JP4474582B2 - 蛍光の検出方法 - Google Patents

蛍光の検出方法 Download PDF

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JP4474582B2
JP4474582B2 JP2004016516A JP2004016516A JP4474582B2 JP 4474582 B2 JP4474582 B2 JP 4474582B2 JP 2004016516 A JP2004016516 A JP 2004016516A JP 2004016516 A JP2004016516 A JP 2004016516A JP 4474582 B2 JP4474582 B2 JP 4474582B2
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ジンメルマン ベルンハルト
ジムビュエルガー エヴァ
ディッキンソン メリー
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

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Description

従来、多重マーキングのなされた、光散乱性の強い試料における個別蛍光色素の検出は、可視領域内での励起波長の選択およびそれに対応する放出用フィルタの使用によって実施できた。
オーバラップする放出スペクトルに対しては、重回帰分析を伴う放出スペクトル捕捉法（Ｓｃｈａｅｆｅｒの特許）が実施できる。そこで使用される検出器は、通例ＰＭＴｓ（点像検出器）であり、光路内にて顕微鏡走査光学系の後に設置される。その場合、可視レーザの浸透深度は、試料の如何によって一部大きく制限される。
光散乱性の強い着色試料における蛍光の生成に多光子レーザを使用すれば、放出信号をフィルタで検出することも可能になる。その場合、放出スペクトルが全く、あるいはごく僅かしかオーバラップしていない蛍光信号であれば互いに分離することができる。
個々の蛍光色素に対する、しかるべき波長による逐次的励起とそれぞれの信号の検出（マルチトラッキング）とを組合せることは、多光子顕微鏡法ではほとんどの場合不可能である。この方法では、複数の蛍光色素が一波長で同時に励起されることが多く、蛍光色素は単光子励起の場合より広い励起スペクトルを示すからである。
それぞれの放出スペクトルが過度にオーバラップする個別蛍光色素に対して多光子顕微鏡法を適用する場合でも、その放出信号は、ＰＭＴアレーおよびそれに前置接続された分散素子（特許文献１）によって、分光学的に捕捉することも、続いて回帰分析によって分離させることもできる。
ＵＳ ６ ４０３ ３３２
従来のいずれの方法でも、光散乱性の強い試料の深部領域について言えば、確かに蛍光信号は生成されるが、しかし検出が十分にできないという問題が発生する。
この問題は特許請求の範囲に記載された特徴によって解消される。
本発明は、同調可能型超短パルスレーザによる励起スペクトルの形成と、様々な波長における単一蛍光色素／複数蛍光色素の強度記録用ノンデスキャン検出器（散乱光検出の最適手段、信号送信は走査光学系にはよらない）の使用とを組合せている。
これらの像は、励起スペクトル形成のために、および多重マーキングのなされた、光散乱性の強い試料における個別蛍光色素の強度比率を回帰分析によって測定するために使用される。超短パルスレーザとノンデスキャン検出器の組合せ使用によって、光散乱性の強い試料においても励起スペクトルの捕捉および過度にオーバラップした放出スペクトルからの信号分離が可能になる。
本発明方法は次のようにして行われる ：
励起スペクトルの捕捉
蛍光性試料を多光子レーザにより照射する。光線はスキャナにより試料上に導かれ、生成された信号が光学素子を通じてノンデスキャン検出器に導かれて、蛍光信号の総強度が捕捉される。超短パルスレーザの波長は所定のステップに従って変更され、各波長毎に総強度が検出器により改めて捕捉されて、それぞれの個別波長で生成された蛍光信号の像が捕捉順に保存される。
励起スペクトルが各蛍光色素毎に測定される。
多重着色試料の蛍光強度が（レーザ強度の設定、走査過程、検出器の調整に関して）同一条件下で測定される。個別蛍光色素の励起スペクトルが、回帰分析（Ｓｃｈａｅｆｅｒの特許）の適用下で、多重マーキングのなされた試料における蛍光色素成分の測定に利用される。
図１には、ダイクロイック・ビームスプリッタＳＴ１およびＸ／Ｙ型走査装置ＳＣを通じて試料Ｐを照射する、同調可能な短パルスレーザＫＰが模式図として描かれている。
共焦点絞りＰＨの後方に配置された波長選択性の検出器ＭＴのほかに、ノンデスキャン検出器ＮＤＴ１およびＮＤＴ２が設置されている。ＮＤＴ１は、ビームスプリッタＳＴ２経由の散乱光も含めた試料Ｐからの励起光のためのもので、ＮＤＴ２は、試料内で生成されて試料を通過した散乱光をも含めた光を対象にしている。この光路には励起光のブロッキング用として励起フィルタＡＦが設置されている。
短パルスレーザの同調は、例えば約７００〜９００ｎｍの波長領域で行われる。その場合、ＮＤＴ１またはＮＤＴ２で測定した強度を対象として、例えば約７５０ｎｍおよび８００ｎｍにおいて励起極大値を示す蛍光体がまず波長別に捕捉される。
この両蛍光体が一つの試料中に存在すれば、レーザの同調時には混合スペクトルが記録されるが、続いて回帰分析により分離することができる。
本発明方法を実施する装置の模式図
符号の説明
ＳＴ１、ＳＴ２ ダイクロイック・ビームスプリッタ
ＳＣ スキャナ
Ｐ 試料
ＫＰ 超短パルスレーザ
ＰＨ 共焦点絞り
ＭＴ ＭＥＴＡ検出器
ＮＤＴ１、ＮＤＴ２ ノンデスキャン検出器
ＡＦ 励起フィルタ

