JP2007093370A - Fluorescence spectroscopic analyzer - Google Patents

Fluorescence spectroscopic analyzer Download PDF

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JP2007093370A
JP2007093370A JP2005282686A JP2005282686A JP2007093370A JP 2007093370 A JP2007093370 A JP 2007093370A JP 2005282686 A JP2005282686 A JP 2005282686A JP 2005282686 A JP2005282686 A JP 2005282686A JP 2007093370 A JP2007093370 A JP 2007093370A
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fluorescence
light
excitation light
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excitation
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Junichi Nishimura
淳一 西村
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Olympus Corp
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Olympus Corp
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Priority to PCT/JP2006/319132 priority patent/WO2007037252A1/en
Priority to EP06810623A priority patent/EP1942334A4/en
Priority to CN2006800362769A priority patent/CN101278191B/en
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Priority to US12/056,962 priority patent/US20080225272A1/en
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent spectroscopic analyzer which is not affected by the measuring error due to cross talk. <P>SOLUTION: The fluorescent spectroscopic analyzer 100 has an exciting optical system 110, an exciting light control part 130, a fluorescence detection part 140, a signal processing part 150, and an arithmetic part 160. The exciting optical system 110 has an exciting light irradiation part 120 for emitting exciting light and the exciting light control part 130 controls the exciting light irradiation part 120 so as to exclusively irradiate a sample S with beams of exciting light different in wavelength so as to shift time. The exciting light irradiation part 120 is constituted so as to irradiate the same region on the sample S with beams of exciting light different in wavelength and the fluorescence detection part 140 is constituted so as to detect the fluorescence emitted from the sample S corresponding to the irradiation with beams of exciting light. The signal processing part 150 processes the signal reflecting the intensity of the fluorescence obtained in the fluorescence detection part 140. The arithmetic part 160 is constituted so as to perform the correlative analysis of the fluctuation of fluorescence at every different wavelength on the basis of a change in the wavelengths of beams of the exciting light thrown on the sample S due to the exciting light control part 130 and the detection result of the fluorescence detection part 140. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、蛍光分光分析装置に関する。   The present invention relates to a fluorescence spectroscopic analyzer.

蛍光相関分光分析法（ＦＣＳ法）は、顕微鏡視野の微小観測領域内で蛍光分子のブラウン運動が作り出す光の揺らぎを解析することにより、蛍光強度の自己相関関数を求め、分子毎の拡散時間や平均分子数を解析する手法であり、例えば非特許文献１に詳述されている。   Fluorescence correlation spectroscopy (FCS method) calculates the autocorrelation function of fluorescence intensity by analyzing the fluctuation of the light generated by the Brownian motion of fluorescent molecules within the microscopic observation region of the microscope field of view. This is a method for analyzing the average number of molecules, which is described in detail in Non-Patent Document 1, for example.

蛍光相互相関分光分析法（ＦＣＣＳ法）は、異なる蛍光信号間の相互相関関数を求めることにより、両者の関連性を解析する手法で、２色の蛍光色素で標識された分子間の相互作用の解析に用いられ、例えば非特許文献２と非特許文献３に詳述されている。蛍光相互相関分光分析法は拡散時間の少ない蛋白質の相互作用などには適している。同様の解析法に共焦点蛍光コインシデンス分析（ＣＦＣＡ法）があり、これは非特許文献４に詳述されている。
「蛍光相関分光法による１分子検出」金城著、蛋白質核酸酵素、1999, vol. 44N09 1431-1438 "Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution", Petra. Schwille et al, Biophysical Journal 1997, 72, 1878-1886 Adynamic 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. U.S.A. 96: 1375-1378, 1999 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. Fluorescence cross-correlation spectroscopy is suitable for protein interactions with a short diffusion time. A similar analysis method is confocal fluorescence coincidence analysis (CFCA method), which is described in detail in Non-Patent Document 4.
"Single molecule detection by fluorescence correlation spectroscopy", Kinjo, Protein Nucleic Acid Enzyme, 1999, vol. 44N09 1431-1438 "Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution", Petra. Schwille et al, Biophysical Journal 1997, 72, 1878-1886 Adynamic 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

