JP4112748B2

JP4112748B2 - Optical measuring device and optical measuring method

Info

Publication number: JP4112748B2
Application number: JP16984499A
Authority: JP
Inventors: 本比呂須山; 克彦河合; 哲家森田; 弘一郎大庭
Original assignee: Hamamatsu Photonics KK
Current assignee: Hamamatsu Photonics KK
Priority date: 1999-06-16
Filing date: 1999-06-16
Publication date: 2008-07-02
Anticipated expiration: 2019-06-16
Also published as: JP2001004443A

Description

【０００１】
【発明の属する技術分野】
本発明は、光計測装置及び光計測方法に関するものである。
【０００２】
【従来の技術】
光量が極端に異なる２つの状況下（例えば昼間と夜間）においても計測を可能とする光計測装置のニーズは高い。このようなニーズに応える光計測装置として、例えば特開昭６１−１３３５４０号公報に記載されている昼夜兼用カメラが挙げられる。かかる昼夜兼用カメラは、光電陰極と光検出器とを備え、光電陰極を透過する光子を光検出器によって検出する昼間モードと光電陰極から放出された光電子を光検出器によって検出する夜間モードとを適宜切り替えることで、日中と夜間という光量が極端に異なる２つの状況下で、入射イメージを検出することができる。
【０００３】
【発明が解決しようとする課題】
上述の如く、上記昼夜兼用カメラは、動作モードを切り替えることにより、昼間と夜間という光量が極端に異なる２つの状況下において単に入射イメージの検出を可能としたものである。しかし、２つの動作モードを切り替えて入射光量を定量的に計測する場合は、それぞれの動作モードにおける感度が異なるため、当該２つの動作モードを切り替えてダイナミックレンジを拡げたとしても、精度の高い計測ができない。
【０００４】
そこで本発明は、広いダイナミックレンジを有し、精度の高い計測が可能な光計測装置及び光計測方法を提供することを課題とする。
【０００５】
【課題を解決するための手段】
上記課題を解決するために、本発明の光計測装置は、光電面と半導体検出器とを有し、上記光電面と上記半導体検出器との間に第１の電圧が印加された第１の動作状態においては、上記光電面への光の入射に伴って上記光電面から放出された光電子を上記半導体検出器によって検出するとともに上記放出された光電子の数に応じた出力信号を出力し、上記光電面と上記半導体検出器との間に上記第１の電圧よりも絶対値が小さい第２の電圧が印加された第２の動作状態においては、上記光電面に入射した光のうち上記光電面を透過する光束を上記半導体検出器によって検出するとともに上記光電面を透過する光束の光量に応じた出力信号を出力し、上記光電面と上記半導体検出器との間に上記第１の電圧よりも絶対値が小さく上記第２の電圧よりも絶対値が大きい第３の電圧が印加された第３の動作状態において、上記光電面への光の入射に伴って上記光電面から放出された光電子を上記半導体検出器によって検出するとともに上記放出された光電子の数に応じた出力信号を出力する光検出手段と、上記光検出手段から出力された出力信号に基づいて、上記光検出手段の上記動作状態を切り替える切替手段と、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第２の動作状態において上記光電面に上記参照光を入射させた場合の出力信号と、上記第３の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第３の動作状態において上記光電面に光を入射させない場合の出力信号と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号と、上記第１の動作状態における上記光検出手段の電流増倍率と、上記第３の動作状態における上記光検出手段の電流増倍率とに基づいて、上記第１の動作状態と上記第２の動作状態とにおける上記半導体検出器の感度差を補償するように、上記第１の動作状態における出力信号または上記第２の動作状態における出力信号を補正する補正手段とを備えたことを特徴としている。
【０００６】
第１の動作状態と第２の動作状態とを有し、かかる動作状態を切り替えることで、ダイナミックレンジを広くすることが可能となる。また、第１の動作状態において光電面に参照光を入射させた場合の出力信号と第２の動作状態において光電面に参照光を入射させた場合の出力信号とに基づいて、第１の動作状態における出力信号または第２の動作状態における出力信号を補正することで、第１の動作状態と第２の動作状態とにおける半導体検出器の感度差を補償することができ、精度の高い計測が可能となる。
【０００７】
第３の動作状態と第２の動作状態との感度差は、第１の動作状態と第２の動作状態との感度差と比較して小さいため、第３の動作状態において光電面に参照光を入射させた場合の出力信号等に基づいて、第１の動作状態における出力信号または第２の動作状態おける出力信号を補正することで、より精度の高い計測が可能となる。
【０００８】
また、本発明の光計測装置においては、上記補正手段は、上記第３の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ３と、上記第３の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ３と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ２と、上記第１の動作状態における上記光検出手段の電流増倍率Ｇ１と、上記第３の動作状態における上記光検出手段の電流増倍率Ｇ３とを用いて、
Ｃ３＝Ｇ・（Ｋ１−１）＋１
Ｇ＝Ｇ１／Ｇ３
Ｋ１＝（Ｉｓ３−Ｉｄ３）／（Ｉｓ２−Ｉｄ２）
で表される係数Ｃ３で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ３を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴とすることが好適である。
【０００９】
また、本発明の光計測装置においては、上記補正手段は、上記第３の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ３と、上記第３の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ３と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ２と、上記第１の動作状態における上記光検出手段の電流増倍率Ｇ１と、上記第３の動作状態における上記光検出手段の電流増倍率Ｇ３とを用いて、
Ｃ４＝Ｇ・Ｋ１
Ｇ＝Ｇ１／Ｇ３
Ｋ１＝（Ｉｓ３−Ｉｄ３）／（Ｉｓ２−Ｉｄ２）
で表される係数Ｃ４で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ４を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００１０】
上記課題を解決するために、本発明の光計測方法は、光電面と半導体検出器とを有し、上記光電面と上記半導体検出器との間に第１の電圧が印加された第１の動作状態においては、上記光電面への光の入射に伴って上記光電面から放出された光電子を上記半導体検出器によって検出するとともに上記放出された光電子の数に応じた出力信号を出力し、上記光電面と上記半導体検出器との間に上記第１の電圧よりも絶対値が小さい第２の電圧が印加された第２の動作状態においては、上記光電面に入射した光のうち上記光電面を透過する光束を上記半導体検出器によって検出するとともに上記光電面を透過する光束の光量に応じた出力信号を出力し、上記光電面と上記半導体検出器との間に上記第１の電圧よりも絶対値が小さく上記第２の電圧よりも絶対値が大きい第３の電圧が印加された第３の動作状態において、上記光電面への光の入射に伴って上記光電面から放出された光電子を上記半導体検出器によって検出するとともに上記放出された光電子の数に応じた出力信号を出力する光計測装置を用いた光計測方法であって、上記出力信号に基づいて、上記動作状態を切り替える切替工程と、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第２の動作状態において上記光電面に上記参照光を入射させた場合の出力信号と、上記第３の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第３の動作状態において上記光電面に光を入射させない場合の出力信号と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号と、上記第１の動作状態における上記光検出手段の電流増倍率と、上記第３の動作状態における上記光検出手段の電流増倍率とに基づいて、上記第１の動作状態と上記第２の動作状態とにおける上記半導体検出器の感度差を補償するように、上記第１の動作状態における出力信号または上記第２の動作状態における出力信号を補正する補正工程とを備えたことを特徴としている。
【００１１】
第１の動作状態と第２の動作状態とを有し、かかる動作状態を切り替えることで、ダイナミックレンジを広くすることが可能となる。また、第１の動作状態において光電面に参照光を入射させた場合の出力信号と第２の動作状態において光電面に参照光を入射させた場合の出力信号とに基づいて、第１の動作状態における出力信号または第２の動作状態における出力信号を補正することで、第１の動作状態と第２の動作状態とにおける半導体検出器の感度差を補償することができ、精度の高い計測が可能となる。
【００１２】
第３の動作状態と第２の動作状態との感度差は、第１の動作状態と第２の動作状態との感度差と比較して小さいため、第３の動作状態において光電面に参照光を入射させた場合の出力信号等に基づいて、第１の動作状態における出力信号または第２の動作状態おける出力信号を補正することで、より精度の高い計測が可能となる。
【００１３】
また、本発明の光計測方法においては、上記補正工程は、上記第３の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ３と、上記第３の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ３と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ２と、上記第１の動作状態における上記光検出手段の電流増倍率Ｇ１と、上記第３の動作状態における上記光検出手段の電流増倍率Ｇ３とを用いて、
Ｃ３＝Ｇ・（Ｋ１−１）＋１
Ｇ＝Ｇ１／Ｇ３
Ｋ１＝（Ｉｓ３−Ｉｄ３）／（Ｉｓ２−Ｉｄ２）
