the emitted light is described as fluorescence. The emitted light may be phosphorescent light, scattered light, chemiluminescence, bioluminescence, etc., and the irradiation light may be light that generates or induces the emitted light.

Configuration of Apparatus FIG. 1 schematically shows a configuration of a multiple light irradiation-multiple light detection type optical analysis apparatus 10 having an optical system of a confocal fluorescence microscope according to one preferred embodiment of the present invention. In the optical analyzer 10, a plurality of different wavelength bands with respect to a sample S (typically a solution injected into a cell (not shown)) containing fluorescently labeled molecules. Alternatively, an excitation optical system (light irradiation means) for irradiating excitation light with wavelength characteristics, and a light detection system (light detection means) for detecting a plurality of mutually different wavelength bands or fluorescence with wavelength characteristics from the sample S An analysis control system for analyzing or analyzing the fluorescence detected by the light detection system, and a measurement control system (measurement control means) for controlling selection of excitation light of the excitation optical system and detection light of the light detection system Composed.

  In the excitation optical system, the excitation light emitted from the plurality of lasers La to Ln is expanded through a beam expander (not shown) and converted into parallel light by the lenses 12a to 12n, and then the dichroic mirrors 14a to 14n. And 16, the objective lens 18 is configured to be focused (condensed) in a certain point region in the sample S (in the figure, three lasers are shown. The number may be arbitrary, as is the case with the following light receiver APD). A point region where the excitation light is focused (usually a region having a width of about the wavelength of the light) serves as an observation or observation region of the optical analyzer of the present embodiment, and when a fluorescently labeled molecule enters the region, The fluorescent label is excited by excitation light, and fluorescence is emitted. The laser may be a YAG laser, an argon laser, a He-Ne laser, or the like of a type normally used in the field of fluorescence spectroscopic analysis or in a confocal fluorescence microscope. , Any value in the range of 400 nm to 600 nm may be used. In the figure, the dichroic mirrors 14a to 14n may be appropriately prepared so as to transmit light arriving from above and reflect light arriving from the right side (14n is a normal mirror). May be.)

  The fluorescence emitted from the sample S by being irradiated with the excitation light as described above is collected by the objective lens 18, passes through the dichroic mirror 16, and then is focused on the pinhole 22 by the lens 20. It is condensed so as to tie. In the pinhole 22, only the imaged light passes therethrough, and light from other than the focal region (observation region) of the objective lens 18 is blocked. Light is essentially only light emitted from the focal region (the principle of confocal microscopy). Thus, after passing through the condenser lens 24, the light transmitted through the pinhole 22 is selectively divided by the dichroic mirrors 26a to 26n and the band pass filters 28a to 28n depending on the wavelength of the fluorescence. The light is directed toward the detectors APDi to n and is incident on the light receiving surfaces (not shown) of the receivers APDi to n. Then, each light receiver APD generates electric signals Da to Dn having a magnitude (as a function of intensity) corresponding to the intensity of the incident light, and sends it to the electronic control unit 50 described below. That is, the optical elements from the objective lens to the light receiver APD constitute a light detection system. Here, since each of the light receivers APDi to n is reflected by the corresponding dichroic mirror 26 and receives a wavelength that passes through the band-pass filter 28, each of the light receivers has a wavelength band different from each other. It should be understood that the light is received. The receiver APD may typically be an avalanche photodiode that is commonly used in fluorescence spectroscopy or confocal fluorescence microscopy.

  The electronic control unit 50 that receives electrical signals (detection signals) from the respective light receivers is a normal type storage device such as a CPU, ROM, RAM, hard disk, etc., connected to each other via a bidirectional common bus, and input / output. The computer may include a port device and a drive circuit thereof, and may operate according to a control program stored in a ROM or other storage device (a user's setting input may be appropriately performed). The electronic control device 50 functions as an analysis control system, and after performing noise removal and / or A / D conversion processing on each of the plurality of received electrical signals, 2 or non-patent documents 1 and 2, as described in various signal analysis / calculation processes, for example, cross-correlation function according to fluorescence cross-correlation spectroscopic analysis, fluctuation coincidence frequency according to confocal coincidence analysis (coincidence value) is calculated (see FIG. 3A).

