JP6564661B2 - Apparatus response function measurement method, fluorescence measurement method, and apparatus response function measurement member - Google Patents
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Description
本発明は、蛍光測定装置の装置応答関数を測定する装置応答関数測定方法、この装置応答関数測定方法により測定された装置応答関数に基づいて蛍光減衰曲線または蛍光寿命値を求める蛍光測定方法、および、この装置応答関数測定方法において用いられる装置応答関数測定用部材に関するものである。 The present invention relates to a device response function measurement method for measuring a device response function of a fluorescence measurement device, a fluorescence measurement method for obtaining a fluorescence decay curve or a fluorescence lifetime value based on the device response function measured by this device response function measurement method, and The present invention relates to a device response function measuring member used in this device response function measuring method.
蛍光測定装置は、励起光を出力して試料に照射する励起光源と、この励起光の照射によって試料中の蛍光色素から発生した蛍光を検出する検出器と、を備える。蛍光測定装置は、蛍光強度を検出する場合や、蛍光画像から蛍光色素の分布を取得する場合の他、蛍光減衰曲線を測定する場合にも用いられ、さらに、この蛍光減衰曲線に基づいて蛍光寿命値を測定する場合にも用いられる。 The fluorescence measuring apparatus includes an excitation light source that outputs excitation light and irradiates the sample, and a detector that detects fluorescence generated from a fluorescent dye in the sample by irradiation of the excitation light. The fluorescence measuring device is used not only to detect fluorescence intensity, to acquire the distribution of the fluorescent dye from the fluorescence image, but also to measure the fluorescence decay curve, and further, based on this fluorescence decay curve, the fluorescence lifetime is used. It is also used when measuring values.
蛍光減衰曲線を測定する場合、理想的には、出力するパルス励起光のパルス幅がゼロである励起光源を用いるとともに、応答時間がゼロである検出器を用いて、励起光源から出力されたパルス励起光を試料に照射し、その試料で発生した蛍光の強度の時間的変化を検出器により測定することが必要である。 When measuring a fluorescence decay curve, ideally, an excitation light source with a pulse width of the output pulse excitation light of zero is used, and a detector with a response time of zero is used to output the pulse output from the excitation light source. It is necessary to irradiate the sample with excitation light and measure the temporal change in the intensity of fluorescence generated in the sample with a detector.
しかし、現実には、そのような励起光源や検出器は存在しないので、有限のパルス幅を有するパルス励起光を出力する励起光源を用いるとともに、有限の応答時間を有する検出器を用いざるを得ない。この場合、歪んだ蛍光減衰曲線が得られることになる。 However, in reality, there are no such excitation light sources and detectors, so it is necessary to use an excitation light source that outputs pulse excitation light having a finite pulse width and a detector that has a finite response time. Absent. In this case, a distorted fluorescence decay curve is obtained.
蛍光測定装置の装置応答関数（Instrument Response Function、ＩＲＦ）は、パルス励起光の時間波形と蛍光測定装置の応答とのコンボリューションで表される。測定される蛍光減衰曲線は、この装置応答関数と蛍光応答とのコンボリューションで表される。したがって、装置応答関数が既知であれば、測定された蛍光減衰曲線に基づいてデコンボリューション計算を行うことにより、正しい蛍光減衰曲線を得ることができ、さらには、正しい蛍光寿命値を得ることができる。その為には、蛍光測定装置の装置応答関数を求めておくことが必要である。 An instrument response function (IRF) of the fluorescence measurement device is represented by a convolution of the time waveform of the pulse excitation light and the response of the fluorescence measurement device. The measured fluorescence decay curve is represented by a convolution of the instrument response function and the fluorescence response. Therefore, if the instrument response function is known, a correct fluorescence decay curve can be obtained by performing a deconvolution calculation based on the measured fluorescence decay curve, and further, a correct fluorescence lifetime value can be obtained. . For this purpose, it is necessary to obtain a device response function of the fluorescence measuring device.
特許文献１および非特許文献１には、蛍光測定装置の装置応答関数を測定する技術が記載されている。非特許文献１に記載された装置応答関数測定技術は、試料で発生する蛍光を観測するときと同じ体積からの散乱を利用するものであり、コロイド懸濁液や、アルミニウムまたは銀のホイル等の散乱反射体を、試料に替えて用いる。特許文献１に記載された装置応答関数測定技術は、パルス励起光の一部を検出器へ導く別の光学系を備える。 Patent Document 1 and Non-Patent Document 1 describe a technique for measuring a device response function of a fluorescence measuring device. The apparatus response function measurement technique described in Non-Patent Document 1 uses scattering from the same volume as when observing fluorescence generated in a sample, such as colloidal suspension, aluminum or silver foil, etc. A scattering reflector is used instead of the sample. The apparatus response function measurement technique described in Patent Document 1 includes another optical system that guides part of the pulsed excitation light to the detector.