相互相関分光分析法（ＦＣＣＳ法）において、蛍光スペクトルに重なりがある２つの蛍光色素を用いた場合、クロストークによる測定誤差の影響を受け、正確に相互相関演算することができない。   In the cross-correlation spectroscopic analysis method (FCCS method), when two fluorescent dyes having overlapping fluorescence spectra are used, the cross-correlation calculation cannot be performed accurately due to measurement error caused by crosstalk.

本発明は、このような実状を考慮してなされたものであり、その目的は、クロストークによる測定誤差の影響を受けずに正確に相互相関演算できる蛍光分光装置を提供することである。   The present invention has been made in consideration of such a situation, and an object of the present invention is to provide a fluorescence spectrometer capable of performing a cross-correlation calculation accurately without being affected by a measurement error due to crosstalk.

本発明による蛍光分光分析装置は、異なる波長の励起光を試料上の同一部位に照射可能な励起光学手段と、前記異なる波長の励起光が時間をずらして排他的に前記試料に照射されるように前記励起光学手段を制御する励起光制御手段と、前記励起光の照射に応じて前記試料から発生する蛍光を検出する蛍光検出手段と、前記励起光制御手段による前記試料に照射される励起光の波長の変化と、前記蛍光検出手段の検出結果とに基づいて、前記異なる波長ごとの前記蛍光のゆらぎの相関分析を行なう演算手段とを備えている。   In the fluorescence spectroscopic analyzer according to the present invention, the excitation optical means capable of irradiating the same part of the sample with excitation light of different wavelengths and the excitation light of the different wavelengths are irradiated exclusively on the sample at different times. Excitation light control means for controlling the excitation optical means, fluorescence detection means for detecting fluorescence generated from the sample in response to irradiation of the excitation light, and excitation light irradiated on the sample by the excitation light control means Computing means for performing a correlation analysis of the fluctuation of the fluorescence for each of the different wavelengths based on the change in wavelength of the light and the detection result of the fluorescence detection means.

本発明によれば、クロストークによる測定誤差の影響を受けずに正確に相互相関演算できる蛍光分光装置が提供される。   According to the present invention, there is provided a fluorescence spectroscopic device that can accurately perform cross-correlation calculation without being affected by a measurement error due to crosstalk.

以下、図面を参照しながら本発明の実施形態について説明する。   Hereinafter, embodiments of the present invention will be described with reference to the drawings.

本発明の実施形態について説明する前に、まず、図７を参照しながら、相互相関分光分析法（ＦＣＣＳ法）におけるクロストーク発生のしくみについて説明する。図７は、蛍光スペクトルに重なりがある２つの蛍光色素においてクロストークが発生している場合の様子を表している。図７において、第１励起スペクトルを持つ第１蛍光色素に第１励起光を照射すると、第１蛍光スペクトルを持つ第１蛍光が第１蛍光色素から発生する。第１蛍光は、フィルターを用いて第１光信号検出範囲内の波長の光を選択的に検出することにより検出される。また、第２励起スペクトルを持つ第２蛍光色素に第２励起光を照射すると、第２蛍光スペクトルを持つ第２蛍光が第２蛍光色素から発生する。第２蛍光は、フィルターを用いて第２光信号検出範囲内の波長の光を選択的に検出することにより検出される。図７から明らかなように、第１蛍光スペクトルの右すそが第２光信号検出範囲に重なっている。このため、第２蛍光を検出する際に第１蛍光も一緒に検出されてしまい、これがクロストークとなる。その結果、相互相関分光分析法（ＦＣＣＳ法）においては、クロストークによる測定誤差の影響を受け、正確に相互相関演算することができない。   Before describing the embodiment of the present invention, first, the mechanism of crosstalk generation in the cross-correlation spectroscopy (FCCS method) will be described with reference to FIG. FIG. 7 shows a state in which crosstalk occurs in two fluorescent dyes having overlapping fluorescence spectra. In FIG. 7, when the first excitation light is irradiated to the first fluorescent dye having the first excitation spectrum, the first fluorescence having the first fluorescence spectrum is generated from the first fluorescent dye. The first fluorescence is detected by selectively detecting light having a wavelength within the first optical signal detection range using a filter. In addition, when the second fluorescent dye having the second excitation spectrum is irradiated with the second excitation light, second fluorescence having the second fluorescence spectrum is generated from the second fluorescent dye. The second fluorescence is detected by selectively detecting light having a wavelength within the second optical signal detection range using a filter. As is apparent from FIG. 7, the right tail of the first fluorescence spectrum overlaps the second optical signal detection range. For this reason, when the second fluorescence is detected, the first fluorescence is also detected, which results in crosstalk. As a result, in the cross-correlation spectroscopy (FCCS method), the cross-correlation calculation cannot be performed accurately due to the influence of measurement errors due to crosstalk.

