JP2004061438A

JP2004061438A - Device and method for measuring changes of chemiluminescence and fluorescence with time

Info

Publication number: JP2004061438A
Application number: JP2002223686A
Authority: JP
Inventors: Shigetoshi Okazaki; 岡崎　茂俊
Original assignee: Hamamatsu Photonics KK; Bio Oriented Technology Research Advancement Institution; Sasaki Co Ltd
Current assignee: Hamamatsu Photonics KK; Bio Oriented Technology Research Advancement Institution; Sasaki Co Ltd
Priority date: 2002-07-31
Filing date: 2002-07-31
Publication date: 2004-02-26
Anticipated expiration: 2022-07-31
Also published as: JP3889334B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide a change-with-time measuring device which measures, in real time, the changes of fluorescence and chemiluminescence, respectively, with elapse of time even if the wavelengths of the fluorescence and chemiluminescence are substantially identical with each other. <P>SOLUTION: The device 1 comprises: an exciting means 10 which repeatedly irradiates excitation light on a sample to be measured pulselikely; a light selecting means 20 which selectively transmits the fluorescence and chemiluminescence, respectively, generated by the irradiation of excitation light; one light detecting means 30 which outputs an electric signal in accordance with the quantity of either both the fluorescent and the chemiluminescence or only the chemiluminescence received; and a change-with-time measuring means 40 which obtains first light intensity data in accordance with the chemiluminescence intensity within a period during which the excitation light is not emitted and second light intensity data in accordance with the fluorescence intensity and the chemiluminescence intensity within a period including a period during which the excitation light is emitted to calculate third light intensity data in accordance with the fluorescence intensity based on the first and second light intensity data, thereby measuring the changes of the fluorescence and chemiluminescence, respectively, with time. <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明の属する技術分野】
本発明は、被測定試料における化学反応に伴い発生する化学発光の経時変化と、その被測定試料への励起光の照射に伴い発生する蛍光の経時変化とを測定する測定装置および測定方法に関するものである。
【０００２】
【従来の技術】
従来から、生体関連物質等の測定においては、蛍光や化学発光を利用した測定方法が用いられている。たとえば、生体内情報伝達に関与していることが知られている細胞内カルシウムイオンは、その濃度を蛍光指示薬で検出、定量することが可能である。また、生体細胞の免疫や老化等に関与していることが知られている活性酸素、特にスーパーオキサイドや一重項酸素は、化学発光試薬による検出が可能であり、免疫系の細胞等が産生するスーパーオキサイドの検出がなされている。そして、このような免疫系細胞からの情報、たとえば細胞内カルシウムイオン濃度と細胞のスーパーオキサイド産生量とを同時に測定しようとした場合、蛍光と化学発光とを同時に検出することが必要となる。
【０００３】
免疫系の細胞として知られている白血球の一種であるヒト好中球により産生されるスーパーオキサイド（Ｏ_２ ⁻）は、人体に侵入した細菌やウイルス等を殺傷するものであり、生体防御において重要な役割を担っている。このスーパーオキサイドの産生には、好中球細胞内のカルシウムイオン濃度が関与していると考えられているが、このカルシウムの役割については不明な点が多い。このスーパーオキサイド産生を担う細胞内カルシウム濃度上昇の実体が解明されれば、例えば、慢性肉芽腫症患者の治療につながる新たな細胞機能診断法や、免疫や炎症等に関与する薬剤の作用機序の解明に役立つと期待される。
【０００４】
スーパーオキサイド産生の測定は、例えば、好中球における化学反応に伴い発生する化学発光を検出することにより可能である。また、細胞内カルシウム濃度変化の測定は、例えば、好中球にカルシウム蛍光指示薬を予め導入しておき、これに励起光を照射して発生した蛍光を検出することにより可能である。
【０００５】
蛍光及び化学発光それぞれを測定する技術としては、特許第３１８３８６３号公報記載の「化学発光および蛍光の経時変化測定装置および方法」がある。この発明は、被測定試料に励起光を繰り返しパルス的に照射して、励起光が被測定試料に照射されていないときに被測定試料で発生した化学発光を検出し、励起光が被測定試料に照射されているときに被測定試料で発生した蛍光を検出するものであり、同一試料で発生した化学発光および蛍光それぞれの経時変化を実時間で検出することができる。また、特開平１１−１４８９００号公報には、単一の検出光学系により蛍光及び化学発光それぞれの測定をすることが可能な「発光パターン読取装置」が記載されている。
【０００６】
【発明が解決しようとする課題】
しかしながら、特許第３１８３８６３号公報に記載の発明においては、蛍光を測定する蛍光検出光学系と、化学発光を測定する化学発光検出光学系とが分離されているため、蛍光及び化学発光それぞれを分光手段により分離する必要がある。このため、蛍光波長及び化学発光波長が互いに同一又は近接する場合には、蛍光と化学発光との分離が困難なため、蛍光および化学発光の測定ができないという問題を有する。