本発明者らは、特許文献１および非特許文献１に記載された技術では、蛍光測定装置の装置応答関数を正確に測定することができないことを見出した。すなわち、コロイド懸濁液を用いる場合には、そのコロイド懸濁液における励起光または蛍光の吸収や多重散乱により測定精度が悪くなる。散乱反射体を用いる場合には、試料における蛍光発生領域より散乱反射体における励起光照射範囲が大きいと、測定精度が悪くなる。特許文献１に記載された技術は、本来の蛍光測定の光学系とは別の光学系を用いて装置応答関数を求めることから、本来の装置応答関数を求めるものではない。 The present inventors have found that the technique described in Patent Literature 1 and Non-Patent Literature 1 cannot accurately measure the device response function of the fluorescence measuring device. That is, when using a colloidal suspension, the measurement accuracy deteriorates due to absorption or multiple scattering of excitation light or fluorescence in the colloidal suspension. When the scattering reflector is used, the measurement accuracy is deteriorated if the excitation light irradiation range in the scattering reflector is larger than the fluorescence generation region in the sample. The technique described in Patent Document 1 does not obtain the original device response function because the device response function is obtained using an optical system different from the original optical system for fluorescence measurement.
本発明は、上記問題点を解消する為になされたものであり、蛍光測定装置の装置応答関数をより正確に測定することができる装置応答関数測定方法を提供することを目的とする。また、本発明は、より正確な蛍光減衰曲線または蛍光寿命値を求めることができる蛍光測定方法を提供することを目的とする。さらに、本発明は、装置応答関数測定方法において用いられる装置応答関数測定用部材を提供することを目的とする。 The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a device response function measurement method capable of measuring the device response function of a fluorescence measurement device more accurately. Another object of the present invention is to provide a fluorescence measurement method capable of obtaining a more accurate fluorescence decay curve or fluorescence lifetime value. Another object of the present invention is to provide a device response function measuring member used in the device response function measuring method.
本発明の装置応答関数測定方法は、パルス励起光を出力して試料に照射する励起光源と、パルス励起光の照射に伴い試料中の蛍光色素から発生した蛍光を検出する検出器と、を備える蛍光測定装置の装置応答関数を測定する方法であって、パルス励起光を透過または鏡面反射させる基板の面の一部領域に拡散反射部を有する装置応答関数測定用部材を、蛍光測定装置において試料が配置されるべき位置に、励起光源から基板へのパルス励起光の照射により生じる鏡面反射光が検出器に入射しない方位となるように設置し、励起光源から出力されたパルス励起光を装置応答関数測定用部材の拡散反射部に照射して、その照射により拡散反射部で生じる拡散反射光を検出器により検出し、検出器による検出結果に基づいて蛍光測定装置の装置応答関数を求める。 An apparatus response function measurement method of the present invention includes an excitation light source that outputs pulsed excitation light and irradiates a sample, and a detector that detects fluorescence generated from a fluorescent dye in the sample accompanying irradiation of the pulsed excitation light. A method for measuring an apparatus response function of a fluorescence measuring apparatus, wherein a device response function measuring member having a diffuse reflection part in a partial region of a surface of a substrate that transmits or mirror-reflects pulse excitation light is sampled in a fluorescence measuring apparatus. Is placed in a position where the specular reflection light generated by the irradiation of the pulsed excitation light from the excitation light source to the substrate is not incident on the detector, and the pulse excitation light output from the excitation light source responds to the device. Irradiate the diffuse reflection part of the function measurement member, detect the diffuse reflection light generated in the diffuse reflection part by the irradiation, and detect the response of the fluorescence measurement device based on the detection result by the detector Determine the number.
本発明では、装置応答関数測定用部材において拡散反射部が設けられる領域を、試料における蛍光発生領域に対応したものとするのが好適である。また、試料における蛍光発生領域を撮像した結果に基づいて、装置応答関数測定用部材において拡散反射部が設けられる領域を決定するのが好適である。 In the present invention, it is preferable that the region where the diffuse reflection portion is provided in the apparatus response function measurement member corresponds to the fluorescence generation region in the sample. Further, it is preferable to determine a region where the diffuse reflection portion is provided in the apparatus response function measurement member based on the result of imaging the fluorescence generation region in the sample.
本発明の蛍光測定方法は、上記の本発明の装置応答関数測定方法により求めた装置応答関数と、試料中の蛍光色素から発生した蛍光について検出器による検出結果とに基づいて、蛍光減衰曲線または蛍光寿命値を求める。 The fluorescence measurement method of the present invention is based on the device response function obtained by the above-described device response function measurement method of the present invention and the detection result by the detector for the fluorescence generated from the fluorescent dye in the sample. Obtain fluorescence lifetime values.
本発明の装置応答関数測定用部材は、パルス励起光を出力して試料に照射する励起光源と、パルス励起光の照射に伴い試料中の蛍光色素から発生した蛍光を検出する検出器と、を備える蛍光測定装置の装置応答関数を測定する方法において用いられ、パルス励起光を透過または鏡面反射させる基板の面の一部領域に拡散反射部を有する。 The apparatus response function measuring member of the present invention includes an excitation light source that outputs pulsed excitation light and irradiates the sample, and a detector that detects fluorescence generated from the fluorescent dye in the sample accompanying irradiation of the pulsed excitation light. It is used in a method for measuring a device response function of a fluorescence measuring device provided, and has a diffuse reflection part in a partial region of the surface of a substrate that transmits or mirror-reflects pulsed excitation light.