＜第一実施形態＞
図１は、本発明の第一実施形態の蛍光分光分析装置を概略的に示している。図１に示されるように、蛍光分光分析装置１００は、励起光学系１１０と蛍光検出部１４０と信号処理部１５０と演算部１６０とを有している。励起光学系１１０は、励起光を生成する励起光照射部１２０と、試料Ｓが載せられるステージ１１２と、対物レンズ１１４と、励起光と蛍光を分離するダイクロイックミラー１１６とを有している。 <First embodiment>
FIG. 1 schematically shows a fluorescence spectroscopic analyzer according to a first embodiment of the present invention. As shown in FIG. 1, the fluorescence spectroscopy analyzer 100 includes an excitation optical system 110, a fluorescence detection unit 140, a signal processing unit 150, and a calculation unit 160. The excitation optical system 110 includes an excitation light irradiation unit 120 that generates excitation light, a stage 112 on which a sample S is placed, an objective lens 114, and a dichroic mirror 116 that separates excitation light and fluorescence.

蛍光分光分析装置１００はさらに、異なる波長の励起光が時間をずらして排他的に試料Ｓに照射されるように励起光照射部１２０を制御する励起光制御部１３０を有している。従って、励起光照射部１２０は、異なる波長の励起光を試料Ｓ上の同一部位に所定のタイミングで繰り返し照射し得る。   The fluorescence spectroscopic analyzer 100 further includes an excitation light control unit 130 that controls the excitation light irradiation unit 120 so that excitation light of different wavelengths is irradiated onto the sample S exclusively at different times. Therefore, the excitation light irradiation unit 120 can repeatedly irradiate the same site on the sample S with excitation light having different wavelengths at a predetermined timing.

試料Ｓは、異なる波長の励起光が照射される部位に、異なる波長の励起光にそれぞれ反応して蛍光を発する異なる色素を含んでいる。異なる色素の蛍光スペクトルは少なくとも一部重なっている。   The sample S includes different dyes that emit fluorescence in response to excitation light of different wavelengths, at sites irradiated with excitation light of different wavelengths. The fluorescence spectra of the different dyes overlap at least partly.

蛍光検出部１４０は、励起光の照射に応じて試料Ｓから発生する蛍光を検出する。   The fluorescence detection unit 140 detects fluorescence generated from the sample S in response to irradiation with excitation light.

信号処理部１５０は、蛍光検出部１４０で得られる蛍光強度を反映した信号を処理する。演算部１６０は、励起光制御部１３０による試料Ｓに照射される励起光の波長の変化と、蛍光検出部１４０の検出結果とに基づいて、異なる波長ごとの蛍光のゆらぎの相関分析を行なう。例えば演算部１６０は、それぞれの蛍光に対応した出力信号同士の比較に基づいて自己相関分析または相互相関分析または共焦点蛍光コインシデンス分析解析する。   The signal processing unit 150 processes a signal reflecting the fluorescence intensity obtained by the fluorescence detection unit 140. The calculation unit 160 performs correlation analysis of fluorescence fluctuations at different wavelengths based on the change in the wavelength of the excitation light applied to the sample S by the excitation light control unit 130 and the detection result of 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 corresponding to the respective fluorescences.