【０００７】
また、特開平１１−１４８９００号公報に記載の発明は、蛍光及び化学発光それぞれを単一の検出器により測定するものであり、蛍光波長及び化学発光波長が互いに同一又は近接する場合であっても、蛍光及び化学発光それぞれの測定を行うことができる。しかし、本装置は、試料に励起光を照射して蛍光のみを測定し、蛍光の測定とは別に励起光の照射を停止して化学発光のみを測定するものであり、同一現象における蛍光及び化学発光それぞれの経時変化を同時に測定することができない。よって、スーパーオキサイド産生と細胞内カルシウム濃度変化との間の微妙な関係を検出することができないという問題を有する。
【０００８】
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、蛍光波長及び化学発光波長が互いに同一又は近接する場合であっても、蛍光及び化学発光それぞれの経時変化を実時間で測定することができる化学発光および蛍光の経時変化測定装置および方法を提供することを目的とする。
【０００９】
【課題を解決するための手段】
本発明に係る化学発光および蛍光の経時変化測定装置は、（１）被測定試料に励起光をパルス的に繰り返し照射する励起手段と、（２）励起手段により励起光が被測定試料に照射されることにより被測定試料で発生した蛍光および被測定試料で発生した化学発光それぞれを選択して透過させる光選択手段と、（３）光選択手段により選択されて透過された蛍光および化学発光の双方又は化学発光を受光し、その受光量に応じて電気信号を出力する一の光検出手段と、（４）光検出手段により出力された電気信号に基づいて、励起手段により励起光が被測定試料に照射されていない期間内において被測定試料で発生した化学発光の光強度に応じた第１光強度データと、励起手段により励起光が被測定試料に照射されているときを含む期間内において被測定試料で発生した蛍光および化学発光の光強度に応じた第２光強度データとを求め、第２光強度データと第１光強度データとに基づいて蛍光の光強度に応じた第３光強度データを算出し、第１光強度データに基づいて化学発光の経時変化を測定し、第３光強度データに基づいて蛍光の経時変化を測定する経時変化測定手段と、を備えることを特徴とする。
【００１０】
本発明に係る化学発光および蛍光の経時変化測定方法は、被測定試料に励起光を励起手段によりパルス的に繰り返し照射して、励起手段により励起光が被測定試料に照射されることにより被測定試料で発生した蛍光および被測定試料で発生した化学発光それぞれを光選択手段により選択して透過させ、光選択手段により選択されて透過された蛍光および化学発光の双方又は化学発光を受光し、その受光量に応じて電気信号を一の光検出手段により出力し、光検出手段により出力された電気信号に基づいて、励起手段により励起光が被測定試料に照射されていない期間内において被測定試料で発生した化学発光の光強度に応じた第１光強度データと、励起手段により励起光が被測定試料に照射されているときを含む期間内において被測定試料で発生した蛍光および化学発光の光強度に応じた第２光強度データとを求め、第２光強度データと第１光強度データとに基づいて蛍光の光強度に応じた第３光強度データを算出し、第１光強度データに基づいて化学発光の経時変化を測定し、第３光強度データに基づいて蛍光の経時変化を測定する、ことを特徴とする。
【００１１】
本発明に係る化学発光および蛍光の経時変化測定装置又は方法によれば、被測定試料は、励起手段により励起光がパルス的に繰り返し照射される。励起手段により励起光が被測定試料に照射されることにより被測定試料で発生した蛍光および被測定試料で発生した化学発光それぞれは、光選択手段により選択されて透過される。そして、光選択手段により選択されて透過された蛍光および化学発光の双方又は化学発光が受光され、その受光量に応じて電気信号が一の光検出手段により出力され、光検出手段により出力された電気信号に基づいて、励起手段により励起光が被測定試料に照射されていない期間内において被測定試料で発生した化学発光の光強度に応じた第１光強度データと、励起手段により励起光が被測定試料に照射されているときを含む期間内において被測定試料で発生した蛍光および化学発光の光強度に応じた第２光強度データとが求められる。さらに、第２光強度データと第１光強度データとに基づいて蛍光の光強度に応じた第３光強度データが算出され、第１光強度データに基づいて化学発光の経時変化が測定され、第３光強度データに基づいて蛍光の経時変化が経時変化測定手段により測定される。このようにして、同一試料で発生した化学発光および蛍光それぞれの経時変化が実時間で検出される。
【００１２】
また、本発明に係る化学発光および蛍光の経時変化測定装置は、被測定試料の温度を制御する温度制御手段を更に備えることを特徴とする。
【００１３】
また、本発明に係る化学発光および蛍光の経時変化測定方法は、化学発光および蛍光それぞれの経時変化を測定するとき更に被測定試料の温度を温度制御手段により制御することを特徴とする。
【００１４】
この場合には、被測定試料の温度が温度制御手段により制御されるので、例えば被測定試料が細胞である場合に好適である。
【００１５】
また、本発明に係る化学発光および蛍光の経時変化測定装置は、被測定試料が液体であって、その被測定試料を攪拌する攪拌手段を更に備えることを特徴とする。
【００１６】
また、本発明に係る化学発光および蛍光の経時変化測定方法は、被測定試料が液体であって、化学発光および蛍光それぞれの経時変化を測定するとき更に被測定試料を攪拌手段により攪拌することを特徴とする。
【００１７】
この場合には、被測定試料が攪拌手段により攪拌されるので、被測定試料が懸濁液である場合に好適である。
【００１８】
【発明の実施の形態】
以下、添付図面を参照して本発明の実施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。
【００１９】
まず、本実施形態に係る化学発光および蛍光の経時変化測定装置の構成について説明する。図１は、本実施形態に係る化学発光および蛍光の経時変化測定装置１の構成図である。経時変化測定装置１は、励起手段１０、光選択手段２０、光検出手段３０、経時変化測定手段４０、温度制御手段５０及び攪拌手段６０を備えている。
【００２０】
励起手段１０は、励起光源１１０、シャッタ１２０、チョッパ１３０、チョッパコントローラ１６０、集光レンズ１４０及び光ファイバ１５０を備え、被測定試料に励起光をパルス的に繰り返し照射する。
【００２１】
励起光源１１０は、被測定試料に予め導入された蛍光指示薬を励起して蛍光を発生させる波長の励起光を出力する。励起光源１１０から出力された励起光はシャッタ１２０、チョッパ１３０、集光レンズ１４０を経て光ファイバ１５０へ導かれる。光ファイバ１５０へ入射した励起光は光ファイバ１５０内を伝播し、サンプルホルダ７００内の被測定試料へ照射される。チョッパ１３０は、チョッパコントローラ１６０により制御されて回転し、励起光源１１０からの励起光の通過又は遮断の制御を行い、励起光を被測定試料にパルス的に照射するためのものである。シャッタ１２０は、開いているときに励起光を通過させる。
【００２２】
光選択手段２０は、フィルタ２１０、レンズ２２０及びレンズ２２１を備え、励起手段１０により励起光が被測定試料に照射されることにより測定試料で発生した蛍光および被測定試料で発生した化学発光それぞれを選択して透過させる。
【００２３】
フィルタ２１０は、バンドリジェクションフィルタ等が好適に用いられ、励起光の波長成分を選択的に除去するとともに、被測定試料で発生した蛍光及び化学発光を選択して透過させる。また、レンズ２２０およびレンズ２２１は、被測定試料で発生した蛍光および化学発光を光電子増倍管３１０の受光面に集光する。
【００２４】