で表される係数Ｃ３で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ３を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することが好適である。
【００１４】
また、本発明の光計測方法においては、上記補正工程は、上記第３の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ３と、上記第３の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ３と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ２と、上記第１の動作状態における上記光検出手段の電流増倍率Ｇ１と、上記第３の動作状態における上記光検出手段の電流増倍率Ｇ３とを用いて、
Ｃ４＝Ｇ・Ｋ１
Ｇ＝Ｇ１／Ｇ３
Ｋ１＝（Ｉｓ３−Ｉｄ３）／（Ｉｓ２−Ｉｄ２）
で表される係数Ｃ４で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ４を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００１５】
本発明の光計測装置は、光電面と半導体検出器とを有し、上記光電面と上記半導体検出器との間に第１の電圧が印加された第１の動作状態においては、上記光電面への光の入射に伴って上記光電面から放出された光電子を上記半導体検出器によって検出するとともに上記放出された光電子の数に応じた出力信号を出力し、上記光電面と上記半導体検出器との間に上記第１の電圧よりも絶対値が小さい第２の電圧が印加された第２の動作状態においては、上記光電面に入射した光のうち上記光電面を透過する光束を上記半導体検出器によって検出するとともに上記光電面を透過する光束の光量に応じた出力信号を出力する光検出手段と、上記光検出手段から出力された出力信号に基づいて、上記光検出手段の上記動作状態を切り替える切替手段と、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号と上記第２の動作状態において上記光電面に上記参照光を入射させた場合の出力信号とに基づいて、上記第１の動作状態と上記第２の動作状態とにおける上記半導体検出器の感度差を補償するように、上記第１の動作状態における出力信号または上記第２の動作状態における出力信号を補正する補正手段とを備えたことを特徴としてもよい。
【００１６】
本発明の光計測装置においては、上記補正手段は、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ１と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２とを用いて
Ｃ１＝Ｉｓ１／Ｉｓ２
で表される係数Ｃ１で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ１を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００１７】
また、本発明の光計測装置においては、上記補正手段は、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第１の動作状態において上記光電面に光を入射させない場合の出力信号と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号とに基づいて、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００１８】
第１の動作状態において光電面に光を入射させない場合の出力信号と、第２の動作状態において光電面に光を入射させない場合の出力信号をも考慮して第１の動作状態における出力信号または第２の動作状態おける出力信号を補正することで、光電面及び半導体検出器の暗電流が出力信号に与える影響を除去することができ、より精度の高い計測が可能となる。
【００１９】
また、本発明の光計測装置においては、上記補正手段は、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ１と、上記第１の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ１と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ２とを用いて
Ｃ２＝（Ｉｓ１−Ｉｄ１）／（Ｉｓ２−Ｉｄ２）
で表される係数Ｃ２で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ２を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００２０】
本発明の光計測方法は、光電面と半導体検出器とを有し、上記光電面と上記半導体検出器との間に第１の電圧が印加された第１の動作状態においては、上記光電面への光の入射に伴って上記光電面から放出された光電子を上記半導体検出器によって検出するとともに上記放出された光電子の数に応じた出力信号を出力し、上記光電面と上記半導体検出器との間に上記第１の電圧よりも絶対値が小さい第２の電圧が印加された第２の動作状態においては、上記光電面に入射した光のうち上記光電面を透過する光束を上記半導体検出器によって検出するとともに上記光電面を透過する光束の光量に応じた出力信号を出力する光計測装置を用いた光計測方法であって、上記出力信号に基づいて、上記動作状態を切り替える切替工程と、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号と上記第２の動作状態において上記光電面に上記参照光を入射させた場合の出力信号とに基づいて、上記第１の動作状態と上記第２の動作状態とにおける上記半導体検出器の感度差を補償するように、上記第１の動作状態における出力信号または上記第２の動作状態における出力信号を補正する補正工程とを備えたことを特徴としてもよい。
【００２１】
また、本発明の光計測方法においては、上記補正工程は、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ１と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２とを用いて
Ｃ１＝Ｉｓ１／Ｉｓ２
で表される係数Ｃ１で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ１を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００２２】
また、本発明の光計測方法においては、上記補正工程は、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第１の動作状態において上記光電面に光を入射させない場合の出力信号と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号とに基づいて、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００２３】
第１の動作状態において光電面に光を入射させない場合の出力信号と、第２の動作状態において光電面に光を入射させない場合の出力信号をも考慮して第１の動作状態における出力信号または第２の動作状態おける出力信号を補正することで、光電面及び半導体検出器の暗電流が出力信号に与える影響を除去することができ、より精度の高い計測が可能となる。
【００２４】
また、本発明の光計測方法においては、上記補正工程は、上記第１の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ１と、上記第１の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ１と、上記第２の動作状態において上記光電面に参照光を入射させた場合の出力信号Ｉｓ２と、上記第２の動作状態において上記光電面に光を入射させない場合の出力信号Ｉｄ２とを用いて
Ｃ２＝（Ｉｓ１−Ｉｄ１）／（Ｉｓ２−Ｉｄ２）
で表される係数Ｃ２で上記第１の動作状態における出力信号を除す、または、上記係数Ｃ２を上記第２の動作状態おける出力信号に乗ずることにより、上記第１の動作状態における出力信号または上記第２の動作状態おける出力信号を補正することを特徴としてもよい。
【００２５】
【発明の実施の形態】
本発明の実施形態に係る光計測装置について図面を参照して説明する。本実施形態にかかる光計測装置は、蛍光色素で標識された試料（被測定対象）に対して励起光を照射し、この励起光によって生ずる蛍光の光量を計測する光計測装置である。まず、本実施形態に係る光計測装置の構成について説明する。図１は本実施形態に係る光計測装置の構成図である。
【００２６】
本実施形態にかかる光計測装置１０は、光源室１２、試料室１４及び計測室１６を備えている。光源室１２には、試料１８（標準試料、被測定対象である試料）に励起光を照射するための光源２０が設けられている。
【００２７】
試料室１４には、試料１８を入れるバイアル２２（容器）と、光源２０から照射された励起光の光路を変更して試料に入射させる反射ミラー２４と、試料１８から発せられた蛍光を集光する集光ミラー２６とが設けられている。集光ミラー２６は、試料１８を入れるバイアル２２を覆うように配置されており、光源２０から照射された励起光を試料１８に入射させるための孔２６ａと、孔２６ａと対向して設けられるとともに試料１８を透過した上記励起光を外部向けて通過させる孔２６ｂと、上記励起光の光軸から離れた部位に設けられるとともに集光された蛍光を出力する出力孔２６ｃとを有している。出力孔２６ｃが上記励起光の光軸から離れた部位に設けられていることから、出力孔２６ｃからは励起光が出力されない。
【００２８】
計測室１６には、光検出手段であるハイブリッドフォトディテクタ（以下、ＨＰＤ２８という）、トランスインピーダンスアンプ（以下、ＴＺアンプ３０という）、Ａ／Ｄ変換器３２、切替手段である電圧切替部３４、補正手段である補正部３６、メモリ３８及びディスプレイ４０が設けられている。以下、各構成要素について詳細に説明する。
【００２９】
ＨＰＤ２８は、Ｓｂ，Ｎａ，Ｋ，Ｃｓを真空中で面板に蒸着して形成したマルチアルカリの光電面２８ａと、半導体検出器であるアバランシェフォトダイオード（以下、ＡＰＤ２８ｂという）とを、真空筐体２８ｃ内に対向配置（３ｍｍ間隔）して構成されている。ここで、ＡＰＤ２８ｂのアノード−カソード間には、電圧切替部３４により、＋１５０Ｖの逆バイアス電圧が印加されている。また、ＨＰＤ２８は、光電面２８ａが上記集光ミラー２６の出力孔２６ｃに対向するように配置され、試料１８から発せられて集光ミラー２６によって集光された蛍光を受光することができるようになっている。
【００３０】