図２は、励起光照射部１２０の構成例を示している。この例では、励起光照射部１２０は、第１光源１２２ａと、第２光源１２２ｂと、ミラー１２４ａと、ダイクロイックミラー１２４ｂと、音響光学素子（ＡＯＴＦ）１２６とから構成されている。第１光源１２２ａと第２光源１２２ｂは互いに波長帯域が異なる第１励起光と第２励起光をそれぞれ発する。ミラー１２４ａは第１光源１２２ａから発せられた第１励起光を図１の対物レンズ１１４に向けて反射する。ダイクロイックミラー１２４ｂは、ミラー１２４ａで反射された第１励起光を透過し、第２光源１２２ｂから発せられた第２励起光を図１の対物レンズ１１４に向けて反射する。第１光源１２２ａと第２光源１２２ｂは連続的に駆動され、第１励起光と第２励起光をそれぞれ発し続ける。音響光学素子１２６は、第１励起光と第２励起光が通過する共通の光路中に配置されていて、通過帯域を制御可能であり、図１の励起光制御部１３０から供給される励起光操作信号に応じて、第１励起光と第２励起光のいずれか一方を選択的に透過する。つまり音響光学素子１２６は、第１光源１２２ａと第２光源１２２ｂの発する異なる波長の光の中から試料Ｓに照射される光を選択する。励起光操作信号は時系列的に変化する信号であり、音響光学素子１２６は第１励起光と第２励起光を交互に透過する。   FIG. 2 shows a configuration example of the excitation light irradiation unit 120. In this example, the excitation light irradiation unit 120 includes a first light source 122a, a second light source 122b, a mirror 124a, a dichroic mirror 124b, and an acousto-optic element (AOTF) 126. The first light source 122a and the second light source 122b respectively emit first excitation light and second excitation light having different wavelength bands. The mirror 124a reflects the first excitation light emitted from the first light source 122a toward the objective lens 114 in FIG. The dichroic mirror 124b transmits the first excitation light reflected by the mirror 124a, and reflects the second excitation light emitted from the second light source 122b toward the objective lens 114 in FIG. The first light source 122a and the second light source 122b are continuously driven and continue to emit the first excitation light and the second excitation light, respectively. The acoustooptic device 126 is disposed in a common optical path through which the first excitation light and the second excitation light pass, and can control the passband. The excitation light supplied from the excitation light control unit 130 in FIG. One of the first excitation light and the second excitation light is selectively transmitted according to the operation signal. That is, the acoustooptic device 126 selects the light to be applied to the sample S from the light of different wavelengths emitted from the first light source 122a and the second light source 122b. The excitation light operation signal is a signal that changes in time series, and the acoustooptic device 126 alternately transmits the first excitation light and the second excitation light.