本発明によれば、蛍光測定装置の装置応答関数をより正確に測定することができ、さらに、より正確な蛍光減衰曲線または蛍光寿命値を求めることができる。 According to the present invention, the apparatus response function of the fluorescence measuring apparatus can be measured more accurately, and a more accurate fluorescence decay curve or fluorescence lifetime value can be obtained.
以下、添付図面を参照して、本発明を実施するための形態を詳細に説明する。なお、図面の説明において同一の要素には同一の符号を付し、重複する説明を省略する。本発明は、これらの例示に限定されるものではなく、特許請求の範囲によって示され、特許請求の範囲と均等の意味および範囲内でのすべての変更が含まれることが意図される。 DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. The present invention is not limited to these exemplifications, but is defined by the scope of the claims, and is intended to include all modifications within the meaning and scope equivalent to the scope of the claims.
先ず、本発明者らが本発明を想到するに至った経緯について説明する。本発明者らは、非特許文献１に記載された技術について検証実験を行った。その結果、測定された蛍光減衰曲線の半値全幅より、得られた装置応答関数の半値全幅が大きくなる場合があった。この傾向は、特に蛍光寿命が短い場合に見られた。このような場合、デコンボリューション計算に基づいて蛍光減衰曲線や蛍光寿命値を正確に求めることができない。すなわち、測定される蛍光減衰曲線の半値全幅より装置応答関数の半値全幅は小さくなければならない。 First, how the present inventors came to come up with the present invention will be described. The present inventors conducted a verification experiment on the technique described in Non-Patent Document 1. As a result, the full width at half maximum of the obtained device response function may be larger than the full width at half maximum of the measured fluorescence decay curve. This tendency was seen especially when the fluorescence lifetime was short. In such a case, the fluorescence decay curve and the fluorescence lifetime value cannot be obtained accurately based on the deconvolution calculation. That is, the full width at half maximum of the device response function must be smaller than the full width at half maximum of the fluorescence decay curve to be measured.
また、本発明者らは、励起光を吸収する黒色基板の面の一部領域に散乱反射体を有する装置応答関数測定用部材を作製し、これを用いて装置応答関数を求めた。このような装置応答関数測定用部材を用いれば、試料における蛍光発生領域と散乱反射体における励起光散乱領域とを互いに同程度とすることができ、より正しい装置応答関数を得ることができると期待された。しかし、この場合においても、測定された蛍光減衰曲線の半値全幅より、得られた装置応答関数の半値全幅が大きくなる場合があった。 In addition, the present inventors produced a device response function measuring member having a scattering reflector in a partial region of the surface of the black substrate that absorbs excitation light, and obtained the device response function using this. If such a device response function measurement member is used, it is expected that the fluorescence generation region in the sample and the excitation light scattering region in the scattering reflector can be approximately equal to each other, and a more accurate device response function can be obtained. It was done. However, even in this case, the full width at half maximum of the obtained device response function may be larger than the full width at half maximum of the measured fluorescence decay curve.
さらに、本発明者らは、上記のような装置応答関数測定用部材を用いた場合の問題発生原因を解明すべく、散乱反射体を設けることなく黒色基板のみを用いて装置応答関数を試みに求めてみた。しかし、この場合においても、測定された蛍光減衰曲線の半値全幅より、得られた装置応答関数の半値全幅が大きくなる場合があった。これは、黒色基板からの拡散反射光が検出器により受光されることに因ると考えられる。 Furthermore, the present inventors tried to make a device response function using only a black substrate without providing a scattering reflector in order to elucidate the cause of the problem when the device response function measuring member as described above is used. I asked for it. However, even in this case, the full width at half maximum of the obtained device response function may be larger than the full width at half maximum of the measured fluorescence decay curve. This is thought to be due to the fact that diffusely reflected light from the black substrate is received by the detector.
本発明者らは、以上のような知見に基づいて本発明を想到した。以下に、蛍光測定装置の構成について説明するとともに、本実施形態の装置応答関数測定方法、蛍光測定方法および装置応答関数測定用部材について説明する。 The present inventors have conceived the present invention based on the above findings. Hereinafter, the configuration of the fluorescence measuring apparatus will be described, and the apparatus response function measuring method, the fluorescence measuring method, and the apparatus response function measuring member of the present embodiment will be described.