ＨＰＤ２８は、電圧切替部３４からの出力電圧により、２つの異なる動作モード（動作状態）で動作するようになっている。すなわち、ＨＰＤ２８は、APD２８ｂ（のアノード）に対して光電面２８ａに−８ｋＶの電圧（第１の電圧）が印加された第１の動作状態（以下、電子照射モードという）においては、光電面２８ａへの光の入射に伴って光電面２８ａから放出された光電子をAPD２８ｂよって検出し、光電面２８ａから放出された光電子の数に応じた出力信号を出力する。一方、ＨＰＤ２８は、APD２８ｂに対して光電面２８ａに上記−８ｋＶよりも絶対値が小さい＋１２Ｖの電圧（第２の電圧）が印加された第２の動作状態（以下、光透過モードという）においては、光電面２８ａに入射した光のうち当該光電面２８ａを透過する光束をAPD２８ｂによって検出し、光電面２８ａを透過する光束の光量に応じた出力信号を出力する。
【００３１】
電子照射モードにおいて、光電面２８ａに光が入射すると、光電面２８ａからは入射した光束の光量に応じた光電子が放出される。かかる光電子は電界の作用によって加速され、APD２８ｂに入射する。APD２８ｂに入射した光電子は、そのエネルギーを失う際に多数の正孔−電子対を生成し、これが初段の増倍率になる。初段の増倍率は、電子の加速電圧（光電面に印加した電圧）に依存し、かかる電圧が−８ｋＶである電子照射モードでは約１２００（電子照射ゲイン）となる。その後、電子はさらに５０倍程度（アバランシェゲイン）にアバランシェ増倍する。その結果、ＨＰＤ２８全体で約６００００倍のゲインが得られる。ここで、初段の増倍率が約１２００と極めて大きいことから、APD２８ｂを用いたＨＰＤ２８の増倍ゆらぎは極めて小さい。
【００３２】
一方、光透過モードにおいて、光電面２８ａに光が入射すると、マルチアルカリの光電面２８ａは可視波長域の光に対して約５０％の透過率を有することから、入射した光束の光量の約５０％が光電面２８ａを透過し、APD２８ｂに入射する。ここで、APD２８ｂは光にも感度を有するため、当該透過光はAPD２８ｂによって検出され、透過光の光量に応じた出力信号がAPD２８ｂから出力される。光透過モードにおいては、APD２８ｂに対する光電面２８ａの電位が正なので光電面２８ａから電子は放出されず、APD２８ｂによる光電変換後のアバランシェゲイン（５０倍）がＨＰＤ２８全体のゲインとなる。
【００３３】
ＴＺアンプ３０は、ＨＰＤ２８のＡＰＤ２８ｂから出力された電流信号を増幅するとともに、電圧に変換して出力する電流電圧変換回路である。かかるＴＺアンプ３０としては、１μＡを５０ｍＶ程度に増幅する増幅率を有し、３００ＭＨｚ程度の帯域を有することが好ましく、例えばＭｉｔｅｑ社のＡＵ−１４９４−３００（商品名）等が好適である。
【００３４】
Ａ／Ｄ変換器３２は、ＴＺアンプ３０から出力された電圧信号をＡ／Ｄ変換した値（以下、Ａ／Ｄ出力信号という）を補正部３６及び電圧切替部に対して出力する。Ａ／Ｄ変換器３２は、入力レンジに対して１２ビット（４０９６階調）の分解能を有している。
【００３５】
電圧切替部３４は、Ａ／Ｄ変換器３２から出力されたＡ／Ｄ出力信号に基づいて、ＨＰＤ２８の動作モードを切り替える。より詳細には、ＨＰＤ２８が電子照射モードで動作している状態、すなわち、電圧切替部３４が光電面２８ａに対して−８ｋＶの電圧を印加している状態において、Ａ／Ｄ出力信号がしきい値（例えば４０９６）以上となった場合、電圧切替部３４は光電面２８ａに印加する電圧を＋１２Ｖに切り替える。光電面２８ａに印加する電圧が＋１２Ｖに切り替わることで、ＨＰＤ２８の動作モードは光透過モードに切り替わる。一方、ＨＰＤ２８が光透過モードで動作している状態、すなわち、電圧切替部３４が光電面２８ａに対して＋１２Ｖの電圧を印加している状態において、Ａ／Ｄ出力信号がしきい値（例えばＡ／Ｄ出力信号が９）以下となった場合、電圧切替部３４は光電面２８ａに印加する電圧を−８ｋＶに切り替える。光電面２８ａに印加する電圧が−８ｋＶに切り替わることで、ＨＰＤ２８の動作モードは電子照射モードに切り替わる。
【００３６】
補正部３６は、電子照射モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号と、電子照射モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号と、光透過モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号と、光透過モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号とに基づいて、光透過モードにおける出力信号を補正する。具体的には、電子照射モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号Ｉｓ１と、電子照射モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号Ｉｄ１と、光透過モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号Ｉｓ２と、光透過モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号Ｉｄ２とを用いて
Ｃ２＝（Ｉｓ１−Ｉｄ１）／（Ｉｓ２−Ｉｄ２）（１）
で表される係数Ｃ２を光透過モードにおけるＡ／Ｄ出力信号に乗ずることにより、当該光透過モードにおけるＡ／Ｄ出力信号を補正する。ここで参照光としては、被測定対象となる試料が発する蛍光とほぼ同一の波長、放射角度分布を有する光が選択される。補正部３６はまた、電子照射モードにおけるＡ／Ｄ出力信号、光透過モードおける補正されたＡ／Ｄ出力信号を必要に応じて入射光量に変換し、出力信号Soutとしてメモリ３８に格納するとともに、ディスプレイ４０に表示する。
【００３７】
試料室１４と計測室１６とを隔てる壁面であって、ＨＰＤ２８の光電面２８ａに対向する位置にはシャッタ４２が設けられており、シャッタ４２を開閉することにより、ＨＰＤ２８の光電面２８ａへの光の入射、遮断を制御することが可能となっている。
【００３８】
続いて、本実施形態にかかる光計測装置１０の動作について説明し、併せて本発明の実施形態にかかる光計測方法について説明する。図２は、光計測装置１０を用いた光計測の手順を示すフローチャートである。光計測装置１０を用いて光計測を行う際は、まず、補正部３６で用いる係数Ｃ２の算出が行われる（Ｓ１）。具体的には、図３に示すフローチャートに従って係数Ｃ２が算出される。
【００３９】
まず、標準試料をバイアル２２に入れ、試料室１４にセットする（Ｓ２１）。ここで標準試料は、係数Ｃ２を算出するために用いる参照光を発する試料である。標準試料は、具体的には、被測定対象である試料１８と同じ色素で標識されており、励起光を照射することで、被測定対象である試料１８とほぼ同じ波長、ほぼ同じ放射角度分布を有する光（参照光）を発する。
【００４０】
標準試料がセットされ、光源２０から標準試料に対して励起光の照射が開始されると、電圧切替部３４により、APD２８ｂのアノード−カソード間に＋１５０Ｖの逆バイアス電圧が印加されるとともに、APD２８ｂ（のアノード）に対して光電面２８ａに＋１２Ｖの電圧が印加される（Ｓ２２）。ここで、シャッタ４２を閉じた状態（ダーク状態）におけるＡ／Ｄ出力信号、すなわち、光透過モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号Ｉｄ２が補正部３６によって取得され、メモリ３８に格納される（Ｓ２３）。同様に、シャッタ４２を開いた状態におけるＡ／Ｄ出力信号、すなわち、光透過モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号Ｉｓ２が補正部３６によって取得され、メモリ３８に格納される（Ｓ２４）。
【００４１】
続いて、電圧切替部３４により、APD２８ｂ（のアノード）に対して光電面２８ａに−８ｋＶの電圧が印加された状態で（Ｓ２５）、上記と同様に、電子照射モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号Ｉｄ１（Ｓ２６）、電子照射モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号Ｉｓ１（Ｓ２７）がそれぞれ補正部３６によって取得され、メモリ３８に格納される。
【００４２】
上記Ｉｓ１，Ｉｓ２，Ｉｄ１，Ｉｄ２が取得された後、補正部３６により、係数Ｃ２が算出される（Ｓ２８）。係数Ｃ２は、上述の（１）式に基づいて算出され、算出された係数Ｃ２は、メモリ３８に格納される。
【００４３】
係数Ｃ２の算出が終わると、図２に示す手順に従って計測が行われる。まず、被測定対象である試料１８をバイアル２２に入れ、試料室１４にセットする（Ｓ２）。ここで、光源２０から試料１８に対して励起光が照射されるとともに、光電面２８ａには、APD２８ｂ（のアノード）に対して−８ｋＶの電圧が電圧切替部３４によって印加される（Ｓ３）。すなわち、ＨＰＤ２８は電子照射モードで動作する状態となる。この状態で、シャッタ４２が開かれると、試料１８から発せられる蛍光がＨＰＤ２８によって検出され、ＴＺアンプ３０によってＩ／Ｖ変換及び増幅がなされた後Ａ／Ｄ変換器３２によってＡ／Ｄ変換され、Ａ／Ｄ出力信号Ｉａｄが取得される（Ｓ４）。ここで、Ａ／Ｄ出力信号Ｉａｄがしきい値（例えば４０９６）以上か否かが判断され（Ｓ５）、Ａ／Ｄ出力信号Ｉａｄがしきい値以上でなければ、補正部３６により外部への出力信号Ｓｏｕｔが算出され（Ｓ６）、メモリ３８に格納されるとともに外部に出力される（Ｓ７）。外部への出力信号Ｓｏｕｔは、Ａ／Ｄ出力信号Ｉａｄと、電子照射モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号Ｉｄ１とを用いて、式（２）に基づいて算出され、必要に応じて光量への変換が行われる。
Ｓｏｕｔ＝Ｉａｄ − Ｉｄ１（２）
【００４４】
一方、Ａ／Ｄ出力信号Ｉａｄがしきい値以上である場合は、電圧切替部３４により光電面２８ａへの印加電圧が＋１２Ｖに切り替えられ、ＨＰＤ２８の動作状態が光透過モードに切り替えられる（Ｓ８）。この状態で、試料から発せられる蛍光がＨＰＤ２８によって検出され、ＴＺアンプ３０によってＩ／Ｖ変換及び増幅がなされた後Ａ／Ｄ変換器３２によってＡ／Ｄ変換され、Ａ／Ｄ出力信号Ｉａｄが取得される（Ｓ９）。Ａ／Ｄ出力信号Ｉａｄが取得されると、外部への出力信号Ｓｏｕｔが算出され（Ｓ１１）、メモリ３８に格納されるとともに外部に出力される（Ｓ７）。ここで、外部への出力信号Ｓｏｕｔは、Ａ／Ｄ出力信号Ｉａｄと、光透過モードにおいて光電面２８ａに光を入射させない場合のＡ／Ｄ出力信号Ｉｄ２と、係数Ｃ２とを用いて、式（３）に基づいて算出され、必要に応じて光量への変換が行われる。
Ｓｏｕｔ＝（Ｉａｄ − Ｉｄ２）× Ｃ２（３）
尚、光透過モードにおいて取得したＡ／Ｄ出力信号がしきい値以下である場合は、電圧切替部３４により光電面２８ａへの印加電圧が−８ｋＶに切り替えられ、ＨＰＤ２８の動作状態が電子照射モードに切り替えられる（Ｓ１０）。
【００４５】