  Furthermore, in the apparatus of the present invention, as described later, in the above-described configuration, from the respective lasers that are controlled by the electronic control unit 50 for the purpose of removing the crosstalk of the light detection value or the measurement value. Shutters 30a to 30n arranged in the outgoing optical path and shutters 32a to 32n arranged in the incident optical path to each light receiver are provided, and an excitation optical system is provided by the electronic control device 50 and the shutters 30a to n and 32a to n. And a control system for controlling propagation of excitation light and fluorescence of the light detection system. The shutters 30a to 32n and the shutters 32a to 32n selectively block the corresponding laser light and light from the sample S toward the corresponding light receivers APDi to n in accordance with a control command (on / off) from the electronic control unit 50. Arranged to be able to. Accordingly, when each shutter is of a type that transmits light only when the control command on is given and blocks light when the control command off is given (or vice versa). The laser beam is irradiated to the sample S only when the control command on is given to the corresponding shutter 30. Similarly, each light receiver APD is given the control command on to the corresponding shutter 32. Only radiated light (fluorescence) from the sample S is received, and a signal corresponding to the intensity of the received light is output. In other words, when the fluorescence is blocked by the corresponding shutter 32, each light receiver is prohibited from detecting light, and during that time, the light receiver transmits an electric signal having a light reception intensity of zero. The shutters 30a to 30n and the shutters 32a to 32n may be any optical element capable of selectively transmitting or blocking light in response to a control command from the electronic control unit. AOTF), a liquid crystal filter, or the like may be employed.

  The selection of the excitation light applied to the sample S and the selection of permission and prohibition of light detection in each light receiver are performed by controlling the light emission operation of the laser and the detection operation of the light receiver, respectively. Also good. In that case, the electronic control unit 50 sends a control command for permitting and prohibiting (on / off) the light emission operation to the laser, as indicated by the dotted arrow in FIG. Sends a control command for permitting or prohibiting the transmission of an electrical signal (for example, switching of connection / insulation of the output line of the electrical signal) or permitting or prohibiting the generation of an electrical signal of the receiver to the receiver. You may come to do. In addition, as shown by the dotted line in the figure, the wavelength of the excitation light is selected by placing an AOTF whose transmission wavelength can be selected in the excitation light path after all of the excitation light has been merged. The transmission wavelength, that is, the wavelength of the light reaching the sample S may be selected based on.

  Thus, in both cases, the selection of the excitation light wavelength and the light detection permission / prohibition control of each light receiver are sent to the control command on / off according to the control program stored in the electronic control device. Can be executed. That is, the measurement control system for controlling the selection of excitation light and fluorescence of the excitation optical system and the light detection system is configured by the operation of the electronic control device 50 and the shutters 30a to 30n and the shutters 32a to 32n.

As described in the "Background Art" and "Disclosure of the Invention" sections for crosstalk in light detection , a multiple light irradiation-multiple light detection type optical analyzer as illustrated in FIG. In order to execute an arbitrary fluorescence analysis at the time of obtaining a detection value or measurement value of one fluorescence intensity corresponding to each excitation light, a sample S corresponding to one excitation light is obtained. The measurement value of the emitted fluorescence is acquired in a state completely separated from the fluorescence emitted from the sample S corresponding to other excitation light, that is, the measurement value of the fluorescence emitted in response to one excitation light. In addition, a state where there is no fluorescence contribution (crosstalk) due to other excitation light may be required. However, in general, the emission wavelengths of fluorescent dyes or fluorescent molecules used in one sample overlap each other, and thus crosstalk is mixed as described above in a certain fluorescence measurement value. .