図１は、蛍光測定装置１０の構成を示す図である。この蛍光測定装置１０は、本実施形態の装置応答関数測定方法により装置応答関数を測定するのに好適なものである。蛍光測定装置１０は、試料室１１、励起光源１５、検出器１６および処理部１７を備える。試料室１１は、内部に試料９０を配置するもので、開口部１２〜１４を有する。開口部１２は、試料室１１外部に配置された励起光源１５から出力されたパルス励起光ＬＥを試料室１１内部に導く。開口部１３，１４については後述する。検出器１６は、パルス励起光ＬＥの照射に伴い試料９０中の蛍光色素から発生した蛍光ＬＦを検出する。処理部１７は、検出器１６による検出結果に基づいて所要の処理（後述）を行う。 FIG. 1 is a diagram showing the configuration of the fluorescence measuring apparatus 10. This fluorescence measuring apparatus 10 is suitable for measuring an apparatus response function by the apparatus response function measuring method of the present embodiment. The fluorescence measurement device 10 includes a sample chamber 11, an excitation light source 15, a detector 16, and a processing unit 17. The sample chamber 11 has a sample 90 disposed therein and has openings 12 to 14. Opening 12 guides the output pulse excitation light L E from the sample chamber 11 the excitation light source 15 disposed outside the inside of the sample chamber 11. The openings 13 and 14 will be described later. Detector 16 detects the fluorescence L F generated from the fluorescent dye in the sample 90 with the irradiation of the pulsed excitation light L E. The processing unit 17 performs a required process (described later) based on the detection result by the detector 16.
図２は、本実施形態の装置応答関数測定用部材２０の構成を示す図である。図２（ａ）は上面図であり、図２（ｂ）は側面図である。装置応答関数測定用部材２０は、蛍光測定装置１０の装置応答関数を測定する際に用いられる。装置応答関数測定用部材２０は、基板２１、拡散反射部２２およびベース部２３を備える。基板２１は、パルス励起光ＬＥを透過または鏡面反射させる。基板２１は、パルス励起光ＬＥを拡散反射させないか、または、拡散反射が極めて小さい。拡散反射部２２は、基板２１の面の一部領域に配置されており、パルス励起光ＬＥを拡散反射させることができる。拡散反射部２２は、蛍光を有さない白色物質や金属粉末が好ましい。装置応答関数測定用部材２０において拡散反射部２２が設けられる領域は、試料９０における蛍光発生領域に対応したものとするのが好ましい。ベース部２３は、基板２１を保持する。 FIG. 2 is a diagram showing a configuration of the device response function measuring member 20 of the present embodiment. 2A is a top view, and FIG. 2B is a side view. The device response function measuring member 20 is used when measuring the device response function of the fluorescence measuring device 10. The apparatus response function measurement member 20 includes a substrate 21, a diffuse reflection part 22, and a base part 23. Substrate 21 and transmits or specular pulsed excitation light L E. Substrate 21 is either not diffuse reflected pulsed excitation light L E, or diffuse reflection is very small. Diffusion reflectors 22 is disposed on a portion of the surface of the substrate 21 can be diffused reflected pulsed excitation light L E. The diffuse reflection part 22 is preferably a white substance or metal powder that does not have fluorescence. It is preferable that the region where the diffuse reflection portion 22 is provided in the device response function measuring member 20 corresponds to the fluorescence generation region in the sample 90. The base part 23 holds the substrate 21.
図３は、蛍光測定装置１０において装置応答関数測定用部材２０を配置した構成を示す図である。本実施形態の装置応答関数測定方法により装置応答関数を測定する際には、装置応答関数測定用部材２０は、蛍光測定装置１０において試料９０が配置されるべき位置に設置される。また、装置応答関数測定用部材２０は、励起光源１５から基板２１へのパルス励起光ＬＥ２の照射により生じる鏡面反射光ＬＲが検出器１６に入射しない方位となるように設置される。 FIG. 3 is a diagram showing a configuration in which the apparatus response function measuring member 20 is arranged in the fluorescence measuring apparatus 10. When the device response function is measured by the device response function measurement method of the present embodiment, the device response function measurement member 20 is installed at a position where the sample 90 is to be placed in the fluorescence measurement device 10. The device response function measuring member 20 is installed so as specularly reflected light L R caused by the irradiation of the pulsed excitation light L E2 from the excitation light source 15 to the substrate 21 is the orientation which is not incident on the detector 16.
パルス励起光ＬＥ２の照射により基板２１で生じる透過光ＬＴは、開口部１３を通過して、試料室１１外部に設けられた光トラップ１８により吸収される。パルス励起光ＬＥ２の照射により基板２１で生じる鏡面反射光ＬＲは、開口部１４を通過して、試料室１１外部に設けられた光トラップ１９により吸収される。これら透過光ＬＴおよび鏡面反射光ＬＲは、検出器１６に入射しない。 Transmitted light L T generated in the substrate 21 by irradiation of the pulsed excitation light L E2 passes through the opening 13, it is absorbed by the sample chamber 11 a light trap 18 provided outside. Specular reflection light L R generated in the substrate 21 by irradiation of the pulsed excitation light L E2 passes through the opening 14, it is absorbed by the sample chamber 11 a light trap 19 provided outside. These transmitted light L T and specularly reflected light L R is not incident on the detector 16.