続いて、本実施形態にかかる光計測装置の作用及び効果について説明する。本実施形態にかかる光計測装置１０は、小さい光量を計測する電子照射モードと大きい光量を計測する光透過モードとを有するＨＰＤ２８を光検出手段として用いることで、ダイナミックレンジを広くすることが可能となる。また、電子照射モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号と光透過モードにおいて光電面２８ａに参照光を入射させた場合のＡ／Ｄ出力信号とを用いて、光透過モードにおけるＡ／Ｄ出力信号を補正することで、電子照射モードと光透過モードとにおけるAPD２８ｂの感度差を補償することができ、精度の高い計測が可能となる。
【００４６】
例えば、５００ｎｍ波長光に対する光電面２８ｂの量子効率を１５％、光電面２８ｂの光透過率を５０％、APD２８ｂの量子効率を８０％、電子照射モードのゲインを６００００、光透過モードのゲインを５０、係数Ｃ２を４５０、ＴＺアンプ３０の増幅率を１Ｖ／μＡ、Ａ／Ｄ変換器３２の入力レンジを４Ｖ、Ａ／Ｄ変換器３２の分解能を１２ビット（４０９６階調）とすると、電子照射モードにおいては、ＨＰＤ２８の光電面２８ａに１ｎＷの光が入射すると３．６μＡの出力電流が得られる。かかる出力電流は、ＴＺアンプ３０によって３．６Ｖの電圧となり、Ａ／Ｄ変換器３２からはＡ／Ｄ出力信号としてデジタル値”３６８６”が出力され、この”３６８６”が外部に対する出力信号となる（暗電流は非常に小さいものとして無視している）。
【００４７】
ここで、入射する光の光量が２ｎＷになると、Ａ／Ｄ変換器３２から出力されるＡ／Ｄ出力信号は”４０９６”以上（飽和）となるため、電圧切替部３４により、ＨＰＤ２８の動作状態が光透過モードに切り替えられる。この場合、ＨＰＤ２８からは１６ｎＡの出力電流が得られ、ＴＺアンプ３０によって１６ｍＶの電圧となり、Ａ／Ｄ変換器３２からはＡ／Ｄ出力信号としてデジタル値”１６”が出力される。このＡ／Ｄ出力信号は、補正部３６によってＣ２（＝４５０）倍され、”７２００”が外部に対する出力信号となる。この場合、分解能が１２ビットであるＡ／Ｄ変換器３２を用いながら、ダイナミックレンジを実質的に４５０倍（２０ビット、１×１０⁶階調）に広げることが可能となる。また、参照光を用いた計測値により算出されたＣ２を用いた補正を行うことで、精度の高い計測が可能である。
【００４８】
また、本実施形態にかかる光計測装置１０は、電子照射モードにおいて光電面に光を入射させない場合のＡ／Ｄ出力信号Ｉｄ１と、光透過モードにおいて光電面に光を入射させない場合のＡ／Ｄ出力信号Ｉｄ２をも考慮し、式（１）に基づいて、光透過モードにおけるＡ／Ｄ出力信号を補正することで、光電面２８ａおよびAPD２８ｂの暗電流がＡ／Ｄ出力信号に与える影響を除去することができ、より精度の高い計測が可能となる。
【００４９】
また、本実施形態にかかる光計測装置１０は、小さな光量と大きな光量との双方をＨＰＤ２８によって計測することで、小さな光量を計測するための光電子増倍管と大きな光量を計測するためのフォトダイオードとを併設する光計測装置と比較して、入射光学系、信号処理回路等を簡素化でき、装置全体を小型化することが可能となる。
【００５０】
また、本実施形態にかかる光計測装置１０は、大きな光量を計測する際は透過光の光量を計測するに光透過モードとするため、光電面２８ａからは光電子が放出されない。従って、ＨＰＤ２８の寿命を長くすることが可能となる。また、上記光透過モードにおいては、電子管に大きい光量の光を入射させたときに一般的に生じるドリフトの影響を受けることが無くなる。ここで、光透過モードにおいては、光電面２８ａの透過率が１００％ではないことから、APD２８ｂに入射する光の光量は減少するが、光透過モードは大きな光量を計測する場合にのみ選択されるため、Ｓ／Ｎ比への影響は少ない。
【００５１】
上記実施形態にかかる光検出装置１０においては、式（１）に基づいて算出される係数Ｃ２を用いてＡ／Ｄ出力信号の補正を行っていたが、光電面２８ａ、APD２８ｂの暗電流が小さい場合は、式（４）に基づいて算出される係数Ｃ１を光透過モードにおけるＡ／Ｄ出力信号に乗ずることによってＡ／Ｄ出力信号の補正を行ってもよい。
Ｃ１＝Ｉｓ１／Ｉｓ２（４）
この場合、電子照射モード、光透過モードにおける外部への出力信号Ｓｏｕｔは、Ａ／Ｄ出力信号Ｉａｄを用いてそれぞれ、式（５）、式（６）に示すようになる。尚、必要に応じて光量への変換が行われる。
Ｓｏｕｔ＝Ｉａｄ（５）
Ｓｏｕｔ＝Ｉａｄ × Ｃ２（６）
【００５２】
また、Ａ／Ｄ出力信号の補正は、以下に示すように行ってもよい。すなわち、電子照射モードと光透過モードとでは、感度差が大きいので、電子照射モードにおいて適当なＡ／Ｄ出力信号が得られるように参照光の強度を決定すると、光透過モードにおいては十分大きいＡ／Ｄ出力信号が得られず、感度の補償が難しくなる場合がある。この場合、光電面２８ａとAPD２８ｂとの間に、電子照射モードにおいて印加される−８ｋＶよりも絶対値が小さく、光透過モードにおいて印加される＋１２Ｖよりも絶対値が大きい−２ｋＶ（第３の電圧）が印加された第２電子照射モード（第３の動作状態）でＨＰＤ２８を動作させ、第２電子照射モードにおいて光電面２８ａに参照光を入射させた場合の出力信号Ｉｓ３と、第２電子照射モードにおいて光電面２８ａに光を入射させない場合の出力信号Ｉｄ３と、光透過モードにおいて光電面２８ａに参照光を入射させた場合の出力信号Ｉｓ２と、光透過モードにおいて光電面２８ａに光を入射させない場合の出力信号Ｉｄ２と、電子照射モードにおけるＨＰＤ２８ｂの電流増倍率Ｇ１と、第２電子照射モードにおけるＨＰＤ２８ｂの電流増倍率Ｇ３とを用いて式（７）〜（９）に基づいて求められた係数Ｃ３を光透過モードにおけるＡ／Ｄ出力信号に乗ずることによってＡ／Ｄ出力信号の補正を行ってもよい。
Ｃ３＝Ｇ・（Ｋ１−１）＋１（７）
Ｇ＝Ｇ１／Ｇ３（８）
Ｋ１＝（Ｉｓ３−Ｉｄ３）／（Ｉｓ２−Ｉｄ２）（９）
ここで、電流増倍率Ｇ１，Ｇ３とは、光電面２８ａから放出される光電子流に対するAPD２８ｂから出力される出力電流の比である。
【００５３】
電流増倍率Ｇ１（Ｇ３についても同様）は、図４に示す手順に従って求められる。すなわち、まず、標準試料をバイアル２２に入れ、試料室１４にセットする（Ｓ３１）。標準試料がセットされ、光源２０から標準試料に対して励起光の照射が開始されると、電圧切替部３４により、APD２８ｂ（のアノード）に対して光電面２８ａに−８ｋＶの電圧が印加され（Ｓ３２）、APD２８ｂのアノード−カソード間が短絡される（Ｓ３３）。ここで、シャッタ４２を閉じた状態（ダーク状態）におけるＡ／Ｄ出力信号Ｉｄ０１（Ｓ３４）、シャッタ４２を開いた状態におけるＡ／Ｄ出力信号Ｉｓ０１（Ｓ３５）が補正部３６によってそれぞれ取得され、メモリ３８に格納される。
【００５４】
続いて、電圧切替部３４により、APD２８ｂのアノード−カソード間に＋１５０Ｖの逆バイアス電圧が印加され（Ｓ３６）、シャッタ４２を閉じた状態（ダーク状態）におけるＡ／Ｄ出力信号Ｉｄ１（Ｓ３７）、シャッタ４２を開いた状態におけるＡ／Ｄ出力信号Ｉｓ１（Ｓ３８）が補正部３６によってそれぞれ取得され、メモリ３８に格納される。
【００５５】
上記Ｉｓ０１，Ｉｄ０１，Ｉｓ１，Ｉｄ１が取得された後、補正部３６により、電流増倍率Ｇ１が算出される（Ｓ３９）。電流増倍率Ｇ１は、式（１０）に基づいて算出され、メモリ３８に格納される。
Ｇ１＝（Ｉｓ１−Ｉｄ１）／（Ｉｓ０１−Ｉｄ０１）（１０）
尚、上記電流増倍率Ｇ１，Ｇ３は、入射光の波長の影響を受けず、APD２８ｂに対する光電面２８ａの電圧、APD２８ｂのアノード−カソード間に印加される逆バイアス電圧によって決定されるため、必ずしも被測定対象である試料と同波長の蛍光を発する標準試料を用いる必要はない。また、電流増倍率Ｇ１，Ｇ３は、ＨＰＤ２８を光計測装置１０に組み込む前に計測されてもよい。
【００５６】
また、上記式（７）において、Ｋ１、及び、Ｇ・（Ｋ１−１）が１と比較して十分大きい場合は、式（１１）に基づいて算出される係数Ｃ４を用いてＡ／Ｄ出力信号の補正を行ってもよい。
Ｃ４＝Ｇ・Ｋ１（１１）
【００５７】
また、第２電子照射モードを係数Ｃ３あるいはＣ４の算出のみならず、実際の光計測に用いることも可能である。すなわち、電子照射モードと第２電子照射モードとの電流増倍率の比、及び、光透過モードと第２電子照射モードとの感度の比をあらかじめ求めておき、Ａ／Ｄ出力信号に基づいてこれらの動作モードを切替え、また、上記電流増倍率の比、あるいは、感度の比を用いてＡ／Ｄ出力信号を補正して外部に出力することも可能である。光電面２８ａとAPD２８ｂとの間に−２ｋＶの電圧が印加された第２電子照射モードにおいては、電子照射ゲインが約６０となり、アバランシェゲインが約５０であることから、ＨＰＤ２８全体のゲインは約３０００となる。
【００５８】
上記第２電子照射モードの如く、電子照射モードと光透過モードとの中間のゲインを有する動作モードを設けることにより、感度の低い領域における分解能を向上させることが可能となる。例えば、Ａ／Ｄ変換器３２の１ビットは、電子照射モードにおいては２．７×１０^-13Ｗ、光透過モードにおいては１．２×１０^- ¹⁰Ｗの光量にそれぞれ相当する。これに対して、第２電子照射モードにおいては、電子照射モードとの感度比が２２であるため、Ａ／Ｄ変換器３２の１ビットは、５．４×１０^-12Ｗの光量に相当する。従って、第２電子照射モードで計測可能な光量である１．１×１０^-9〜２．２×１０^-8Ｗの範囲における分解能を向上させることができる。
【００５９】
上記実施形態にかかる光計測装置１０においては、半導体検出器としてAPD２８ｂを用いていたが、これは、フォトダイオードなどでもよい。
【００６０】
また、上記実施形態にかかる光計測装置は、被測定対象である試料から発せられた蛍光を計測するものであったが、これは、化学発光その他の光を計測するものであってもよい。
【００６１】
【発明の効果】
本発明の光計測装置及び光計測方法は、第１の動作状態と第２の動作状態とを有し、かかる動作状態を切り替えることで、ダイナミックレンジを広くすることが可能となる。また、第１の動作状態において光電面に参照光を入射させた場合の出力信号と第２の動作状態において光電面に参照光を入射させた場合の出力信号とに基づいて、第１の動作状態における出力信号または第２の動作状態における出力信号を補正することで、第１の動作状態と第２の動作状態とにおける半導体検出器の感度差を補償することができ、精度の高い計測が可能となる。
【図面の簡単な説明】
【図１】光計測装置の構成図である。
【図２】光計測の手順を示すフローチャートである。
【図３】係数Ｃ２を求める手順を示すフローチャートである。
【図４】係数Ｇ１を求める手順を示すフローチャートである。
【符号の説明】
１０…光計測装置、１２…光源室、１４…試料室、１６…計測室、１８…試料、２０…光源、２２…バイアル、２４…反射ミラー、２６…集光ミラー、２８…ＨＰＤ、２８ａ…光電面、２８ｂ…APD、３０…ＴＺアンプ、３２…Ａ／Ｄ変換器、３４…電圧切替部、３６…補正部、３８…メモリ、４０…ディスプレイ、４２…シャッタ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an optical measurement device and an optical measurement method.