図３は、励起光照射部１２０の別の構成例を示している。この例では、励起光照射部１２０は、第１光源１２２ａと、第２光源１２２ｂと、ミラー１２４ａと、ダイクロイックミラー１２４ｂと、切替器１２８とから構成されている。第１光源１２２ａと第２光源１２２ｂとミラー１２４ａとダイクロイックミラー１２４ｂの機能は図２の例と同様である。さらに第１光源１２２ａと第２光源１２２ｂはオンオフ制御可能である。切替器１２８は、図１の励起光制御部１３０から供給される励起光操作信号に応じて、第１光源１２２ａと第２光源１２２ｂのいずれか一方を選択的にオンにし、他方を選択的にオフにする。つまり切替器１２８は、発光する光源を選択する。励起光操作信号は時系列的に変化する信号であり、第１光源１２２ａと第２光源１２２ｂが交互にオンされる。その結果、試料Ｓに第１励起光と第２励起光が交互に照射される。つまり切替器１２８は、第１光源１２２ａと第２光源１２２ｂの発する異なる波長の光の中から試料Ｓに照射される光を選択する。   FIG. 3 shows another configuration example of the excitation light irradiation unit 120. In this example, the excitation light irradiation unit 120 includes a first light source 122a, a second light source 122b, a mirror 124a, a dichroic mirror 124b, and a switch 128. The functions of the first light source 122a, the second light source 122b, the mirror 124a, and the dichroic mirror 124b are the same as in the example of FIG. Further, the first light source 122a and the second light source 122b can be controlled on and off. The switch 128 selectively turns on one of the first light source 122a and the second light source 122b and selectively turns the other according to the excitation light operation signal supplied from the excitation light control unit 130 of FIG. Turn off. That is, the switch 128 selects a light source that emits light. The excitation light operation signal is a signal that changes in time series, and the first light source 122a and the second light source 122b are alternately turned on. As a result, the sample S is irradiated with the first excitation light and the second excitation light alternately. That is, the switch 128 selects the light irradiated to the sample S from the light of different wavelengths emitted from the first light source 122a and the second light source 122b.

図１に戻り、蛍光検出部１４０は、ダイクロイックミラー１４２と、第１蛍光フィルター１４４ａと、第２蛍光フィルター１４４ｂと、第１受光素子１４６ａと、第２受光素子１４６ｂとから構成されている。第１蛍光フィルター１４４ａは第１蛍光を選択的に透過し、第２蛍光フィルター１４４ｂは第２蛍光を選択的に透過する。第１受光素子１４６ａは第１蛍光の波長帯域に感度を有し、第２受光素子１４６ｂは第２蛍光の波長帯域に感度を有している。つまり、第１受光素子１４６ａと第２受光素子１４６ｂは、異なる波長帯域に感度を有している。   Returning to FIG. 1, the fluorescence detection unit 140 includes a dichroic mirror 142, a first fluorescence filter 144a, a second fluorescence filter 144b, a first light receiving element 146a, and a second light receiving element 146b. The first fluorescence filter 144a selectively transmits the first fluorescence, and the second fluorescence filter 144b selectively transmits the second fluorescence. The first light receiving element 146a has sensitivity in the wavelength band of the first fluorescence, and the second light receiving element 146b has sensitivity in the wavelength band of the second fluorescence. That is, the first light receiving element 146a and the second light receiving element 146b have sensitivity in different wavelength bands.

以下、図４のフローチャートを参照しながら、本実施形態の蛍光分光分析装置の動作について説明する。   Hereinafter, the operation of the fluorescence spectrometer of the present embodiment will be described with reference to the flowchart of FIG.

励起光制御部１３０は励起光操作信号を生成し、これを励起光照射部１２０に出力する。励起光操作信号は、図５に示されるように、周期的に変化する「０」と「１」の二値信号である。   The excitation light control unit 130 generates an excitation light operation signal and outputs it to the excitation light irradiation unit 120. As shown in FIG. 5, the excitation light operation signal is a binary signal of “0” and “1” that periodically changes.

励起光照射部１２０が図２の構成の場合、音響光学素子１２６は、励起光操作信号が「０」のときに第１励起光を選択的に透過し、励起光操作信号が「１」のときに第２励起光を選択的に透過する。   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.

また励起光照射部１２０が図３の構成の場合、切替器１２８は、励起光操作信号が「０」のときに第１光源１２２ａを選択的にオンし、励起光操作信号が「１」のときに第２光源１２２ｂを選択的にオンにする。   When the excitation light irradiation unit 120 has the configuration shown in FIG. 3, the switch 128 selectively turns on the first light source 122a when the excitation light operation signal is “0”, and the excitation light operation signal is “1”. Sometimes the second light source 122b is selectively turned on.