光検出手段３０は、光電子増倍管３１０、高電圧電源３１１及びシャッタ３２０を備え、光選択手段２０により選択されて透過された蛍光および化学発光の双方又は化学発光の光子の受光面への入射に応じたパルスを出力する。
【００２５】
シャッタ３２０は、開閉し、開いているときに被測定試料で発生した蛍光および化学発光それぞれを光電子増倍管３１０の受光面に入射させる。光電子増倍管３１０は、高電圧電源３１１から供給された高電圧により駆動され、蛍光又は化学発光の光子の受光面への入射に応じてパルスを出力する。
【００２６】
経時変化測定手段４０は、フォトンカウンタ４１０及びコンピュータ４２０を備え、光検出手段３０により出力されたパルスに基づいて、励起光が被測定試料に照射されていない期間内において被測定試料で発生した化学発光の光強度に応じた第１光強度データと、励起光が被測定試料に照射されているときを含む期間内において被測定試料で発生した蛍光および化学発光の光強度に応じた第２光強度データとを求め、第２光強度データと第１光強度データとに基づいて蛍光の光強度に応じた第３光強度データを算出し、第１光強度データに基づいて化学発光の経時変化を測定し、第３光強度データに基づいて蛍光の経時変化を測定する。
【００２７】
フォトンカウンタ４１０は、光電子増倍管３１０より出力されたパルスが入力される。また、フォトンカウンタ４１０は、ゲート回路を有し、このゲート回路に計測期間を制御する信号を入力することにより任意の計測期間を設定することができる。本実施形態においては、このゲート回路にチョッパコントローラ１６０による励起光の通過又は遮断の制御信号を入力する。そして、フォトンカウンタ４１０は、これらの信号に基づいて、励起光が被測定試料に照射されていない期間内において被測定試料で発生した化学発光の光子が光電子増倍管３１０の受光面に入射したという事象の数に応じたパルス数を計数して第１光強度データを求める。また、励起光が被測定試料に照射されているときを含む期間内において被測定試料で発生した蛍光及び化学発光の光子が光電子増倍管３１０の受光面に入射したという事象の数に応じたパルス数を計数して第２光強度データを求める。さらに、フォトンカウンタ４１０は、第１光強度データ及び第２光強度データそれぞれをコンピュータ４２０へ出力する。
【００２８】
コンピュータ４２０は、フォトンカウンタ４１０から出力された第１光強度データ及び第２光強度データを入力し、第２光強度データから第１光強度データを減算し第３光強度データを算出する。そして、第１光強度データに基づいて化学発光の経時変化を測定し、第３光強度データに基づいて蛍光の経時変化を測定する。
【００２９】
温度制御手段５０は、サーモバス５１０、配管５２０及び配管５２１を備え、被測定試料の温度を適切に制御する。
【００３０】
攪拌手段６０は、マグネティックスターラ及びマグネティックスターラコントローラ６１０を備え、マグネティックスターラは、サンプルホルダ７００に容れられた液体状の被測定試料を攪拌する。
【００３１】
さらに、被測定試料を容れるサンプルホルダ７００は、サーモバス５１０と配管５２０及び配管５２１を介して接続されている。そしてサンプルホルダ７００に容れられた被測定試料の温度は、サーモバス５１０により所定温度に制御される。また、被測定試料は、マグネティックスターラコントローラ６１０により制御されるマグネティックスターラにより攪拌される。さらに、サンプルホルダ７００は、被測定試料や試薬を導入するためのサンプルディスペンサ７１０と接続されている。
【００３２】
図２は、本実施形態における励起光照射、第１光強度データ測定及び第２光強度データ測定それぞれの動作タイミングを示す図である。図２に基づいて励起光照射、第１光強度データ測定及び第２光強度データ測定それぞれの動作タイミングを詳細に説明する。励起光は、チョッパ１３０により通過又は遮断の制御を受けて被測定試料に照射される。図２では、チョッパ１３０による励起光の通過時間と遮断時間との割合は１対９である。この時間割合は必要に応じて変化させることが出来る。この励起光が照射されている時間帯を除いて、第１光強度データが測定される。より詳細には、被測定試料に導入された蛍光指示薬の蛍光寿命時間（約５ｎｓ）、装置の応答時間（約１０ｎｓ）及びチョッパ１３０が励起光を遮断する際に励起光のビームを通過するのに必要な時間（約２０μｓ）の経過後から第１光強度データの測定を開始し、チョッパ１３０が回転することにより次に励起光がチョッパ１３０を通過する時刻の２０μｓ前に第１光強度データの測定を終了する。これにより、第１光強度データは、励起光の影響をまったく受けることなく化学発光の光強度のみを反映した値となる。
【００３３】
また、第２光強度データは、励起光照射を行っているときを含む期間内において測定され、第２光強度データの測定時間と第１光強度データの測定時間とを等しくすることにより、第２光強度データは、第１光強度データと同じ化学発光の光強度および蛍光の光強度を反映した値となる。よって、チョッパ１３０の通過／遮断周期における同一周期内において第２光強度データから第１光強度データを減算し第３光強度データを算出することにより蛍光強度のみを抽出することが出来る。
【００３４】
次に、本実施形態に係る化学発光および蛍光の経時変化測定装置１の動作について説明すると共に、化学発光および蛍光の経時変化測定方法についても説明する。以下の説明では具体的な実施例として被測定試料がヒト好中球様細胞である場合について説明する。ここで、ヒト好中球様細胞は、前骨髄芽球系株細胞ＨＬ−６０　を１．２％ＤＭＳＯを含むＧＩＴ　ｍｅｄｉｕｍで細胞密度を３×１０^５細胞／ｍｌに合わせ、これを４〜６日間３７℃で５％ＣＯ_２存在下で培養したものである。
【００３５】
被測定試料である好中球様細胞は、予め、カルシウム蛍光指示薬ｆｌｕｏ−３（１−［２−ａｍｉｎｏ−５−（２，７−ｄｉｃｈｌｏｒｏ−６−ｈｙｄｒｏｘｙ−３−ｏｘｙ−９−ｘａｎｔｈｅｎｙｌ）ｐｈｅｎｏｘｙｌ］−２−（２−ａｍｉｎｏ−５−ｍｅｔｈｙｌｐｈｅｎｏｘｙ）ｅｔｈａｎｅ−Ｎ，Ｎ，Ｎ’，Ｎ’−ｔｅｔｒａａｃｅｔｉｃ　ａｃｉｄ　）で処理され、また、化学発光試薬ＣＬＡ（ウミホタルルシフェリン誘導体、２−ｍｅｔｈｙｌ−６−ｐｈｅｎｙｌ−３，７−ｄｉｈｙｄｒｏｉｍｉｄａｚｏ［１，２−ａ］ｐｙｒａｚｉｎ−３−ｏｎｅ）が添加されて、懸濁液とされる。この被測定試料は、ポリメチルメタクリレート製のセルに容れられてサンプルホルダ７００にセットされる。サンプルホルダ７００にセットされた被測定試料は、サーモバス５１０により一定温度３７℃に維持され、マグネティックスターラコントローラ６１０により制御されたマグネティックスターラにより攪拌される。
【００３６】
以上の測定準備が終了すると、チョッパ１３０はチョッパコントローラ１６０により制御されて一定速度で回転し、シャッタ１２０は開く。チョッパ１３０による励起光の通過、遮断の周期は、５０Ｈｚ〜２ｋＨｚ程度であり、特に１００〜５００Ｈｚが好ましい。本実施例では２５０Ｈｚとしている。
【００３７】
励起光源１１０から出力された励起光は、チョッパ１３０に入射する。そして、チョッパ１３０に入射した励起光は、チョッパ１３０により通過又は遮断の制御を受け、集光レンズ１４０、光ファイバ１５０を通過して、サンプルホルダ７００に容れられた被測定試料にパルス的に照射される。励起光源１１０は、波長４７３ｎｍのレーザ光を出力するネオジウムドープヤグレーザ光源が用いられる。
【００３８】
被測定試料で発生した化学発光（波長３８５ｎｍ）は、レンズ２２０により集光され、フィルタ２１０、レンズ２２１およびシャッタ３２０を通過し、光電子増倍管３１０の受光面に入射する。この化学発光は、スーパーオキサイドと化学発光試薬ＣＬＡとが化学反応してＣＬＡ酸化物が生成される際に発生する化学発光である。すなわち、化学発光強度は、スーパーオキサイド産生量を表している。
【００３９】