[0002]
[Prior art]
There is a great need for an optical measuring device that can perform measurement even in two situations where the amount of light is extremely different (for example, daytime and nighttime). As an optical measuring device that meets such needs, for example, a day / night camera described in Japanese Patent Laid-Open No. 61-133540 can be cited. Such a day / night camera includes a photocathode and a photodetector, and includes a daytime mode in which photons transmitted through the photocathode are detected by a photodetector and a night mode in which photoelectrons emitted from the photocathode are detected by a photodetector. By switching appropriately, an incident image can be detected under two situations in which the light intensity is extremely different between daytime and nighttime.
[0003]
[Problems to be solved by the invention]
As described above, the daytime / nighttime camera can simply detect an incident image by switching the operation mode under two situations in which the light amounts of daytime and nighttime are extremely different. However, when measuring the amount of incident light quantitatively by switching between two operation modes, the sensitivity in each operation mode is different, so even if the dynamic range is expanded by switching between the two operation modes, high-accuracy measurement I can't.
[0004]
Therefore, an object of the present invention is to provide an optical measurement device and an optical measurement method that have a wide dynamic range and can perform highly accurate measurement.
[0005]
[Means for Solving the Problems]
  In order to solve the above-described problem, an optical measurement device of the present invention includes a photocathode and a semiconductor detector, and a first voltage is applied between the photocathode and the semiconductor detector. In the operating state, photoelectrons emitted from the photocathode with the incidence of light on the photocathode are detected by the semiconductor detector and output signals corresponding to the number of emitted photoelectrons are output. In a second operating state in which a second voltage having a smaller absolute value than the first voltage is applied between the photocathode and the semiconductor detector, the photocathode out of the light incident on the photocathode. The semiconductor detector detects the light beam that passes through the light source and outputs an output signal corresponding to the amount of light beam that passes through the photocathode.In a third operating state in which a third voltage having an absolute value smaller than the first voltage and larger than the second voltage is applied between the photocathode and the semiconductor detector. The semiconductor detector detects photoelectrons emitted from the photocathode with the incidence of light on the photocathode and outputs an output signal corresponding to the number of emitted photoelectrons.Reference light is incident on the photocathode in the first operation state, the light detection means, the switching means for switching the operation state of the light detection means based on the output signal output from the light detection means If the output signal and,An output signal when the reference light is incident on the photocathode in the second operation state;The output signal when the reference light is incident on the photocathode in the third operation state, the output signal when no light is incident on the photocathode in the third operation state, and the second operation An output signal when no light is incident on the photocathode in the state, a current multiplication factor of the light detection means in the first operation state, and a current multiplication factor of the light detection means in the third operation stateBased on the output signal in the first operating state or the output in the second operating state so as to compensate for the difference in sensitivity of the semiconductor detector between the first operating state and the second operating state. And correction means for correcting the signal.
[0006]
By having the first operating state and the second operating state and switching the operating state, the dynamic range can be widened. Further, based on the output signal when the reference light is incident on the photocathode in the first operation state and the output signal when the reference light is incident on the photocathode in the second operation state, the first operation is performed. By correcting the output signal in the state or the output signal in the second operation state, the difference in sensitivity of the semiconductor detector between the first operation state and the second operation state can be compensated, and high-precision measurement can be performed. It becomes possible.
[0007]
  Since the difference in sensitivity between the third operating state and the second operating state is small compared to the sensitivity difference between the first operating state and the second operating state, the reference light is applied to the photocathode in the third operating state. By correcting the output signal in the first operation state or the output signal in the second operation state based on the output signal or the like when the light is incident, it is possible to perform measurement with higher accuracy.
[0008]
  In the optical measurement device of the present invention, the correction means includes an output signal Is3 when reference light is incident on the photocathode in the third operation state, and the photocathode in the third operation state. Output signal Id3 when light is not incident on the light, output signal Is2 when reference light is incident on the photocathode in the second operation state, and light is incident on the photocathode in the second operation state. Using the output signal Id2 when not, the current multiplication factor G1 of the light detection means in the first operation state, and the current multiplication factor G3 of the light detection means in the third operation state,
  C3 = G · (K1-1) +1
    G = G1 / G3
    K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C3 expressed by the following equation, or multiplying the output signal in the second operation state by the coefficient C3, the output signal in the first operation state or It is preferable that the output signal in the second operation state is corrected.
[0009]
  In the optical measurement device of the present invention, the correction means includes an output signal Is3 when reference light is incident on the photocathode in the third operation state, and the photocathode in the third operation state. Output signal Id3 when light is not incident on the light, output signal Is2 when reference light is incident on the photocathode in the second operation state, and light is incident on the photocathode in the second operation state. Using the output signal Id2 when not, the current multiplication factor G1 of the light detection means in the first operation state, and the current multiplication factor G3 of the light detection means in the third operation state,
  C4 = G ・ K1
    G = G1 / G3
    K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C4 expressed by the following, or multiplying the output signal in the second operation state by the coefficient C4, the output signal in the first operation state or The output signal in the second operation state may be corrected.
[0010]
  In order to solve the above problems, an optical measurement method of the present invention includes a photocathode and a semiconductor detector, and a first voltage is applied between the photocathode and the semiconductor detector. In the operating state, photoelectrons emitted from the photocathode with the incidence of light on the photocathode are detected by the semiconductor detector and output signals corresponding to the number of emitted photoelectrons are output. In a second operating state in which a second voltage having a smaller absolute value than the first voltage is applied between the photocathode and the semiconductor detector, the photocathode out of the light incident on the photocathode. The semiconductor detector detects the light beam that passes through the light source and outputs an output signal corresponding to the amount of light beam that passes through the photocathode.In a third operating state in which a third voltage having an absolute value smaller than the first voltage and larger than the second voltage is applied between the photocathode and the semiconductor detector. The semiconductor detector detects photoelectrons emitted from the photocathode with the incidence of light on the photocathode and outputs an output signal corresponding to the number of emitted photoelectrons.An optical measurement method using an optical measurement device, the switching step of switching the operation state based on the output signal, and an output signal when reference light is incident on the photocathode in the first operation state When,An output signal when the reference light is incident on the photocathode in the second operation state;The output signal when the reference light is incident on the photocathode in the third operation state, the output signal when no light is incident on the photocathode in the third operation state, and the second operation An output signal when no light is incident on the photocathode in the state, a current multiplication factor of the light detection means in the first operation state, and a current multiplication factor of the light detection means in the third operation stateBased on the output signal in the first operating state or the output in the second operating state so as to compensate for the difference in sensitivity of the semiconductor detector between the first operating state and the second operating state. And a correction step for correcting the signal.
[0011]
  By having the first operating state and the second operating state and switching the operating state, the dynamic range can be widened. Further, based on the output signal when the reference light is incident on the photocathode in the first operation state and the output signal when the reference light is incident on the photocathode in the second operation state, the first operation is performed. By correcting the output signal in the state or the output signal in the second operation state, the difference in sensitivity of the semiconductor detector between the first operation state and the second operation state can be compensated, and high-precision measurement can be performed. It becomes possible.
[0012]
  Since the difference in sensitivity between the third operating state and the second operating state is small compared to the sensitivity difference between the first operating state and the second operating state, the reference light is applied to the photocathode in the third operating state. By correcting the output signal in the first operation state or the output signal in the second operation state based on the output signal or the like when the light is incident, it is possible to perform measurement with higher accuracy.