検出器１６は、励起光源１５から拡散反射部２２へのパルス励起光ＬＥ１の照射により生じる拡散反射光ＬＳを検出する。処理部１７は、拡散反射光ＬＳを受光した検出器１６から出力される電気信号に基づいて装置応答関数を求め、更に蛍光減衰曲線や蛍光寿命値を求める。 The detector 16 detects the diffuse reflected light L S generated by the irradiation of the pulse excitation light L E1 from the excitation light source 15 to the diffuse reflection unit 22. Processor 17 obtains a device response function based on the electric signal output from the detector 16 which receives the diffuse reflection light L S, further obtains the fluorescence decay curves and fluorescence lifetime values.
このような構成とすることで、試料９０における蛍光発生領域に対応した領域に設けられた拡散反射部２２からの拡散反射光ＬＳのみが検出器１６により受光されるので、正確な装置応答関数を求めることができる。 With such a configuration, since only the diffuse reflection light L S from the diffuse reflection part 22 provided in the region corresponding to the fluorescence generation region in the sample 90 is received by the detector 16, an accurate device response function is obtained. Can be requested.
なお、上記の構成では光トラップ１８，１９により透過光ＬＴおよび鏡面反射光ＬＲを吸収することとしたが、透過光ＬＴおよび鏡面反射光ＬＲが弱い場合には、開口部１３，１４に替えて暗幕や黒色材を設置して、これにより透過光ＬＴおよび鏡面反射光ＬＲを吸収することとしてもよい。 In the configuration described above it was to absorb the transmitted light L T and specularly reflected light L R by an optical trap 18 and 19, but if a weak transmitted light L T and specularly reflected light L R, the opening 13, It established the blackout curtain or black material instead of 14, thereby it is also possible to absorb the transmitted light L T and specularly reflected light L R.
次に、装置応答関数測定用部材２０において拡散反射部２２が設けられる領域を決める方法の例について、図４および図５を用いて説明する。 Next, an example of a method for determining a region where the diffuse reflection portion 22 is provided in the apparatus response function measurement member 20 will be described with reference to FIGS. 4 and 5.
図４は、パルス励起光の大部分が試料を透過するくらい光学的に試料が希薄である場合における拡散反射部２２の設置領域の決定方法を説明する図である。図４（ａ）に示されるように、容器３１内の試料３２にパルス励起光ＬＥを入射させたときにパルス励起光ＬＥの大部分が試料３２を透過する場合、試料３２中のパルス励起光の光路の全領域において蛍光が発生することになる。そこで、図４（ｂ）に示されるように、基板３３上において拡散反射部３４は、パルス励起光の光路の全領域に亘って設けられる。基板３３は図２における基板２１に相当し、拡散反射部３４は図２における拡散反射部２２に相当する。 FIG. 4 is a diagram for explaining a method of determining the installation region of the diffuse reflector 22 when the sample is optically thin enough that most of the pulsed excitation light passes through the sample. As shown in FIG. 4 (a), if a majority of the pulsed excitation light L E when the sample 32 in the container 31 is made incident pulsed excitation light L E is transmitted through the sample 32, pulse in the sample 32 Fluorescence is generated in the entire region of the optical path of the excitation light. Therefore, as shown in FIG. 4B, the diffuse reflector 34 is provided over the entire region of the optical path of the pulse excitation light on the substrate 33. The substrate 33 corresponds to the substrate 21 in FIG. 2, and the diffuse reflector 34 corresponds to the diffuse reflector 22 in FIG.
図５は、パルス励起光の試料を透過できないくらい光学的に試料が濃厚である場合における拡散反射部２２の設置領域の決定方法を説明する図である。図５（ａ）に示されるように、容器４１内の試料４２にパルス励起光ＬＥを入射させたときに、パルス励起光ＬＥが途中で吸収されてしまい、試料４２中でパルス励起光が吸収されるまでの光路の領域において蛍光が発生することになる。そこで、図５（ｂ）に示されるように、基板４３上において拡散反射部４４は、パルス励起光が吸収されるまでの光路の領域に設けられる。基板４３は図２における基板２１に相当し、拡散反射部４４は図２における拡散反射部２２に相当する。 FIG. 5 is a diagram for explaining a method for determining the installation region of the diffuse reflector 22 when the sample is optically thick enough to not pass through the pulse excitation light sample. As shown in FIG. 5 (a), when the sample 42 in the container 41 is made incident pulsed excitation light L E, pulsed excitation light L E is absorbed in the middle, pulsed excitation light in the sample 42 Fluorescence is generated in the region of the optical path until is absorbed. Therefore, as shown in FIG. 5B, the diffuse reflector 44 is provided on the substrate 43 in the region of the optical path until the pulsed excitation light is absorbed. The substrate 43 corresponds to the substrate 21 in FIG. 2, and the diffuse reflection portion 44 corresponds to the diffuse reflection portion 22 in FIG.