Claims (1)

  1. 蛍光性試料内で短パルスレーザによって生成された光の検出および評価のための方法であって、
    少なくとも第１と第２の蛍光体および／または自己蛍光性試料が様々な波長で別々に照射されて、
    試料から放出された光が、波長別に二個のノンデスキャン検出器により参照スペクトルとして記録され、
    試料から放出された光が、蛍光検出器によって測定スペクトルとして検出され、
    その場合、少なくとも２つの蛍光体および／または自己蛍光性試料の照射と同時に、測定スペクトルと参照スペクトルからの回帰分析により個別の蛍光スペクトルに分離され、
    散乱光を含む試料からの励起光が第１のノンデスキャン検出器により検出され、試料内で生成され、かつ該試料を通過した散乱光を含む光が第２のノンデスキャン検出器により検出される、方法。
JP2004016516A 2003-01-27 2004-01-26 蛍光の検出方法 Expired - Fee Related JP4474582B2 (ja)

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JP4835730B2 (ja) * 2009-08-06 2011-12-14 横河電機株式会社 蛍光量または吸光量の測定方法および測定装置
US20130087718A1 (en) * 2010-06-28 2013-04-11 Ge Healthcare Bio-Sciences Corp. Confocal fluorescence lifetime imaging system
US10027855B2 (en) * 2010-06-30 2018-07-17 Ge Healthcare Bio-Science Corp. System for synchronization in a line scanning imaging microscope
US9222870B2 (en) * 2010-12-10 2015-12-29 The Regents Of The University Of California Method and device for multi-parameter imaging within a single fluorescent channel
CN102778301B (zh) * 2012-07-31 2014-10-01 中国科学院上海光学精密机械研究所 自参考光谱干涉飞秒激光脉冲的实时测量装置
DE102016210357A1 (de) * 2016-06-10 2017-12-14 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtung zur Erfassung einer Belegung einer Oberfläche mittels induzierter Fluoreszenz

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JP2004233351A (ja) 2004-08-19
EP1441218A3 (de) 2004-10-06
US7119898B2 (en) 2006-10-10
DE10303404A1 (de) 2004-08-05
EP1441218A2 (de) 2004-07-28

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