[0013]
  In the optical measurement method of the present invention, the correction step includes an output signal Is3 when reference light is incident on the photocathode in the third operation state, and the photocathode in the third operation state. Output signal Id3 when light is not incident on the light, output signal Is2 when reference light is incident on the photocathode in the second operation state, and light is incident on the photocathode in the second operation state. Using the output signal Id2 when not, the current multiplication factor G1 of the light detection means in the first operation state, and the current multiplication factor G3 of the light detection means in the third operation state,
  C3 = G · (K1-1) +1
    G = G1 / G3
    K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C3 expressed by the following equation, or multiplying the output signal in the second operation state by the coefficient C3, the output signal in the first operation state or It is preferable to correct the output signal in the second operation state.
[0014]
  In the optical measurement method of the present invention, the correction step includes an output signal Is3 when reference light is incident on the photocathode in the third operation state, and the photocathode in the third operation state. Output signal Id3 when light is not incident on the light, output signal Is2 when reference light is incident on the photocathode in the second operation state, and light is incident on the photocathode in the second operation state. Using the output signal Id2 when not, the current multiplication factor G1 of the light detection means in the first operation state, and the current multiplication factor G3 of the light detection means in the third operation state,
  C4 = G ・ K1
    G = G1 / G3
    K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C4 expressed by the following, or multiplying the output signal in the second operation state by the coefficient C4, the output signal in the first operation state or The output signal in the second operation state may be corrected.
[0015]
  The optical measuring device of the present invention has a photocathode and a semiconductor detector, and in the first operating state in which a first voltage is applied between the photocathode and the semiconductor detector, the photocathode The photoelectrons emitted from the photocathode with the incidence of light onto the photocathode are detected by the semiconductor detector and output signals corresponding to the number of photoelectrons emitted, and the photocathode, the semiconductor detector, In a second operation state in which a second voltage having an absolute value smaller than the first voltage is applied during the period, a light beam transmitted through the photocathode out of light incident on the photocathode is detected by the semiconductor. A light detection means for detecting an output by the detector and outputting an output signal corresponding to the amount of the light beam transmitted through the photocathode, and the operation state of the light detection means based on the output signal output from the light detection means. Switching means for switching Based on the output signal when the reference light is incident on the photocathode in the first operation state and the output signal when the reference light is incident on the photocathode in the second operation state, The output signal in the first operating state or the output signal in the second operating state is corrected so as to compensate for the difference in sensitivity of the semiconductor detector between the first operating state and the second operating state. And a correction means.Good.
[0016]
  In the optical measurement device of the present invention, the correction means refers to the output signal Is1 when reference light is incident on the photocathode in the first operation state, and refers to the photocathode in the second operation state. Using the output signal Is2 when light is incident
  C1 = Is1 / Is2
The output signal in the first operation state is divided by the coefficient C1 represented by the following equation, or the output signal in the second operation state is multiplied by the coefficient C1 or Correcting the output signal in the second operating state;May.
[0017]
  In the optical measurement device of the present invention, the correction means outputs an output signal when reference light is incident on the photocathode in the first operation state, and applies to the photocathode in the first operation state. An output signal when light is not incident, an output signal when reference light is incident on the photocathode in the second operation state, and a case where light is not incident on the photocathode in the second operation state The output signal in the first operation state or the output signal in the second operation state may be corrected based on the output signal.
[0018]
  In consideration of also the output signal when light is not incident on the photocathode in the first operation state and the output signal when light is not incident on the photocathode in the second operation state, the output signal in the first operation state or By correcting the output signal in the second operation state, the influence of the dark current of the photocathode and the semiconductor detector on the output signal can be removed, and measurement with higher accuracy is possible.
[0019]
  In the optical measurement device of the present invention, the correction means includes an output signal Is1 when reference light is incident on the photocathode in the first operation state, and the photocathode in the first operation state. The output signal Id1 when no light is incident on the light, the output signal Is2 when the reference light is incident on the photocathode in the second operation state, and the light incident on the photocathode in the second operation state When the output signal Id2 is not used
  C2 = (Is1-Id1) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C2 represented by the following, or by multiplying the output signal in the second operation state by the coefficient C2, the output signal in the first operation state or Correcting the output signal in the second operating state;May.
[0020]
  The optical measurement method of the present invention includes a photocathode and a semiconductor detector, and the photocathode in the first operating state in which a first voltage is applied between the photocathode and the semiconductor detector. The photoelectrons emitted from the photocathode with the incidence of light onto the photocathode are detected by the semiconductor detector and output signals corresponding to the number of photoelectrons emitted, and the photocathode, the semiconductor detector, In a second operation state in which a second voltage having an absolute value smaller than the first voltage is applied during the period, a light beam transmitted through the photocathode out of light incident on the photocathode is detected by the semiconductor. An optical measurement method using an optical measurement device that detects an optical signal and outputs an output signal corresponding to the amount of light flux that passes through the photocathode, and switches the operation state based on the output signal; , The first motion The first operation state based on the output signal when the reference light is incident on the photocathode in the state and the output signal when the reference light is incident on the photocathode in the second operation state And a correction step of correcting the output signal in the first operating state or the output signal in the second operating state so as to compensate for the difference in sensitivity of the semiconductor detector between the first operating state and the second operating state. As a featureGood.
[0021]
  In the optical measurement method of the present invention, the correction step includes an output signal Is1 when reference light is incident on the photocathode in the first operation state, and the photocathode in the second operation state. Using the output signal Is2 when the reference light is incident on
  C1 = Is1 / Is2
The output signal in the first operation state is divided by the coefficient C1 represented by the following equation, or the output signal in the second operation state is multiplied by the coefficient C1 or Correcting the output signal in the second operating state;May.
[0022]
  In the optical measurement method of the present invention, the correction step includes an output signal when reference light is incident on the photocathode in the first operation state, and an output signal on the photocathode in the first operation state. An output signal when light is not incident, an output signal when reference light is incident on the photocathode in the second operation state, and a case where light is not incident on the photocathode in the second operation state The output signal in the first operation state or the output signal in the second operation state may be corrected based on the output signal.
[0023]
  In consideration of also the output signal when light is not incident on the photocathode in the first operation state and the output signal when light is not incident on the photocathode in the second operation state, the output signal in the first operation state or By correcting the output signal in the second operation state, the influence of the dark current of the photocathode and the semiconductor detector on the output signal can be removed, and measurement with higher accuracy is possible.
[0024]
  In the optical measurement method of the present invention, the correction step includes an output signal Is1 when reference light is incident on the photocathode in the first operation state, and the photocathode in the first operation state. The output signal Id1 when no light is incident on the light, the output signal Is2 when the reference light is incident on the photocathode in the second operation state, and the light incident on the photocathode in the second operation state When the output signal Id2 is not used
  C2 = (Is1-Id1) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C2 represented by the following, or by multiplying the output signal in the second operation state by the coefficient C2, the output signal in the first operation state or Correcting the output signal in the second operating state;May.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
An optical measurement apparatus according to an embodiment of the present invention will be described with reference to the drawings. The optical measurement apparatus according to the present embodiment is an optical measurement apparatus that irradiates a sample (measurement target) labeled with a fluorescent dye with excitation light and measures the amount of fluorescence generated by the excitation light. First, the configuration of the optical measurement device according to the present embodiment will be described. FIG. 1 is a configuration diagram of an optical measurement device according to the present embodiment.
[0026]
The optical measurement device 10 according to the present embodiment includes a light source chamber 12, a sample chamber 14, and a measurement chamber 16. The light source chamber 12 is provided with a light source 20 for irradiating the sample 18 (standard sample, sample to be measured) with excitation light.
[0027]
In the sample chamber 14, a vial 22 (container) for storing the sample 18, a reflection mirror 24 that changes the optical path of the excitation light emitted from the light source 20 and makes it incident on the sample, and the fluorescence emitted from the sample 18 are collected. And a condensing mirror 26 is provided. The condensing mirror 26 is disposed so as to cover the vial 22 into which the sample 18 is placed, and is provided opposite to the hole 26a for allowing the excitation light irradiated from the light source 20 to enter the sample 18 and the hole 26a. It has a hole 26b that allows the excitation light that has passed through the sample 18 to pass outward, and an output hole 26c that is provided at a position away from the optical axis of the excitation light and that outputs the condensed fluorescence. Since the output hole 26c is provided at a position away from the optical axis of the excitation light, no excitation light is output from the output hole 26c.
[0028]
The measurement chamber 16 includes a hybrid photodetector (hereinafter referred to as HPD 28) that is a light detection means, a transimpedance amplifier (hereinafter referred to as TZ amplifier 30), an A / D converter 32, a voltage switching unit 34 that is switching means, and a correction means. A correction unit 36, a memory 38, and a display 40 are provided. Hereinafter, each component will be described in detail.
[0029]
The HPD 28 includes a multi-alkali photocathode 28a formed by evaporating Sb, Na, K, and Cs on a face plate in a vacuum, and an avalanche photodiode (hereinafter referred to as APD 28b) that is a semiconductor detector. It is configured to be opposed to each other (3 mm interval). Here, a reverse bias voltage of +150 V is applied between the anode and the cathode of the APD 28 b by the voltage switching unit 34. The HPD 28 is arranged so that the photocathode 28a faces the output hole 26c of the condenser mirror 26 so that it can receive the fluorescence emitted from the sample 18 and condensed by the condenser mirror 26. It has become.