試料４２が濃厚である場合、実際には、パルス励起光ＬＥは、試料４２への入射位置で最も強く、試料４２中を進むに従って徐々に弱くなる。実用上は、試料４２への入射位置でのパルス励起光の強度に対し概ね１／２の強度になる位置までの領域に拡散反射部４４を設ければよい。この場合、試料４２の１ｃｍ当りの光学濃度をＯＤとすると、光量が１／２になる距離ｄ（ｃｍ）は下記（１）式で近似的に表される。例えば、ＯＤ＝３である場合、ｄ＝０.１ｃｍであるから、約１ｍｍの大きさの拡散反射部４４を設ければよい。 If the sample 42 is thick, in fact, pulsed excitation light L E is strongest at the incident position of the sample 42, and gradually becomes weaker as the process proceeds the sample 42 inside. Practically, the diffuse reflection part 44 may be provided in a region up to a position where the intensity is approximately ½ of the intensity of the pulse excitation light at the incident position on the sample 42. In this case, assuming that the optical density per 1 cm of the sample 42 is OD, the distance d (cm) at which the light quantity is halved is approximately expressed by the following equation (1). For example, when OD = 3, since d = 0.1 cm, the diffuse reflection part 44 having a size of about 1 mm may be provided.
なお、検出器１６に替えて撮像器を配置し、試料９０における蛍光発生領域を撮像器により撮像した結果に基づいて、装置応答関数測定用部材２０において拡散反射部２２が設けられる領域を決定するのも好ましい。 Note that an image pickup device is arranged instead of the detector 16, and the region where the diffuse reflection portion 22 is provided in the device response function measurement member 20 is determined based on the result of imaging the fluorescence generation region in the sample 90 by the image pickup device. It is also preferable.
次に、本実施形態の蛍光測定方法について説明する。本実施形態の蛍光測定方法は、上記のような装置応答関数測定方法により求めた装置応答関数と、試料９０中の蛍光色素から発生した蛍光について検出器１６による検出結果とに基づいて、蛍光減衰曲線または蛍光寿命値を求めるものであり、処理部１７により以下のようにして行われる。 Next, the fluorescence measurement method of this embodiment will be described. The fluorescence measurement method of the present embodiment is based on the apparatus response function obtained by the apparatus response function measurement method as described above and the fluorescence decay generated from the fluorescent dye in the sample 90 based on the detection result by the detector 16. A curve or a fluorescence lifetime value is obtained, and is performed by the processing unit 17 as follows.
装置応答関数測定方法により測定された装置応答関数をＥ(t)とする。試料９０中の蛍光色素から発生した蛍光について検出器１６により測定された蛍光減衰曲線をＦ(ｔ)とする。ｔは時間変数である。 The device response function measured by the device response function measurement method is defined as E (t). Let F (t) be the fluorescence decay curve measured by the detector 16 for the fluorescence generated from the fluorescent dye in the sample 90. t is a time variable.
また、試料９０中にｎ種類の蛍光分子種が存在する可能性があるとし、これらｎ種類の蛍光分子種のうち第ｉの蛍光分子種の成分量をＡｉとするとともに蛍光寿命値をτｉとする。なお、蛍光分子種とは、試料中に存在する蛍光を発する個々の分子種を意味する。試料中で蛍光色素が単一の分子種として存在する場合、単純に蛍光色素そのものを蛍光分子種と見なすことができる。一方、例えば、タンパク質に結合した蛍光色素のように、蛍光色素が様々な分子種として存在し得る場合は、１つの蛍光色素であっても複数の蛍光分子種を持つ。これらｎ種類の蛍光分子種から発生する蛍光の減衰曲線Ｇ(ｔ)は、下記（２）式で表わされる。 Also, n types of fluorescent molecular species may exist in the sample 90, and the component amount of the i-th fluorescent molecular species among these n types of fluorescent molecular species is A i and the fluorescence lifetime value is τ. Let i . The fluorescent molecular species means individual molecular species that emit fluorescence present in the sample. When a fluorescent dye exists as a single molecular species in a sample, the fluorescent dye itself can be simply regarded as a fluorescent molecular species. On the other hand, for example, when a fluorescent dye can exist as various molecular species such as a fluorescent dye bonded to a protein, even one fluorescent dye has a plurality of fluorescent molecular species. A decay curve G (t) of fluorescence generated from these n kinds of fluorescent molecular species is expressed by the following equation (2).
装置応答関数がＥ(ｔ)である蛍光測定装置を用いて蛍光減衰曲線Ｇ(ｔ)を測定したとき、その測定により得られる蛍光減衰曲線Ｉ(ｔ)は、Ｅ(ｔ)とＧ(ｔ)とのコンボリューションにより下記（３）式で表される。この式中のＣは装置の暗雑音を表す。 When the fluorescence decay curve G (t) is measured using a fluorescence measurement device whose instrument response function is E (t), the fluorescence decay curve I (t) obtained by the measurement is E (t) and G (t ) And the following equation (3). C in this equation represents the background noise of the device.