  FIG. 2A shows the relationship between the excitation light wavelength, the emission wavelength of the fluorescent molecule, and the detection wavelength band of the light receiver when two types of fluorescent molecules are present in the sample S. FIG. Referring to the figure, for example, as shown in the drawing, one fluorescent molecule A (having excitation / emission spectra λex1 and λem1) in the sample is excited by the light of laser La, and the fluorescence is shown in the drawing. Detection or measurement is performed by a light receiver APPD having a detection area, and the other fluorescent molecule B (having excitation and emission spectra λex2 and λem2) is excited with the light of the laser Lb, and the fluorescence is shown in the figure. It is assumed that detection or measurement is performed by a light receiver APDb having such a detection area. In that case, when the sample S is excited by the excitation light La and Lb, the light receiver APDa receives and detects only the emission wavelength λem1 of the fluorescent molecule A, but the detection region of the light receiver APDb includes the fluorescent molecule B. Since not only the emission wavelength λem2 but also a part on the longer wavelength side of the emission wavelength λem1 of the fluorescent molecule A enters, the detection value or measurement value of the light receiver APDb contributes to the fluorescence of the fluorescent molecule A, that is, Cross talk will be mixed. Then, when signal analysis, for example, calculation of the FCCS cross-correlation function and the frequency of simultaneous occurrence of fluctuations in CFCA, is performed using the detection values of the light receivers APDa and APDb, the values of these analysis results are received. Since the fluorescence contribution of the fluorescent molecule A (crosstalk) is mixed in the detection value of the detector APDb, the accuracy is lowered (the value is apparently increased).

  In order to eliminate the influence of the crosstalk as described above from the result of optical analysis, in the methods of Patent Documents 1 and 2, as already described, a plurality of excitation lights are sequentially applied to the sample S one by one. The fluorescence is detected in a state where the excitation light that excites the sample S at one time is combined into one. In this case, since the period during which each excitation light irradiates the sample S is grasped, the excitation light corresponding to each light receiver is selected from the detection values obtained in time series at the stage of signal analysis or arithmetic processing. And the crosstalk is eliminated from the detected value (for example, the receiver APDb receives the fluorescence of the fluorescent molecule A during the period when the laser La is selected. The detected value is excluded at the stage of signal analysis.)

  According to such a method, the influence of crosstalk is certainly excluded from the analysis result. However, as described above, the calculation process of specifying and extracting the detection value in the period when the corresponding excitation light is selected from the detection values of a certain time-series photoreceiver is a signal. The burden of processing increases. Therefore, in the apparatus of the present invention, while one excitation light is irradiated on the sample S, the detection of light is allowed only for one light receiver, and in synchronization with the sequential switching of the excitation light, The measurement control system is configured so that light receivers that are permitted to detect light are also sequentially switched. For example, in the case of the example of FIG. 2A, in the present invention, as illustrated in FIGS. 2B and 2C, when the excitation light La is selected, only the light receiver APPDa. However, when the light detection is possible (ON) and the excitation light Lb is selected, only the light receiver APDb is in the light detectable state (ON). The permission / prohibition of the detection of is controlled. According to such a control method of the present invention, the excitation light given to the sample S at one time is one kind, and the number of light receivers that are allowed to detect light at the same time is limited to one, Since the detection of light from other light receivers is prohibited, mixing of crosstalk as described above is avoided in the output of each light receiver.

Selection and switching control of excitation light and light receiver As described above, in the apparatus of the present invention, a light receiver that is allowed to detect light in synchronization with sequential switching of excitation light under the control of the electronic control device. In addition, the light transmission / blocking of the shutters 30a to 30n corresponding to the lasers La to Ln and the shutters 32a to 32n corresponding to the light receivers APDa to n is controlled so as to be sequentially switched. FIG. 3A shows, in the form of functional blocks, the configuration of the electronic control unit 50 that executes such shutter switching control and processing and analysis of signals detected by the light receiver. Such a configuration may be achieved by executing a control program in the electronic control unit 50. The illustrated configuration is activated when light measurement for optical analysis is performed in accordance with a user instruction. Although not shown for simplicity, in the electronic control unit 50, various settings of the optical analyzer, such as the wavelength band of the excitation light or the detection light, the laser to be used, and the light reception in a known manner. There are provided a part for selecting a measuring instrument, setting a measurement time of one fluorescence, the number of times of measurement, etc. based on a user input, a part for inputting parameters for various spectroscopic analyses, and the like.