[0030]
The HPD 28 operates in two different operation modes (operation states) according to the output voltage from the voltage switching unit 34. That is, the HPD 28 has a photocathode 28a in a first operation state (hereinafter referred to as an electron irradiation mode) in which a voltage (first voltage) of −8 kV is applied to the photocathode 28a with respect to the APD 28b (the anode thereof). The photoelectrons emitted from the photocathode 28a with the incidence of light on the APD 28b are detected by the APD 28b, and an output signal corresponding to the number of photoelectrons emitted from the photocathode 28a is output. On the other hand, the HPD 28 is in a second operation state (hereinafter referred to as a light transmission mode) in which a voltage (second voltage) of +12 V having an absolute value smaller than −8 kV is applied to the photocathode 28a with respect to the APD 28b. The light beam transmitted through the photocathode 28a out of the light incident on the photocathode 28a is detected by the APD 28b, and an output signal corresponding to the amount of the light beam transmitted through the photocathode 28a is output.
[0031]
When light is incident on the photocathode 28a in the electron irradiation mode, photoelectrons corresponding to the amount of incident light flux are emitted from the photocathode 28a. Such photoelectrons are accelerated by the action of the electric field and enter the APD 28b. When the photoelectrons incident on the APD 28b lose their energy, they generate a large number of hole-electron pairs, which become the first stage multiplication factor. The multiplication factor in the first stage depends on the acceleration voltage (voltage applied to the photocathode) of electrons, and is about 1200 (electron irradiation gain) in the electron irradiation mode where the voltage is −8 kV. Thereafter, the electrons are further avalanche multiplied by about 50 times (avalanche gain). As a result, a gain of about 60000 times is obtained in the entire HPD 28. Here, since the multiplication factor at the first stage is as large as about 1200, the multiplication fluctuation of the HPD 28 using the APD 28b is extremely small.
[0032]
On the other hand, in the light transmission mode, when light is incident on the photocathode 28a, the multi-alkali photocathode 28a has a transmittance of about 50% with respect to light in the visible wavelength range, so that the light quantity of the incident light beam is about 50%. % Passes through the photocathode 28a and enters the APD 28b. Here, since the APD 28b has sensitivity to light, the transmitted light is detected by the APD 28b, and an output signal corresponding to the amount of transmitted light is output from the APD 28b. In the light transmission mode, since the potential of the photocathode 28a with respect to the APD 28b is positive, electrons are not emitted from the photocathode 28a, and the avalanche gain (50 times) after photoelectric conversion by the APD 28b becomes the gain of the entire HPD 28.
[0033]
The TZ amplifier 30 is a current-voltage conversion circuit that amplifies the current signal output from the APD 28b of the HPD 28, converts the current signal into a voltage, and outputs the voltage. Such a TZ amplifier 30 has an amplification factor for amplifying 1 μA to about 50 mV, and preferably has a band of about 300 MHz. For example, AU-1494-300 (trade name) manufactured by Miteq is suitable.
[0034]
The A / D converter 32 outputs a value obtained by A / D converting the voltage signal output from the TZ amplifier 30 (hereinafter referred to as an A / D output signal) to the correction unit 36 and the voltage switching unit. The A / D converter 32 has a resolution of 12 bits (4096 gradations) with respect to the input range.
[0035]
The voltage switching unit 34 switches the operation mode of the HPD 28 based on the A / D output signal output from the A / D converter 32. More specifically, when the HPD 28 is operating in the electron irradiation mode, that is, when the voltage switching unit 34 is applying a voltage of −8 kV to the photocathode 28a, the A / D output signal is threshold. When the value (for example, 4096) or more is reached, the voltage switching unit 34 switches the voltage applied to the photocathode 28a to + 12V. When the voltage applied to the photocathode 28a is switched to + 12V, the operation mode of the HPD 28 is switched to the light transmission mode. On the other hand, in a state in which the HPD 28 is operating in the light transmission mode, that is, in a state in which the voltage switching unit 34 applies a voltage of +12 V to the photocathode 28a, the A / D output signal is a threshold value (for example, A When the / D output signal is 9) or less, the voltage switching unit 34 switches the voltage applied to the photocathode 28a to -8 kV. When the voltage applied to the photocathode 28a is switched to -8 kV, the operation mode of the HPD 28 is switched to the electron irradiation mode.
[0036]
The correction unit 36 has an A / D output signal when the reference light is incident on the photocathode 28a in the electron irradiation mode, an A / D output signal when no light is incident on the photocathode 28a in the electron irradiation mode, Based on the A / D output signal when the reference light is incident on the photocathode 28a in the transmission mode and the A / D output signal when no light is incident on the photocathode 28a in the light transmission mode, Correct the output signal. Specifically, the A / D output signal Is1 when the reference light is incident on the photocathode 28a in the electron irradiation mode, and the A / D output signal Id1 when no light is incident on the photocathode 28a in the electron irradiation mode. Using the A / D output signal Is2 when the reference light is incident on the photocathode 28a in the light transmission mode, and the A / D output signal Id2 when no light is incident on the photocathode 28a in the light transmission mode
C2 = (Is1-Id1) / (Is2-Id2) (1)
Is multiplied by the A / D output signal in the light transmission mode to correct the A / D output signal in the light transmission mode. Here, light having substantially the same wavelength and radiation angle distribution as the fluorescence emitted from the sample to be measured is selected as the reference light. The correction unit 36 also converts the A / D output signal in the electron irradiation mode and the corrected A / D output signal in the light transmission mode into an incident light amount as necessary, and stores it in the memory 38 as an output signal Sout. This is displayed on the display 40.
[0037]
A shutter 42 is provided on a wall surface separating the sample chamber 14 and the measurement chamber 16 and facing the photocathode 28a of the HPD 28. By opening and closing the shutter 42, light to the photocathode 28a of the HPD 28 is provided. It is possible to control the incidence and interception.
[0038]
Then, operation | movement of the optical measuring device 10 concerning this embodiment is demonstrated, and the optical measuring method concerning embodiment of this invention is demonstrated collectively. FIG. 2 is a flowchart showing a procedure of optical measurement using the optical measurement device 10. When optical measurement is performed using the optical measurement device 10, first, the coefficient C2 used in the correction unit 36 is calculated (S1). Specifically, the coefficient C2 is calculated according to the flowchart shown in FIG.
[0039]
First, a standard sample is put in the vial 22 and set in the sample chamber 14 (S21). Here, the standard sample is a sample that emits reference light used to calculate the coefficient C2. Specifically, the standard sample is labeled with the same dye as that of the sample 18 to be measured, and is irradiated with excitation light so that it has substantially the same wavelength and the same radiation angle distribution as the sample 18 to be measured. Emits light (reference light).
[0040]
When the standard sample is set and irradiation of excitation light from the light source 20 to the standard sample is started, a reverse bias voltage of +150 V is applied between the anode and the cathode of the APD 28b by the voltage switching unit 34, and the APD 28b ( A voltage of +12 V is applied to the photocathode 28a (S22). Here, an A / D output signal in a state where the shutter 42 is closed (dark state), that is, an A / D output signal Id2 in a case where no light is incident on the photocathode 28a in the light transmission mode, is acquired by the correction unit 36. It is stored in the memory 38 (S23). Similarly, an A / D output signal in a state where the shutter 42 is opened, that is, an A / D output signal Is2 when the reference light is incident on the photocathode 28a in the light transmission mode is acquired by the correction unit 36, and the memory 38 (S24).
[0041]
Subsequently, with the voltage switching unit 34 applying a voltage of −8 kV to the photocathode 28a with respect to the APD 28b (the anode) (S25), light is applied to the photocathode 28a in the electron irradiation mode in the same manner as described above. The A / D output signal Id1 (S26) when not incident, and the A / D output signal Is1 (S27) when reference light is incident on the photocathode 28a in the electron irradiation mode are respectively acquired by the correction unit 36, and the memory 38 Stored in
[0042]
After the above Is1, Is2, Id1, and Id2 are acquired, the coefficient C2 is calculated by the correction unit 36 (S28). The coefficient C2 is calculated based on the above equation (1), and the calculated coefficient C2 is stored in the memory 38.
[0043]
When the calculation of the coefficient C2 is completed, measurement is performed according to the procedure shown in FIG. First, the sample 18 to be measured is placed in the vial 22 and set in the sample chamber 14 (S2). Here, the sample 18 is irradiated with excitation light from the light source 20, and a voltage of -8 kV is applied to the photocathode 28a with respect to the APD 28b (the anode thereof) by the voltage switching unit 34 (S3). That is, the HPD 28 is in a state of operating in the electron irradiation mode. In this state, when the shutter 42 is opened, the fluorescence emitted from the sample 18 is detected by the HPD 28, subjected to I / V conversion and amplification by the TZ amplifier 30, and then A / D converted by the A / D converter 32, An A / D output signal Iad is acquired (S4). Here, it is determined whether or not the A / D output signal Iad is greater than or equal to a threshold value (for example, 4096) (S5). The output signal Sout is calculated (S6), stored in the memory 38, and output to the outside (S7). The output signal Sout to the outside is calculated based on the equation (2) using the A / D output signal Iad and the A / D output signal Id1 when no light is incident on the photocathode 28a in the electron irradiation mode. Conversion to light quantity is performed as necessary.
Sout = Iad−Id1 (2)
[0044]
On the other hand, when the A / D output signal Iad is equal to or greater than the threshold value, the voltage switching unit 34 switches the applied voltage to the photocathode 28a to + 12V, and the operation state of the HPD 28 is switched to the light transmission mode (S8). . In this state, fluorescence emitted from the sample is detected by the HPD 28, subjected to I / V conversion and amplification by the TZ amplifier 30, and then A / D converted by the A / D converter 32 to obtain an A / D output signal Iad. (S9). When the A / D output signal Iad is acquired, an output signal Sout to the outside is calculated (S11), stored in the memory 38, and output to the outside (S7). Here, the output signal Sout to the outside uses the A / D output signal Iad, the A / D output signal Id2 when light is not incident on the photocathode 28a in the light transmission mode, and the coefficient C2. 3) and is converted into a light amount as necessary.