このＩ(ｔ)をＦ(ｔ)にフィッティングすることにより、試料９０中に存在する第ｉの蛍光分子種の成分量Ａｉおよび蛍光寿命値τｉを求める。すなわち、非線形最適化法により、下記（４）式で表されるχ２値が最小となるときの成分量Ａｉおよび蛍光寿命値τｉを求める。ここで、ｍ１およびｍ２は、フィッティングを行う時間範囲を表す。 By fitting this I (t) to F (t), the component amount A i and the fluorescence lifetime value τ i of the i-th fluorescent molecular species present in the sample 90 are obtained. That is, the component amount A i and the fluorescence lifetime value τ i when the χ 2 value represented by the following equation (4) is minimized are obtained by the nonlinear optimization method. Here, m1 and m2 represent time ranges for performing fitting.
次に、比較例と対比しつつ実施例について説明する。実施例および比較例において、蛍光色素チオフラビンＴ（ＴｈＴ）で染色したＡβアミロイドを試料として用いた。この試料におけるＴｈＴ濃度は５μＭであった。励起波長４０５ｎｍにおける試料の光学濃度ＯＤ値は０.６である。濃度５μＭのＴｈＴの蛍光減衰曲線の半値全幅は２１５ｐｓである。 Next, examples will be described in comparison with comparative examples. In Examples and Comparative Examples, Aβ amyloid stained with the fluorescent dye thioflavin T (ThT) was used as a sample. The ThT concentration in this sample was 5 μM. The optical density OD value of the sample at an excitation wavelength of 405 nm is 0.6. The full width at half maximum of the fluorescence decay curve of ThT at a concentration of 5 μM is 215 ps.
実施例では、上記（１）式にＯＤ＝０.６を代入して得られるｄ＝５ｍｍの大きさの拡散反射部２２を有する装置応答関数測定用部材２０を作製し、この装置応答関数測定用部材２０を図３に示されるように蛍光測定装置１０に配置して装置応答関数を測定した。基板２１はガラス基板であり、拡散反射部２２はアルミドットであった。比較例では、非特許文献１に記載されている散乱体溶液であるＲｕｄｏｘ懸濁液（シリカの懸濁水溶液）を用いて装置応答関数を測定した。 In the embodiment, a device response function measuring member 20 having a diffuse reflection part 22 having a size of d = 5 mm obtained by substituting OD = 0.6 into the above equation (1) is manufactured, and this device response function measurement is performed. The member 20 was placed on the fluorescence measuring device 10 as shown in FIG. 3, and the device response function was measured. The substrate 21 was a glass substrate, and the diffuse reflection part 22 was an aluminum dot. In the comparative example, the instrument response function was measured using a Rudox suspension (silica suspension) which is a scatterer solution described in Non-Patent Document 1.
図６は、実施例および比較例それぞれで得られた装置応答関数を示す図である。この図に示されるように、実施例で得られた装置応答関数の半値全幅（２０４ｐｓ）は、比較例で得られた装置応答関数の半値全幅（２３１ｐｓ）より小さい。 FIG. 6 is a diagram showing device response functions obtained in each of the example and the comparative example. As shown in this figure, the full width at half maximum (204 ps) of the device response function obtained in the example is smaller than the full width at half maximum (231 ps) of the device response function obtained in the comparative example.
試料を図１に示されるように蛍光測定装置１０に配置して、パルス励起光照射により試料で発生する蛍光を検出器１６により検出した。蛍光を受光した検出器１６から出力された電気信号を処理部１７へ送り、蛍光減衰曲線を得た。そして、試料中に４種類の蛍光分子種が含まれると仮定して、上記の蛍光測定方法により４種類の蛍光分子種それぞれの成分量および蛍光寿命値を求めた。 The sample was placed in the fluorescence measuring apparatus 10 as shown in FIG. 1, and the fluorescence generated in the sample by pulse excitation light irradiation was detected by the detector 16. The electrical signal output from the detector 16 that received the fluorescence was sent to the processing unit 17 to obtain a fluorescence decay curve. Then, assuming that the sample includes four types of fluorescent molecular species, the component amounts and fluorescence lifetime values of the four types of fluorescent molecular species were determined by the above-described fluorescence measurement method.
図７は、実施例および比較例で得られた４種類の蛍光分子種それぞれの成分量および蛍光寿命値を纏めた表である。Ｒｕｄｏｘ懸濁液を用いた比較例では、得られた装置応答関数が不正確であることから、フィッティングの程度の目安であるχ２値が１０以上と大きく、フィッティングとしては不適合であり、或る蛍光分子種の成分量が負値になるなど、信頼性が低い結果となった。これに対して、本実施形態の装置応答関数測定用部材を用いた実施例では、正確な装置応答関数を測定することができたことにより、χ２値が１.４と小さく、各蛍光分子種の成分量が妥当であり、信頼性が高い結果が得られた。 FIG. 7 is a table summarizing the component amounts and fluorescence lifetime values of the four types of fluorescent molecular species obtained in the examples and comparative examples. In the comparative example using the Rudox suspension, since the obtained device response function is inaccurate, the χ 2 value, which is a measure of the degree of fitting, is as large as 10 or more, which is incompatible as a fitting. The results showed low reliability, such as the amount of components of fluorescent molecular species being negative. On the other hand, in the example using the apparatus response function measuring member of the present embodiment, the accurate apparatus response function could be measured, so that the χ 2 value was as small as 1.4, and each fluorescent molecule The amount of seed component was reasonable and reliable results were obtained.