一方、被測定試料で発生した蛍光（波長５２３ｎｍ）は、同様にレンズ２２０により集光され、フィルタ２１０、レンズ２２１およびシャッタ３２０を透過し、光電子増倍管３１０の受光面に入射する。この蛍光は、好中球様細胞内の遊離カルシウムイオンとカルシウム蛍光指示薬ｆｌｕｏ−３とが結合して生成された錯体に励起光が照射されて発生する蛍光である。すなわち、蛍光強度は、好中球様細胞内の遊離カルシウムイオンの濃度を表している。なお、励起光の散乱光は、フィルタ２１０により遮断されるので、光電子増倍管３１０の受光面に入射することはない。
【００４０】
光電子増倍管３１０からは、蛍光および化学発光の双方又は化学発光の光子の受光面への入射に応じたパルスが出力される。
【００４１】
光電子増倍管３１０から出力されたパルスは、フォトンカウンタ４１０に入力される。また、チョッパコントローラ１６０による励起光の通過、遮断の制御信号もフォトンカウンタ４１０に入力される。そして、フォトンカウンタ４１０により、励起光が被測定試料に照射されていない期間内において被測定試料で発生した化学発光の光子数が計数されて第１光強度データが求められ、励起光が被測定試料に照射されているときを含む期間内において被測定試料で発生した蛍光および化学発光の光子数が計数されて第２光強度データが求められる。そしてさらに、第２光強度データから第１光強度データが減算されて第３光強度データが算出される。
【００４２】
以上のように励起光照射、化学発光強度測定および蛍光強度測定を行いながら、サンプルディスペンサ７１０より刺激薬が被測定試料（好中球様細胞）へ滴下される。刺激薬としては、例えば好中球遊走性ペプチドｆＭＬＰが用いられる。そして、コンピュータ４２０により、刺激薬の被測定試料への滴下、化学発光強度（スーパーオキサイド産生量）および蛍光強度（細胞内カルシウム濃度）の間の因果関係や時間的関係が解析される。
【００４３】
図３は、「化学発光強度」並びに「化学発光強度及び蛍光強度」それぞれの経時変化の測定結果を示す図である。被測定試料は、上述したようなカルシウム蛍光指示薬ｆｌｕｏ−３で処理され化学発光試薬ＣＬＡが添加された好中球様細胞を、１ｍＭの細胞外カルシウム溶液内に容れて懸濁液としたものである。測定開始後の２５０秒の時点で１μＭのｆＭＬＰで被測定試料を刺激したものである。「化学発光強度」並びに「化学発光強度及び蛍光強度」それぞれの経時変化が実時間で検出できていることがわかる。
【００４４】
図４は、「化学発光強度」及び「蛍光強度」それぞれの経時変化の測定結果を示す図である。図４における「蛍光強度」は、図３における「化学発光強度及び蛍光強度」から「化学発光強度」を減算することにより算出した。
【００４５】
図４から判るように、ｆＭＬＰで被測定試料が刺激されると、直ちに蛍光強度が上昇し、その後に数秒遅れて、化学発光強度が上昇している。このことから、ｆＭＬＰ刺激により、細胞内カルシウム濃度が直ちに上昇し、その後に数秒遅れてスーパーオキサイドが産生されることが確認された。
【００４６】
以上、本実施形態に係る化学発光および蛍光の経時変化測定装置及び方法によれば、単一の光検出手段により化学発光及び蛍光それぞれの経時変化を同時に測定することができるため、蛍光波長及び化学発光波長が互いに同一又は近接する場合であっても同一現象における蛍光及び化学発光それぞれの経時変化を実時間で測定することができる。さらに、化学発光及び蛍光それぞれを分光手段により分離する必要が無いことにより光学系を単純化することができるため、装置の小型化及び光の利用効率の向上を図ることができる。
【００４７】
本発明は、上記実施形態に限定されるものではなく種々の変形が可能である。上記実施形態では、ヒト好中球様細胞を被測定試料として、被測定試料においてスーパーオキサイド産生に伴い発生した化学発光およびカルシウム濃度変化に伴い発生した蛍光を測定する場合について説明したが、これに限られるものではない。
【００４８】
【発明の効果】
以上、詳細に説明したとおり、本発明によれば、蛍光波長及び化学発光波長が同一又は近接する場合であっても蛍光及び化学発光それぞれの経時変化を実時間で測定することができる化学発光および蛍光の経時変化測定装置および方法を提供することができる。
【図面の簡単な説明】
【図１】本実施形態に係る化学発光および蛍光の経時変化測定装置の構成図である。
【図２】本実施形態における励起光照射、第１光強度データ測定及び第２光強度データ測定それぞれの動作タイミングを示す図である。
【図３】「化学発光強度」並びに「化学発光強度及び蛍光強度」それぞれの経時変化の測定結果を示す図である。
【図４】「化学発光強度」及び「蛍光強度」それぞれの経時変化の測定結果を示す図である。
【符号の説明】
１…経時変化測定装置、１０…励起手段、２０…光選択手段、３０…光検出手段、４０…経時変化測定手段、５０…温度制御手段、６０…攪拌手段、１１０…励起光源、１２０…シャッタ、１３０…チョッパ、１４０…集光レンズ、１５０…光ファイバ、１６０…チョッパコントローラ、２１０…フィルタ、２２０，２２１…レンズ、３１０…光電子増倍管、３１１…高電圧電源、３２０…シャッタ、４１０…フォトンカウンタ、４２０…コンピュータ、５１０…サーモバス、５２０，５２１…配管、７００…サンプルホルダ、７１０…サンプルディスペンサ。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a measuring device and a measuring method for measuring a temporal change of chemiluminescence caused by a chemical reaction in a sample to be measured and a temporal change of fluorescence generated by irradiation of the sample to be measured with excitation light. It is.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, measurement methods using fluorescence or chemiluminescence have been used for measuring biologically related substances and the like. For example, the concentration of intracellular calcium ions known to be involved in in vivo signal transmission can be detected and quantified with a fluorescent indicator. In addition, active oxygen known to be involved in immunity and aging of living cells, particularly superoxide and singlet oxygen, can be detected by a chemiluminescent reagent, and cells of the immune system are produced. Superoxide has been detected. When it is intended to simultaneously measure information from such immune system cells, for example, the intracellular calcium ion concentration and the amount of superoxide produced by the cells, it is necessary to simultaneously detect fluorescence and chemiluminescence.