  Referring to FIG. 3A, in electronic control unit 50, for switching control of the shutter, excitation light wavelength selection control unit 50a, excitation light transmission / cutoff control command determination unit 50b, detection light A transmission / cutoff control command determination unit 50c is provided. The excitation light wavelength selection control unit 50a sequentially selects the excitation light of the lasers La to Ln to be irradiated on the sample S according to a predetermined scheme, and instructs the excitation light transmission / cutoff control command determination unit 50b. And simultaneously transmitted to the detection light transmission / blocking control command determination unit 50c. For example, when excitation light of two lasers La and Lb is used in the measurement, either La or Lb is selected so that these excitation lights alternately irradiate the sample every predetermined time Which excitation light is used is instructed simultaneously to the two control command determining units 50b and 50c. The control command determination units 50b and 50c respectively select one of the shutters 30a and 30b for the excitation light paths of the lasers La and Lb, which corresponds to the designated excitation light, and the shutters 32a and 32b for the light reception light paths of the light receivers APDi to n. The one corresponding to the instructed excitation light is opened, and the other shutters are closed to block light transmission. Thus, since the excitation light wavelength selection control unit 50a alternately selects La and Lb as described above, the shutters 30a, b, 32a, and b are alternately arranged as illustrated in FIG. It will be switched (ON is open, OFF is closed).

  On the other hand, during the switching between the excitation light and the detection light as described above, the output corresponding to the fluorescence intensity detected by the light receivers APDi to n is input to the electronic control unit 50 and is normally executed in this field. After noise removal or background subtraction and A / D conversion are performed, the signal is sent to a signal analysis unit that performs signal analysis designated by the user (in addition, it is not necessary to perform analysis in real time during measurement. ). At that time, since the crosstalk is not mixed in principle in each output of the light receivers APDi to n, the signal analysis unit is information on when the excitation light is switched from the excitation light wavelength selection control unit. Therefore, it is not necessary to prepare programming for that purpose. Further, since the processing for removing the crosstalk is not executed, it is expected that the amount of signal processing is reduced and the time required for the analysis processing is also reduced.

FIG. 1 schematically shows an optical system of a fluorescence spectroscopy analyzer of a multiple light irradiation-multiple light detection type, which is one preferred embodiment of the optical analyzer of the present invention. In the figure, three lasers La to Ln and three light receivers APDi to n are shown, but the number thereof may be arbitrary. FIG. 2A shows the relationship between the excitation wavelength (one-dot chain line), the fluorescence wavelength (solid thick line), the excitation light La to b, and the detection area of the light receivers APDi to b when two types of fluorescent molecules are excited simultaneously. FIG. λex1 and λem1 are excitation and fluorescence wavelength spectra of one fluorescent molecule, respectively, and λex2 and λem2 are excitation and fluorescence wavelength spectra of the other fluorescent molecule, respectively. In the APDb detection area, crosstalk occurs in the band where the long wavelength of λem1 enters (hatched area). FIGS. 2B and 2C are diagrams similar to FIG. 2A when selection control of excitation light and detection light according to the present invention is executed. In addition, the excitation spectrum of the fluorescent molecule is omitted. (B) is a case where the excitation light La is selected. At this time, only APDA is allowed to detect light (ON), and therefore, in λem1, a band indicated by a solid line in the detection area of APDa The component that receives the fluorescence and enters the detection area of APDb is not reflected in the output of APDb. (C) shows the case where the excitation light Lb is selected. Among λem2, only APDb is allowed to detect light (ON), and the fluorescence in the band indicated by the solid line in the detection area of APDb is received. The FIG. 3A shows a part of the internal configuration of the electronic control unit of the optical analyzer of the present invention in the form of a control function block. FIG. 3B illustrates an operation scheme of the shutters 30a and 30b and the shutters 32a and 32b controlled by the configuration illustrated in FIG. 3A when the lasers La to b and the light receivers APDi to b are used for measurement. (Time change). ON represents opening (light transmission), and OFF represents closing (light blocking).