その結果、試料Ｓに照射される励起光は、図５に示されるように、第１励起光と第２励起光が交互に切り替わるものとなる。   As a result, as shown in FIG. 5, the excitation light applied to the sample S is alternately switched between the first excitation light and the second excitation light.

励起光の照射に応じて試料Ｓから蛍光が発生する。第１受光素子１４６ａは、第１励起光の照射に応じて試料Ｓから発生する第１蛍光を検出し、図５に示される第１蛍光検出信号を信号処理部１５０に出力する。第２受光素子１４６ｂは、第２励起光の照射に応じて試料Ｓから発生する第２蛍光を検出し、図５に示される第２蛍光検出信号を信号処理部１５０に出力する。   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. 5 to the signal processing unit 150. The second light receiving element 146 b 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. 5 to the signal processing unit 150.

信号処理部１５０は、蛍光検出部１４０の第１受光素子１４６ａと第２受光素子１４６ｂからそれぞれ供給される第１蛍光検出信号と第２蛍光検出信号を一定時間ごとの蛍光強度信号に変換し、その蛍光強度信号と励起光制御信号を最適な形で組み合わせて演算用データを生成し、これを演算部１６０に出力する。   The signal processing unit 150 converts the first fluorescence detection signal and the second fluorescence detection signal respectively supplied from the first light receiving element 146a and the second light receiving element 146b of the fluorescence detection unit 140 into a fluorescence intensity signal for each predetermined time, The fluorescence intensity signal and the excitation light control signal are combined in an optimal form to generate calculation data, which is output to the calculation unit 160.

演算部１６０は、信号処理部１５０から供給される演算用データに基づいて自己相関分析または相互相関分析または共焦点蛍光コインシデンス分析を実施する
本実施形態では、異なる波長の第１励起光と第２励起光とが時間をずらして排他的に試料Ｓに照射される。第１励起光と第２励起光の照射に応じて試料Ｓからそれぞれ発生する第１蛍光と第２蛍光は、それぞれ、蛍光検出部１４０の第１受光素子１４６ａと第２受光素子１４６ｂによって検出される。このため、クロストークによる測定誤差の影響を受けずに正確な相互相関演算を行なえる。 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 this embodiment, the first excitation light and the second excitation light having different wavelengths The sample S is irradiated exclusively with the excitation light at different times. The first fluorescence and the second fluorescence generated from the sample S in response to the irradiation of the first excitation light and the second excitation light are respectively detected by the first light receiving element 146a and the second light receiving element 146b of the fluorescence detection unit 140. The Therefore, an accurate cross-correlation calculation can be performed without being affected by measurement errors due to crosstalk.

＜第二実施形態＞
図６は、本発明の第二実施形態の蛍光分光分析装置を概略的に示している。本実施形態の蛍光分光分析装置２００は、蛍光検出部２４０のほかは、第一実施形態の蛍光分光分析装置１００と同様である。 <Second embodiment>
FIG. 6 schematically shows a fluorescence spectroscopic analyzer according to the second embodiment of the present invention. The fluorescence spectroscopic analysis apparatus 200 of this embodiment is the same as the fluorescence spectroscopic analysis apparatus 100 of the first embodiment except for the fluorescence detection unit 240.

蛍光検出部２４０は、マルチバンドフィルター２４２と受光素子２４４とから構成されている。マルチバンドフィルター２４２は第１蛍光と第２蛍光を選択的に透過する。受光素子２４４は第１蛍光と第２蛍光を検出可能な受光帯域を有している。つまり、受光素子２４４は、異なる励起光による異なる波長の蛍光を検出可能な受光帯域を有している。   The fluorescence detection unit 240 includes a multiband filter 242 and a light receiving element 244. The multiband filter 242 selectively transmits the first fluorescence and the second fluorescence. The light receiving element 244 has a light receiving band capable of detecting the first fluorescence and the second fluorescence. That is, the light receiving element 244 has a light receiving band capable of detecting fluorescence of different wavelengths by different excitation light.