[0003]
Superoxide (O ₂ ⁻ ) produced by human neutrophils, a type of white blood cell known as cells of the immune system, kills bacteria and viruses that have invaded the human body and is important in biological defense. Role. It is thought that the concentration of calcium ions in neutrophil cells is involved in the production of superoxide, but there are many unclear points about the role of this calcium. If the substance of the increase in intracellular calcium concentration responsible for the production of superoxide is elucidated, for example, a new diagnostic method for cell function leading to treatment of patients with chronic granulomatosis, and the mechanism of action of drugs involved in immunity and inflammation It is expected to help elucidate.
[0004]
Superoxide production can be measured, for example, by detecting chemiluminescence generated by a chemical reaction in neutrophils. Further, the change in intracellular calcium concentration can be measured, for example, by introducing a calcium fluorescent indicator into neutrophils in advance, and irradiating the neutrophil with excitation light to detect fluorescence generated.
[0005]
As a technique for measuring each of fluorescence and chemiluminescence, there is “Apparatus and method for measuring change with time of chemiluminescence and fluorescence” described in Japanese Patent No. 3183864. The present invention repeatedly irradiates a sample to be measured with excitation light in a pulsed manner, detects chemiluminescence generated in the sample to be measured when the sample to be measured is not irradiated with the excitation light, and emits the excitation light to the sample to be measured. This is to detect the fluorescence generated in the sample to be measured when the sample is irradiated, and it is possible to detect, in real time, the time-dependent changes of the chemiluminescence and the fluorescence generated in the same sample. Japanese Patent Application Laid-Open No. H11-148900 describes a "light emission pattern reader" capable of measuring fluorescence and chemiluminescence with a single detection optical system.
[0006]
[Problems to be solved by the invention]
However, in the invention described in Japanese Patent No. 3183863, since a fluorescence detection optical system for measuring fluorescence and a chemiluminescence detection optical system for measuring chemiluminescence are separated, each of the fluorescence and the chemiluminescence is separated by spectral means. Need to be separated by For this reason, when the fluorescence wavelength and the chemiluminescence wavelength are the same or close to each other, it is difficult to separate the fluorescence and the chemiluminescence, so that there is a problem that the fluorescence and the chemiluminescence cannot be measured.
[0007]
Further, the invention described in JP-A-11-148900 measures fluorescence and chemiluminescence with a single detector. Even when the fluorescence wavelength and the chemiluminescence wavelength are the same or close to each other, , Fluorescence and chemiluminescence can be measured. However, this device irradiates the sample with excitation light and measures only the fluorescence, and stops the excitation light irradiation separately from the fluorescence measurement to measure only the chemiluminescence. It is not possible to simultaneously measure the time-dependent change of each light emission. Therefore, there is a problem that a delicate relationship between superoxide production and a change in intracellular calcium concentration cannot be detected.
[0008]
The present invention has been made in order to solve the above problems, and even when the fluorescence wavelength and the chemiluminescence wavelength are the same or close to each other, the change with time of the fluorescence and the chemiluminescence is measured in real time. It is an object of the present invention to provide an apparatus and a method for measuring the change with time of chemiluminescence and fluorescence which can be performed.
[0009]
[Means for Solving the Problems]
The apparatus for measuring the change with time of chemiluminescence and fluorescence according to the present invention comprises: (1) an excitation means for repeatedly irradiating a sample to be measured with excitation light in a pulsed manner; and (2) an excitation means for irradiating the sample to be measured with excitation light. Light selecting means for selectively transmitting each of the fluorescence generated in the sample to be measured and the chemiluminescence generated in the sample to be measured, and (3) both the fluorescence and the chemiluminescence selected and transmitted by the light selecting means. Alternatively, one of the light detecting means for receiving the chemiluminescence and outputting an electric signal in accordance with the amount of the received light, and (4) the excitation light is supplied to the sample to be measured by the exciting means based on the electric signal output from the light detecting means. The first light intensity data corresponding to the light intensity of the chemiluminescence generated in the sample to be measured during a period in which the sample is not irradiated with the light, and a period including when the sample to be measured is irradiated with the excitation light by the excitation means. Second light intensity data corresponding to the light intensity of the fluorescence and chemiluminescence generated in the sample to be measured, and a third light intensity corresponding to the light intensity of the fluorescence is determined based on the second light intensity data and the first light intensity data. Time change measuring means for calculating light intensity data, measuring a change in chemiluminescence with time based on the first light intensity data, and measuring a change in fluorescence with time based on the third light intensity data. And
[0010]
The method for measuring the change with time of chemiluminescence and fluorescence according to the present invention is characterized in that a sample to be measured is repeatedly irradiated with excitation light in a pulsed manner by an excitation means, and the excitation light is irradiated on the sample to be measured by the excitation means. The fluorescence generated in the sample and the chemiluminescence generated in the sample to be measured are respectively selected and transmitted by the light selection means, and both the fluorescence and the chemiluminescence or the chemiluminescence selected and transmitted by the light selection means are received. An electric signal is output by the one light detecting means in accordance with the amount of received light, and based on the electric signal output by the light detecting means, the sample to be measured is not irradiated with the excitation light by the exciting means on the sample to be measured. The first light intensity data corresponding to the light intensity of the chemiluminescence generated in the step (a), and the sample to be measured within the period including when the sample to be measured is irradiated with the excitation light by the excitation means. Second light intensity data according to the generated light intensity of fluorescence and chemiluminescence is obtained, and third light intensity data corresponding to the light intensity of fluorescence is calculated based on the second light intensity data and the first light intensity data. Then, a time-dependent change in chemiluminescence is measured based on the first light intensity data, and a time-dependent change in fluorescence is measured based on the third light intensity data.
[0011]
According to the apparatus or method for measuring the change with time in chemiluminescence and fluorescence according to the present invention, the sample to be measured is repeatedly irradiated with excitation light in a pulsed manner by the excitation means. The fluorescence generated in the sample to be measured and the chemiluminescence generated in the sample to be measured when the sample to be measured is irradiated with the excitation light by the excitation means are selected and transmitted by the light selection means. Then, both the fluorescence and the chemiluminescence or the chemiluminescence selected and transmitted by the light selection means are received, and an electric signal is output by one of the light detection means and output by the light detection means according to the amount of the received light. On the basis of the electric signal, the first light intensity data corresponding to the light intensity of the chemiluminescence generated in the sample to be measured within a period in which the sample is not irradiated with the excitation light by the excitation means, and the excitation light is generated by the excitation means. Second light intensity data corresponding to the light intensity of the fluorescence and chemiluminescence generated in the sample to be measured during the period including when the sample to be measured is irradiated is obtained. Further, third light intensity data corresponding to the light intensity of the fluorescence is calculated based on the second light intensity data and the first light intensity data, and a change with time of the chemiluminescence is measured based on the first light intensity data, The temporal change of the fluorescence is measured by the temporal change measuring means based on the third light intensity data. In this way, the time-dependent changes in chemiluminescence and fluorescence generated in the same sample are detected in real time.