このような本実施例の効果は、装置応答関数を測定する際に用いられる装置応答関数測定用部材において拡散反射部が設けられる領域が試料における蛍光発生領域に対応したものであること、および、拡散反射部以外の他の箇所からの反射光が検出器１６に入射しないようにしたことにより、実現されたものである。 The effect of this embodiment is that the region where the diffuse reflection portion is provided in the device response function measurement member used when measuring the device response function corresponds to the fluorescence generation region in the sample, and This is realized by preventing the reflected light from other places other than the diffuse reflection part from entering the detector 16.
１０…蛍光測定装置、１１…試料室、１２〜１４…開口部、１５…励起光源、１６…検出器、１７…処理部、１８，１９…光トラップ、２０…装置応答関数測定用部材、２１…基板、２２…拡散反射部、２３…ベース部、３１…容器、３２…試料、３３…基板、３４…拡散反射部、４１…容器、４２…試料、４３…基板、４４…拡散反射部、９０…試料、ＬＥ，ＬＥ１，ＬＥ２…パルス励起光、ＬＦ…蛍光、ＬＲ…鏡面反射光、ＬＳ…拡散反射光、ＬＴ…透過光。 DESCRIPTION OF SYMBOLS 10 ... Fluorescence measuring apparatus, 11 ... Sample chamber, 12-14 ... Opening part, 15 ... Excitation light source, 16 ... Detector, 17 ... Processing part, 18, 19 ... Optical trap, 20 ... Device response function measurement member, 21 DESCRIPTION OF SYMBOLS ... Board | substrate, 22 ... Diffuse reflection part, 23 ... Base part, 31 ... Container, 32 ... Sample, 33 ... Substrate, 34 ... Diffuse reflection part, 41 ... Container, 42 ... Sample, 43 ... Substrate, 44 ... Diffuse reflection part, 90 ... sample, L E, L E1, L E2 ... pulsed excitation light, L F ... fluorescent, L R ... specular reflection light, L S ... diffuse reflected light, L T ... transmitted light.
Claims (5)
前記パルス励起光を透過または鏡面反射させる基板の面の一部領域に拡散反射部を有する装置応答関数測定用部材を、前記蛍光測定装置において試料が配置されるべき位置に、前記励起光源から前記基板へのパルス励起光の照射により生じる鏡面反射光が前記検出器に入射しない方位となるように設置し、
前記励起光源から出力されたパルス励起光を前記装置応答関数測定用部材の前記拡散反射部に照射して、その照射により前記拡散反射部で生じる拡散反射光を前記検出器により検出し、
前記検出器による検出結果に基づいて前記蛍光測定装置の装置応答関数を求める、
装置応答関数測定方法。 An apparatus response function of a fluorescence measuring apparatus comprising: an excitation light source that outputs pulsed excitation light and irradiates the sample; and a detector that detects fluorescence generated from the fluorescent dye in the sample upon irradiation with the pulsed excitation light. A method of measuring,
An apparatus response function measurement member having a diffuse reflection part in a partial region of the surface of the substrate through which the pulse excitation light is transmitted or specularly reflected is moved from the excitation light source to the position where the sample is to be placed in the fluorescence measurement apparatus. Installed so that the specular reflection light generated by the irradiation of the pulse excitation light to the substrate is in the direction not incident on the detector,
The pulse excitation light output from the excitation light source is applied to the diffuse reflection part of the device response function measurement member, and the diffuse reflection light generated in the diffuse reflection part due to the irradiation is detected by the detector.
Obtaining a device response function of the fluorescence measurement device based on a detection result by the detector,
Device response function measurement method.
請求項１に記載の装置応答関数測定方法。 In the device response function measurement member, a region where the diffuse reflection portion is provided corresponds to a fluorescence generation region in the sample.
The apparatus response function measuring method according to claim 1.
請求項２に記載の装置応答関数測定方法。 Based on the result of imaging the fluorescence generation region in the sample, determine the region where the diffuse reflection portion is provided in the device response function measurement member,
The apparatus response function measuring method according to claim 2.
蛍光測定方法。 Fluorescence decay based on the device response function obtained by the device response function measurement method according to any one of claims 1 to 3 and the detection result by the detector for the fluorescence generated from the fluorescent dye in the sample Find a curve or fluorescence lifetime value,
Fluorescence measurement method.
前記パルス励起光を透過または鏡面反射させる基板の面の一部領域に拡散反射部を有する、
装置応答関数測定用部材。 An apparatus response function of a fluorescence measuring apparatus comprising: an excitation light source that outputs pulsed excitation light and irradiates the sample; and a detector that detects fluorescence generated from the fluorescent dye in the sample upon irradiation with the pulsed excitation light. Used in the method of measuring,
Having a diffuse reflection part in a partial region of the surface of the substrate that transmits or mirror-reflects the pulse excitation light;
Device response function measurement member.
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