Explanation of symbols

La-n ... Laser APDa-n ... Light receivers 14a-n, 16, 26a ... Dichroic mirror 18 ... Objective lens 22 ... Pinhole 28a-n ... Bandpass filters 30a-n, 32a-n ... Shutter 50 ... Electronic control device

Claims (9)

An optical analyzer that irradiates a sample with irradiation light having a plurality of wavelength bands or wavelength characteristics and measures and analyzes radiation light having a plurality of wavelength bands or wavelength characteristics emitted from the sample in response to the plurality of irradiation lights. There,
Light irradiation means for selectively irradiating the sample with the plurality of irradiation lights;
A plurality of light detection means for selectively detecting each of the radiation beams of the plurality of wavelength bands or wavelength characteristics;
A measurement control means for performing the measurement of the radiated light by selecting the irradiation light to irradiate the sample,
The measurement control means selects the irradiation light emitted from the sample, and the light detection permission means selectively permits the detection of the emitted light by each of the plurality of light detection means. And when the irradiation light selection means selects one of the plurality of irradiation lights to be emitted to the sample, the light detection permission means selects the plurality of irradiation lights. An apparatus for permitting detection of radiated light of the light detection means for detecting the radiated light to be emitted from the sample in response to the irradiated light being applied.   The apparatus according to claim 1, wherein the irradiation light selection unit selects the plurality of irradiation lights to be sequentially irradiated on the sample one by one, and the light detection permission unit selects the irradiation light selection unit. The detection of the radiated light of the light detecting means for detecting the radiated light to be emitted from the sample corresponding to the selected irradiating light is permitted in synchronization with the selection of the irradiating light of Device to do.   The apparatus according to claim 1, wherein the light irradiation unit includes a plurality of light sources each generating irradiation light of a specific wavelength, and the irradiation light selection unit selectively selects any one of the plurality of light sources. An apparatus wherein one of the light sources is selectively brought into a state in which the irradiation light can be generated so that irradiation light from the light source can be applied to the sample.   2. The apparatus according to claim 1, wherein the irradiation light selection unit includes an optical element that selectively transmits light in a wavelength band of any one of the plurality of irradiation lights.   The apparatus according to claim 1, wherein the light detection permission unit selectively allows the radiation light to enter the light detection unit by selectively opening an optical path of the radiation light from the sample to the light detection unit. An apparatus comprising shutter means.   2. The apparatus according to claim 1, wherein the light detection permission means selectively selects the light detection means for detecting the radiated light to be emitted from the sample in response to the selected irradiation light. A device characterized in that radiation light can be detected.   2. The apparatus according to claim 1, wherein the sample includes a plurality of fluorescent molecules having different emission wavelength characteristics from each other, and each of the plurality of irradiation lights selectively excites one of the plurality of fluorescent molecules. An apparatus, wherein each of the plurality of emitted lights is one of fluorescence emitted from the plurality of fluorescent molecules.   2. The apparatus according to claim 1, wherein the optical system from the light irradiation means to the position of the sample and from the position of the sample to the light detection means constitutes an optical system of a confocal microscope. apparatus.   9. The apparatus according to claim 8, wherein the plurality of irradiation lights are applied to the sample, and the plurality of emitted lights from the sample emitted in response to the plurality of irradiation lights are measured to perform fluorescence cross-correlation spectroscopy analysis. A device characterized by performing.

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