[0012]
Further, the apparatus for measuring the change over time of chemiluminescence and fluorescence according to the present invention is characterized by further comprising a temperature control means for controlling the temperature of the sample to be measured.
[0013]
Further, the method for measuring the change over time of chemiluminescence and fluorescence according to the present invention is characterized in that the temperature of the sample to be measured is further controlled by the temperature control means when measuring the change over time of each of the chemiluminescence and fluorescence.
[0014]
In this case, since the temperature of the sample to be measured is controlled by the temperature control means, it is suitable, for example, when the sample to be measured is a cell.
[0015]
Further, the apparatus for measuring the change with time of chemiluminescence and fluorescence according to the present invention is characterized in that the sample to be measured is a liquid, and further provided with a stirring means for stirring the sample to be measured.
[0016]
Further, the method for measuring the change with time of chemiluminescence and fluorescence according to the present invention is that the sample to be measured is a liquid, and when the change with time of each of the chemiluminescence and fluorescence is measured, the sample to be measured is further stirred by a stirring means. Features.
[0017]
In this case, since the sample to be measured is stirred by the stirring means, it is suitable when the sample to be measured is a suspension.
[0018]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.
[0019]
First, the configuration of the chemiluminescence and fluorescence temporal change measurement apparatus according to the present embodiment will be described. FIG. 1 is a configuration diagram of an apparatus 1 for measuring a change with time of chemiluminescence and fluorescence according to the present embodiment. The temporal change measuring apparatus 1 includes an excitation unit 10, a light selecting unit 20, a light detecting unit 30, a temporal change measuring unit 40, a temperature control unit 50, and a stirring unit 60.
[0020]
The excitation means 10 includes an excitation light source 110, a shutter 120, a chopper 130, a chopper controller 160, a condenser lens 140, and an optical fiber 150, and repeatedly irradiates the sample to be measured with excitation light in a pulsed manner.
[0021]
The excitation light source 110 outputs excitation light having a wavelength that excites a fluorescent indicator previously introduced into the sample to be measured to generate fluorescence. The excitation light output from the excitation light source 110 is guided to the optical fiber 150 via the shutter 120, the chopper 130, and the condenser lens 140. The excitation light that has entered the optical fiber 150 propagates through the optical fiber 150 and irradiates the sample to be measured in the sample holder 700. The chopper 130 is rotated by being controlled by the chopper controller 160, controls the passage or cutoff of the excitation light from the excitation light source 110, and irradiates the sample to be measured with the excitation light in a pulsed manner. The shutter 120 allows the excitation light to pass when it is open.
[0022]
The light selection unit 20 includes a filter 210, a lens 220, and a lens 221. The excitation unit 10 irradiates the sample with the excitation light with the excitation light to emit fluorescence generated in the measurement sample and chemiluminescence generated in the sample. Select and transmit.
[0023]
As the filter 210, a band rejection filter or the like is suitably used. The filter 210 selectively removes a wavelength component of the excitation light, and selectively transmits fluorescence and chemiluminescence generated in the sample to be measured. The lens 220 and the lens 221 converge the fluorescence and the chemiluminescence generated in the sample to be measured on the light receiving surface of the photomultiplier tube 310.
[0024]
The light detecting means 30 includes a photomultiplier tube 310, a high-voltage power supply 311 and a shutter 320, and is used to make both photons of fluorescence and chemiluminescence or chemiluminescence selected and transmitted by the light selection means 20 incident on the light receiving surface. Output a pulse corresponding to.
[0025]
The shutter 320 opens and closes, and causes each of the fluorescence and chemiluminescence generated in the sample to be measured to enter the light receiving surface of the photomultiplier tube 310 when opened and closed. The photomultiplier tube 310 is driven by the high voltage supplied from the high voltage power supply 311 and outputs a pulse in response to the incidence of fluorescent or chemiluminescent photons on the light receiving surface.
[0026]
The temporal change measuring means 40 includes a photon counter 410 and a computer 420. Based on the pulse output from the light detecting means 30, the chemical change generated in the sample to be measured within a period in which the sample to be measured is not irradiated with the excitation light. First light intensity data corresponding to the light intensity of the emitted light, and second light corresponding to the light intensity of the fluorescence and chemiluminescence generated in the measured sample during a period including a time when the sample is irradiated with the excitation light. Intensity data is obtained, third light intensity data corresponding to the light intensity of the fluorescence is calculated based on the second light intensity data and the first light intensity data, and a change with time of the chemiluminescence based on the first light intensity data. Is measured, and the change with time of the fluorescence is measured based on the third light intensity data.
[0027]
The pulse output from the photomultiplier tube 310 is input to the photon counter 410. The photon counter 410 has a gate circuit, and an arbitrary measurement period can be set by inputting a signal for controlling the measurement period to the gate circuit. In the present embodiment, a control signal for passing or blocking the excitation light by the chopper controller 160 is input to the gate circuit. Then, based on these signals, the photon counter 410 causes the chemiluminescence photons generated in the sample to be measured to enter the light receiving surface of the photomultiplier tube 310 during a period in which the sample to be measured is not irradiated with the excitation light. The first light intensity data is obtained by counting the number of pulses corresponding to the number of the above events. In addition, it depends on the number of events in which fluorescence and chemiluminescence photons generated in the sample to be measured have been incident on the light receiving surface of the photomultiplier tube 310 during a period including when the sample to be measured is irradiated with the excitation light. The number of pulses is counted to obtain second light intensity data. Further, the photon counter 410 outputs the first light intensity data and the second light intensity data to the computer 420.
[0028]
The computer 420 receives the first light intensity data and the second light intensity data output from the photon counter 410, and subtracts the first light intensity data from the second light intensity data to calculate third light intensity data. Then, the change with time of the chemiluminescence is measured based on the first light intensity data, and the change with time of the fluorescence is measured based on the third light intensity data.
[0029]
The temperature control unit 50 includes a thermobus 510, a pipe 520, and a pipe 521, and appropriately controls the temperature of the sample to be measured.
[0030]
The stirring means 60 includes a magnetic stirrer and a magnetic stirrer controller 610, and the magnetic stirrer stirs the liquid sample to be measured contained in the sample holder 700.