本実施形態では、異なる波長の第１励起光と第２励起光とが時間をずらして排他的に試料Ｓに照射される。第１励起光と第２励起光の照射に応じて試料Ｓからそれぞれ発生する第１蛍光と第２蛍光は、ひとつの蛍光検出部２４０の受光素子２４４によって時分割に検出される。このため、クロストークによる測定誤差の影響を受けずに正確な相互相関演算を行なえる。   In the present embodiment, the sample S is exclusively irradiated with the first excitation light and the second excitation light having different wavelengths while shifting the time. The first fluorescence and the second fluorescence respectively generated from the sample S in response to the irradiation of the first excitation light and the second excitation light are detected in a time division manner by the light receiving element 244 of one fluorescence detection unit 240. Therefore, an accurate cross-correlation calculation can be performed without being affected by measurement errors due to crosstalk.

これまで、図面を参照しながら本発明の実施形態を述べたが、本発明は、これらの実施形態に限定されるものではなく、その要旨を逸脱しない範囲において様々な変形や変更が施されてもよい。   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.

本発明の第一実施形態の蛍光分光分析装置を概略的に示している。1 schematically shows a fluorescence spectrometer of a first embodiment of the present invention. 図１に示された励起光照射部の構成例を示している。2 illustrates a configuration example of an excitation light irradiation unit illustrated in FIG. 1. 図１に示された励起光照射部の別の構成例を示している。3 shows another configuration example of the excitation light irradiation unit shown in FIG. 1. 図１に示された蛍光分光分析装置の動作のフローチャートを示している。2 shows a flowchart of the operation of the fluorescence spectrometer shown in FIG. 図１に示された蛍光分光分析装置における信号のタイムチャートを示している。2 is a time chart of signals in the fluorescence spectroscopic analyzer shown in FIG. 本発明の第二実施形態の蛍光分光分析装置を概略的に示している。1 schematically shows a fluorescence spectroscopic analyzer of a second embodiment of the present invention. 蛍光スペクトルに重なりがある２つの蛍光色素においてクロストークが発生する様子を表している。This shows how crosstalk occurs in two fluorescent dyes with overlapping fluorescence spectra.

符号の説明Explanation of symbols

１００…蛍光分光分析装置、１１０…励起光学系、１１２…ステージ、１１４…対物レンズ、１１６…ダイクロイックミラー、１２０…励起光照射部、１２２ａ…光源、１２２ｂ…光源、１２４ａ…ミラー、１２４ｂ…ダイクロイックミラー、１２６…音響光学素子、１２８…切替器、１３０…励起光制御部、１４０…蛍光検出部、１４２…ダイクロイックミラー、１４４ａ…蛍光フィルター、１４４ｂ…蛍光フィルター、１４６ａ…受光素子、１４６ｂ…受光素子、１５０…信号処理部、１６０…演算部、２００…蛍光分光分析装置、２４０…蛍光検出部、２４２…マルチバンドフィルター、２４４…受光素子。 DESCRIPTION OF SYMBOLS 100 ... Fluorescence spectroscopy analyzer, 110 ... Excitation optical system, 112 ... Stage, 114 ... Objective lens, 116 ... Dichroic mirror, 120 ... Excitation light irradiation part, 122a ... Light source, 122b ... Light source, 124a ... Mirror, 124b ... Dichroic mirror 126 ... acousto-optic device, 128 ... switch, 130 ... excitation light control unit, 140 ... fluorescence detection unit, 142 ... dichroic mirror, 144a ... fluorescence filter, 144b ... fluorescence filter, 146a ... light receiving device, 146b ... light receiving device, DESCRIPTION OF SYMBOLS 150 ... Signal processing part, 160 ... Operation part, 200 ... Fluorescence spectroscopy analyzer, 240 ... Fluorescence detection part, 242 ... Multiband filter, 244 ... Light receiving element.