[0031]
Further, the sample holder 700 that holds the sample to be measured is connected to the thermobus 510 via the pipe 520 and the pipe 521. Then, the temperature of the sample to be measured contained in the sample holder 700 is controlled to a predetermined temperature by the thermo bus 510. The sample to be measured is stirred by a magnetic stirrer controlled by a magnetic stirrer controller 610. Further, the sample holder 700 is connected to a sample dispenser 710 for introducing a sample to be measured and a reagent.
[0032]
FIG. 2 is a diagram illustrating operation timings of the excitation light irradiation, the first light intensity data measurement, and the second light intensity data measurement in the present embodiment. The operation timings of the excitation light irradiation, the first light intensity data measurement, and the second light intensity data measurement will be described in detail with reference to FIG. The excitation light is irradiated on the sample to be measured under the control of passing or blocking by the chopper 130. In FIG. 2, the ratio between the passage time of the excitation light by the chopper 130 and the cutoff time is 1: 9. This time ratio can be changed as needed. Except for the time period during which the excitation light is irradiated, the first light intensity data is measured. More specifically, the fluorescence lifetime (about 5 ns) of the fluorescent indicator introduced into the sample to be measured, the response time of the apparatus (about 10 ns), and the fact that the chopper 130 passes the beam of excitation light when the excitation light is blocked. Measurement of the first light intensity data is started after a lapse of time (about 20 μs) necessary for the first light intensity data, and the first light intensity data is measured 20 μs before the next time when the excitation light passes through the chopper 130 by the rotation of the chopper 130. Measurement is completed. Thereby, the first light intensity data is a value reflecting only the light intensity of the chemiluminescence without being affected by the excitation light at all.
[0033]
Further, the second light intensity data is measured during a period including a time when the excitation light irradiation is performed, and the second light intensity data is measured by making the measurement time of the second light intensity data equal to the measurement time of the first light intensity data. The two light intensity data is a value reflecting the same light intensity of chemiluminescence and fluorescence as the first light intensity data. Therefore, only the fluorescence intensity can be extracted by subtracting the first light intensity data from the second light intensity data and calculating the third light intensity data within the same period of the pass / block period of the chopper 130.
[0034]
Next, the operation of the chemical luminescence and fluorescence temporal change measurement apparatus 1 according to the present embodiment will be described, and a method of measuring the chemical luminescence and fluorescence temporal change will also be described. In the following description, a case where the sample to be measured is human neutrophil-like cells will be described as a specific example. Here, human neutrophil-like cells were prepared by adjusting the promyeloblast cell line HL-60 to a cell density of 3 × 10 ⁵ cells / ml with GIT medium containing 1.2% DMSO, and adjusting the cell density to 4 to 6 cells. Cultured at 37 ° C. for 5 days in the presence of 5% CO ₂ .
[0035]
The neutrophil-like cells, which are the samples to be measured, were previously prepared with the calcium fluorescent indicator fluo-3 (1- [2-amino-5- (2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl) phenoxyl). ]-(2-amino-5-methylphenoxy) ethane-N, N, N ', N'-tetraacetic acid, and a chemiluminescent reagent CLA (Renilla luciferin derivative, 2-methyl-6-phenyl) -3,7-dihydroimidazo [1,2-a] pyrazin-3-one) is added to form a suspension. The sample to be measured is set in a sample holder 700 while being held in a cell made of polymethyl methacrylate. The sample to be measured set in the sample holder 700 is maintained at a constant temperature of 37 ° C. by the thermo bath 510, and is stirred by the magnetic stirrer controlled by the magnetic stirrer controller 610.
[0036]
When the above measurement preparation is completed, the chopper 130 is controlled by the chopper controller 160 to rotate at a constant speed, and the shutter 120 opens. The period of passage and cutoff of the excitation light by the chopper 130 is about 50 Hz to 2 kHz, and particularly preferably 100 to 500 Hz. In this embodiment, the frequency is 250 Hz.
[0037]
The excitation light output from the excitation light source 110 enters the chopper 130. The excitation light incident on the chopper 130 is controlled to pass or block by the chopper 130, passes through the condenser lens 140 and the optical fiber 150, and irradiates the sample to be measured held in the sample holder 700 in a pulsed manner. Is done. As the excitation light source 110, a neodymium-doped yag laser light source that outputs laser light having a wavelength of 473 nm is used.
[0038]
Chemiluminescence (wavelength: 385 nm) generated in the sample to be measured is condensed by the lens 220, passes through the filter 210, the lens 221, and the shutter 320, and enters the light receiving surface of the photomultiplier tube 310. This chemiluminescence is chemiluminescence generated when a superoxide and a chemiluminescent reagent CLA chemically react to generate a CLA oxide. That is, the chemiluminescence intensity indicates a superoxide production amount.
[0039]
On the other hand, the fluorescence (wavelength 523 nm) generated from the sample to be measured is similarly condensed by the lens 220, passes through the filter 210, the lens 221 and the shutter 320, and enters the light receiving surface of the photomultiplier tube 310. This fluorescence is fluorescence generated by irradiating excitation light to a complex formed by binding of free calcium ions in neutrophil-like cells and the calcium fluorescent indicator fluo-3. That is, the fluorescence intensity represents the concentration of free calcium ions in neutrophil-like cells. Since the scattered light of the excitation light is blocked by the filter 210, it does not enter the light receiving surface of the photomultiplier tube 310.
[0040]
The photomultiplier tube 310 outputs a pulse in accordance with the incidence of both fluorescent and chemiluminescent or chemiluminescent photons on the light receiving surface.
[0041]
The pulse output from the photomultiplier tube 310 is input to the photon counter 410. In addition, a control signal for passing or blocking the excitation light by the chopper controller 160 is also input to the photon counter 410. The photon counter 410 counts the number of photons of chemiluminescence generated in the sample under measurement during a period in which the sample is not irradiated with the excitation light to obtain first light intensity data. The number of photons of fluorescence and chemiluminescence generated in the sample to be measured during the period including the time when the sample is being irradiated is counted, and second light intensity data is obtained. Further, the first light intensity data is subtracted from the second light intensity data to calculate third light intensity data.
[0042]
As described above, the stimulant is dropped from the sample dispenser 710 onto the sample to be measured (neutrophil-like cells) while performing the excitation light irradiation, the chemiluminescence intensity measurement, and the fluorescence intensity measurement. As a stimulant, for example, neutrophil chemotactic peptide fMLP is used. Then, the computer 420 analyzes the causal relationship and the temporal relationship between the stimulant dropped onto the sample to be measured, the chemiluminescence intensity (superoxide production amount), and the fluorescence intensity (intracellular calcium concentration).
[0043]
FIG. 3 is a diagram showing the measurement results of the change over time of “chemiluminescence intensity” and “chemiluminescence intensity and fluorescence intensity”. The sample to be measured was a neutrophil-like cell treated with the above-described calcium fluorescent indicator fluo-3 and added with the chemiluminescent reagent CLA, and was placed in a 1 mM extracellular calcium solution to form a suspension. is there. At 250 seconds after the start of the measurement, the sample to be measured was stimulated with 1 μM fMLP. It can be seen that the changes over time of “chemiluminescence intensity” and “chemiluminescence intensity and fluorescence intensity” were detected in real time.
[0044]
FIG. 4 is a diagram showing the results of measuring the changes over time of “chemiluminescence intensity” and “fluorescence intensity”. “Fluorescence intensity” in FIG. 4 was calculated by subtracting “chemiluminescence intensity” from “chemiluminescence intensity and fluorescence intensity” in FIG.
[0045]
As can be seen from FIG. 4, when the sample to be measured is stimulated with fMLP, the fluorescence intensity increases immediately, and then, several seconds later, the chemiluminescence intensity increases. From this, it was confirmed that the intracellular calcium concentration was immediately increased by fMLP stimulation, and thereafter, superoxide was produced several seconds later.
[0046]
As described above, according to the apparatus and method for measuring the change over time of chemiluminescence and fluorescence according to the present embodiment, it is possible to simultaneously measure the change over time of each of chemiluminescence and fluorescence with a single photodetector, and thus the fluorescence wavelength and the chemical Even when the emission wavelengths are the same or close to each other, the time-dependent changes of fluorescence and chemiluminescence in the same phenomenon can be measured in real time. Furthermore, since there is no need to separate each of chemiluminescence and fluorescence by the spectroscopic means, the optical system can be simplified, so that the size of the apparatus can be reduced and the light use efficiency can be improved.
[0047]
The present invention is not limited to the above embodiment, and various modifications are possible. In the above embodiment, a case where human neutrophil-like cells are used as a sample to be measured, and a case where the chemiluminescence generated due to superoxide production and the fluorescence generated due to a change in calcium concentration in the sample to be measured are measured, It is not limited.
[0048]
【The invention's effect】
As described in detail above, according to the present invention, even if the fluorescence wavelength and the chemiluminescence wavelength are the same or close to each other, it is possible to measure the change with time of each of the fluorescence and the chemiluminescence in real time. An apparatus and a method for measuring a change with time of fluorescence can be provided.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of an apparatus for measuring a change with time of chemiluminescence and fluorescence according to an embodiment.
FIG. 2 is a diagram showing operation timings of excitation light irradiation, first light intensity data measurement, and second light intensity data measurement in the present embodiment.
FIG. 3 is a diagram showing measurement results of changes over time in “chemiluminescence intensity” and “chemiluminescence intensity and fluorescence intensity”.
FIG. 4 is a diagram showing measurement results of changes over time in “chemiluminescence intensity” and “fluorescence intensity”.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Time change measuring device, 10 ... Exciting means, 20 ... Light selection means, 30 ... Light detecting means, 40 ... Time change measuring means, 50 ... Temperature control means, 60 ... Stirring means, 110 ... Excitation light source, 120 ... Shutter , 130 chopper, 140 condensing lens, 150 optical fiber, 160 chopper controller, 210 filter, 220, 221 lens, 310 photomultiplier tube, 311 high voltage power supply, 320 shutter, 410 Photon counter, 420 computer, 510 thermo bus, 520, 521 piping, 700 sample holder, 710 sample dispenser.

Claims

Excitation means for repeatedly irradiating the sample under measurement with excitation light in a pulsed manner,
Light selection means for selecting and transmitting each of fluorescence generated in the measurement sample and chemiluminescence generated in the measurement sample by irradiating the measurement light with the excitation light by the excitation means,
One light detection unit that receives both the fluorescence and the chemiluminescence selected by the light selection unit and the chemiluminescence or the chemiluminescence, and outputs an electric signal according to the amount of received light,
On the basis of the electric signal output by the light detection unit, the excitation unit responds to the light intensity of the chemiluminescence generated in the sample under measurement during a period in which the sample is not irradiated with the excitation light by the excitation unit. The first light intensity data and the light intensity of the fluorescence and the chemiluminescence generated in the sample to be measured during a period including when the sample to be measured is irradiated with the excitation light by the excitation means. Calculating second light intensity data, calculating third light intensity data corresponding to the light intensity of the fluorescence based on the second light intensity data and the first light intensity data,
A temporal change measuring unit that measures a temporal change of the chemiluminescence based on the first light intensity data, and measures a temporal change of the fluorescence based on the third light intensity data;
An apparatus for measuring the change over time of chemiluminescence and fluorescence, comprising:

Further comprising a temperature control means for controlling the temperature of the sample to be measured,
The apparatus for measuring the change with time of chemiluminescence and fluorescence according to claim 1, characterized in that:

The sample to be measured is a liquid, further comprising a stirring means for stirring the sample to be measured,
The apparatus for measuring the change with time of chemiluminescence and fluorescence according to claim 1, characterized in that:

The sample to be measured is irradiated with excitation light repeatedly in a pulsed manner by the excitation means,
The excitation means irradiates the sample to be measured with the excitation light to select and transmit each of the fluorescence generated in the sample to be measured and the chemiluminescence generated in the sample to be measured by light selection means,
Receiving both the fluorescence and the chemiluminescence or the chemiluminescence selected and transmitted by the light selection means, and outputting an electric signal according to the amount of received light by one light detection means,
On the basis of the electric signal output by the light detection unit, the excitation unit responds to the light intensity of the chemiluminescence generated in the sample under measurement during a period in which the sample is not irradiated with the excitation light by the excitation unit. The first light intensity data and the light intensity of the fluorescence and the chemiluminescence generated in the sample to be measured during a period including when the sample to be measured is irradiated with the excitation light by the excitation means. Calculating second light intensity data, calculating third light intensity data according to the light intensity of the fluorescence based on the second light intensity data and the first light intensity data,
Measuring the change over time of the chemiluminescence based on the first light intensity data, and measuring the change over time of the fluorescence based on the third light intensity data;
A method for measuring the change over time of chemiluminescence and fluorescence.

The temperature of the sample to be measured is further controlled by a temperature control unit when measuring the change with time of the chemiluminescence and the fluorescence.
5. The method according to claim 4, wherein the change in chemiluminescence and fluorescence with time is measured.

The sample to be measured is a liquid, and the sample to be measured is further stirred by a stirring unit when measuring the change with time of the chemiluminescence and the fluorescence.
5. The method according to claim 4, wherein the change in chemiluminescence and fluorescence with time is measured.