Claims (8)

異なる波長の励起光を試料上の同一部位に照射可能な励起光学手段と、
前記異なる波長の励起光が時間をずらして排他的に前記試料に照射されるように前記励起光学手段を制御する励起光制御手段と、
前記励起光の照射に応じて前記試料から発生する蛍光を検出する蛍光検出手段と、
前記励起光制御手段による前記試料に照射される励起光の波長の変化と、前記蛍光検出手段の検出結果とに基づいて、前記異なる波長ごとの前記蛍光のゆらぎの相関分析を行なう演算手段とを具備することを特徴とする蛍光分光分析装置。 Excitation optical means capable of irradiating the same site on the sample with excitation light of different wavelengths;
Excitation light control means for controlling the excitation optical means so that the excitation light of the different wavelengths is irradiated to the sample exclusively at different times, and
Fluorescence detecting means for detecting fluorescence generated from the sample in response to irradiation of the excitation light;
An arithmetic means for performing a correlation analysis of the fluctuations of the fluorescence for the different wavelengths based on a change in the wavelength of the excitation light applied to the sample by the excitation light control means and a detection result of the fluorescence detection means; A fluorescence spectroscopic analyzer characterized by comprising. 前記試料は、前記異なる波長の励起光が照射される部位（前記同一部位）に、前記異なる波長の励起光にそれぞれ反応して蛍光を発する異なる色素を含んでおり、当該異なる色素の蛍光スペクトルは少なくとも一部重なっていることを特徴とする請求項１に記載の蛍光分光分析装置。   The sample includes a different dye that emits fluorescence in response to the excitation light of the different wavelength at the site irradiated with the excitation light of the different wavelength (the same site), and the fluorescence spectrum of the different dye is The fluorescence spectroscopic analyzer according to claim 1, wherein at least partly overlaps. 前記励起光学手段は、異なる波長の励起光を所定のタイミングで繰り返し前記試料に対して照射することを特徴とする請求項１に記載の蛍光分光分析装置。   2. The fluorescence spectroscopic analyzer according to claim 1, wherein the excitation optical unit repeatedly irradiates the sample with excitation light having different wavelengths at a predetermined timing. 前記励起光学手段は、異なる波長の光を発する複数の光源と、当該光源の発する異なる波長の光の中から前記試料に照射される光を選択する手段と、を含むことを特徴とする請求項１に記載の蛍光分光分析装置。   The excitation optical unit includes a plurality of light sources that emit light of different wavelengths, and a unit that selects light irradiated on the sample from light of different wavelengths emitted by the light sources. The fluorescence spectroscopic analyzer according to 1. 前記照射される光を選択する手段は、前記異なる波長の複数の光が通過する共通の光路中に配置されることを特徴とする請求項４に記載の蛍光分光装置。   The fluorescence spectroscopic apparatus according to claim 4, wherein the means for selecting the irradiated light is arranged in a common optical path through which the plurality of lights having different wavelengths pass. 前記照射される光を選択する手段は、発光する光源を選択する光源切替手段または通過帯域を制御可能な音響光学素子であることを特徴とする請求項４に記載の蛍光分光装置。   5. The fluorescence spectroscopic apparatus according to claim 4, wherein the means for selecting the irradiated light is a light source switching means for selecting a light source to emit light or an acousto-optic element capable of controlling a pass band. 前記蛍光検出手段は、異なる波長帯域に感度を有する複数の受光素子で構成されていることを特徴とする請求項１に記載の蛍光分光分析装置。   2. 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. 前記蛍光検出手段は、前記異なる励起光による異なる波長の蛍光を検出可能な受光帯域を有するひとつの受光素子で構成されていることを特徴とする請求項１に記載の蛍光分光装置。   2. The fluorescence spectroscopic apparatus according to claim 1, wherein the fluorescence detection unit is configured by one light receiving element having a light receiving band capable of detecting fluorescence of different wavelengths by the different excitation light.
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