Sout = (Iad−Id2) × C2 (3)
When the A / D output signal acquired in the light transmission mode is equal to or lower than the threshold value, the voltage switching unit 34 switches the applied voltage to the photocathode 28a to -8 kV, and the operation state of the HPD 28 is the electron irradiation mode. (S10).
[0045]
Then, the effect | action and effect of the optical measuring device concerning this embodiment are demonstrated. The optical measurement device 10 according to the present embodiment can widen the dynamic range by using the HPD 28 having an electron irradiation mode for measuring a small amount of light and a light transmission mode for measuring a large amount of light as a light detection unit. Become. Also, using an A / D output signal when the reference light is incident on the photocathode 28a in the electron irradiation mode and an A / D output signal when the reference light is incident on the photocathode 28a in the light transmission mode, By correcting the A / D output signal in the light transmission mode, the sensitivity difference of the APD 28b between the electron irradiation mode and the light transmission mode can be compensated, and high-precision measurement is possible.
[0046]
For example, the quantum efficiency of the photocathode 28b with respect to 500 nm wavelength light is 15%, the light transmittance of the photocathode 28b is 50%, the quantum efficiency of the APD 28b is 80%, the electron irradiation mode gain is 60000, and the light transmission mode gain is 50. When the coefficient C2 is 450, the amplification factor of the TZ amplifier 30 is 1 V / μA, the input range of the A / D converter 32 is 4 V, and the resolution of the A / D converter 32 is 12 bits (4096 gradations), electron irradiation In the mode, when 1 nW of light is incident on the photocathode 28a of the HPD 28, an output current of 3.6 μA is obtained. The output current becomes a voltage of 3.6 V by the TZ amplifier 30, and the digital value “3686” is output from the A / D converter 32 as an A / D output signal. This “3686” becomes an output signal to the outside. (The dark current is ignored as it is very small.)
[0047]
Here, when the amount of incident light is 2 nW, the A / D output signal output from the A / D converter 32 becomes “4096” or more (saturated). Is switched to the light transmission mode. In this case, an output current of 16 nA is obtained from the HPD 28, a voltage of 16 mV is obtained by the TZ amplifier 30, and a digital value “16” is output from the A / D converter 32 as an A / D output signal. This A / D output signal is multiplied by C2 (= 450) by the correction unit 36, and “7200” becomes an output signal to the outside. In this case, using the A / D converter 32 having a resolution of 12 bits, the dynamic range is substantially 450 times (20 bits, 1 × 10 6⁶(Gradation). In addition, highly accurate measurement is possible by performing correction using C2 calculated from the measured value using the reference light.
[0048]
In addition, the optical measurement apparatus 10 according to the present embodiment has an A / D output signal Id1 when light is not incident on the photocathode in the electron irradiation mode, and an A / D when light is not incident on the photocathode in the light transmission mode. Considering the output signal Id2 and correcting the A / D output signal in the light transmission mode based on the equation (1), the influence of the dark current of the photocathode 28a and the APD 28b on the A / D output signal is eliminated. Therefore, measurement with higher accuracy is possible.
[0049]
In addition, the optical measuring device 10 according to the present embodiment measures both a small light amount and a large light amount by the HPD 28, so that a photomultiplier tube for measuring the small light amount and a photodiode for measuring the large light amount. Can be simplified, and the entire apparatus can be reduced in size.
[0050]
In addition, since the optical measuring device 10 according to the present embodiment is in the light transmission mode for measuring the amount of transmitted light when measuring a large amount of light, no photoelectrons are emitted from the photocathode 28a. Therefore, the life of the HPD 28 can be extended. Further, in the light transmission mode, it is not affected by a drift that generally occurs when a large amount of light is incident on the electron tube. Here, in the light transmission mode, since the transmittance of the photocathode 28a is not 100%, the amount of light incident on the APD 28b is reduced, but the light transmission mode is selected only when measuring a large amount of light. Therefore, the influence on the S / N ratio is small.
[0051]
In the photodetector 10 according to the above embodiment, the A / D output signal is corrected using the coefficient C2 calculated based on the equation (1), but the dark currents on the photocathode 28a and the APD 28b are small. In this case, the A / D output signal may be corrected by multiplying the coefficient C1 calculated based on Expression (4) by the A / D output signal in the light transmission mode.
C1 = Is1 / Is2 (4)
In this case, the output signal Sout to the outside in the electron irradiation mode and the light transmission mode is as shown in Expression (5) and Expression (6) using the A / D output signal Iad, respectively. Note that conversion to a light amount is performed as necessary.
Sout = Iad (5)
Sout = Iad × C2 (6)
[0052]
Further, the correction of the A / D output signal may be performed as described below. That is, since there is a large sensitivity difference between the electron irradiation mode and the light transmission mode, if the intensity of the reference light is determined so that an appropriate A / D output signal can be obtained in the electron irradiation mode, A sufficiently large in the light transmission mode. / D output signal cannot be obtained, and compensation of sensitivity may be difficult. In this case, between the photocathode 28a and the APD 28b, an absolute value is smaller than −8 kV applied in the electron irradiation mode, and an absolute value larger than +12 V applied in the light transmission mode is −2 kV (third voltage). ) Is applied in the second electron irradiation mode (third operation state), the output signal Is3 when the reference light is incident on the photocathode 28a in the second electron irradiation mode, and the second electron irradiation. Output signal Id3 when light is not incident on the photocathode 28a in the mode, output signal Is2 when reference light is incident on the photocathode 28a in the light transmission mode, and light is not incident on the photocathode 28a in the light transmission mode Output signal Id2, current multiplication factor G1 of HPD 28b in the electron irradiation mode, and current of HPD 28b in the second electron irradiation mode It may be corrected in the A / D output signal by multiplying the magnification G3 and coefficient C3 obtained based on the equation (7) to (9) using the A / D output signal in the light transmission mode.
C3 = G · (K1-1) +1 (7)
G = G1 / G3 (8)
K1 = (Is3-Id3) / (Is2-Id2) (9)
Here, the current multiplication factors G1 and G3 are ratios of the output current output from the APD 28b to the photoelectron current emitted from the photocathode 28a.
[0053]
The current multiplication factor G1 (same for G3) is obtained according to the procedure shown in FIG. That is, first, a standard sample is put in the vial 22 and set in the sample chamber 14 (S31). When the standard sample is set and irradiation of excitation light from the light source 20 to the standard sample is started, the voltage switching unit 34 applies a voltage of −8 kV to the photocathode 28a to the APD 28b (the anode) ( S32), the anode and cathode of the APD 28b are short-circuited (S33). Here, the A / D output signal Id01 (S34) in the state where the shutter 42 is closed (dark state) and the A / D output signal Is01 (S35) in the state where the shutter 42 is opened are respectively acquired by the correction unit 36 and stored in the memory. 38.
[0054]
Subsequently, a reverse bias voltage of +150 V is applied between the anode and the cathode of the APD 28b by the voltage switching unit 34 (S36), the A / D output signal Id1 (S37) when the shutter 42 is closed (dark state), and the shutter The A / D output signal Is1 (S38) in a state in which 42 is opened is acquired by the correction unit 36 and stored in the memory 38.
[0055]
After obtaining Is01, Id01, Is1, and Id1, the current multiplication factor G1 is calculated by the correction unit 36 (S39). The current multiplication factor G1 is calculated based on the equation (10) and stored in the memory 38.
G1 = (Is1-Id1) / (Is01-Id01) (10)
The current multiplication factors G1 and G3 are not affected by the wavelength of incident light and are determined by the voltage of the photocathode 28a with respect to the APD 28b and the reverse bias voltage applied between the anode and the cathode of the APD 28b. It is not necessary to use a standard sample that emits fluorescence of the same wavelength as the sample to be measured. Further, the current multiplication factors G1 and G3 may be measured before the HPD 28 is incorporated into the optical measurement device 10.
[0056]
In the above equation (7), when K1 and G · (K1-1) are sufficiently larger than 1, the A / D output is performed using the coefficient C4 calculated based on the equation (11). Signal correction may be performed.
C4 = G · K1 (11)
[0057]
The second electron irradiation mode can be used not only for calculating the coefficient C3 or C4 but also for actual optical measurement. That is, the ratio of the current multiplication factor between the electron irradiation mode and the second electron irradiation mode and the ratio of the sensitivity between the light transmission mode and the second electron irradiation mode are obtained in advance, and these are obtained based on the A / D output signal. It is also possible to switch the operation mode and to correct the A / D output signal by using the current multiplication ratio or the sensitivity ratio and output it to the outside. In the second electron irradiation mode in which a voltage of −2 kV is applied between the photocathode 28a and the APD 28b, the electron irradiation gain is about 60 and the avalanche gain is about 50. Therefore, the gain of the entire HPD 28 is about 3000. It becomes.
[0058]
By providing an operation mode having a gain intermediate between the electron irradiation mode and the light transmission mode like the second electron irradiation mode, it is possible to improve the resolution in a low sensitivity region. For example, one bit of the A / D converter 32 is 2.7 × 10 in the electron irradiation mode.^-13W, 1.2 × 10 in light transmission mode^- ^TenEach corresponds to the amount of W light. On the other hand, in the second electron irradiation mode, since the sensitivity ratio to the electron irradiation mode is 22, 1 bit of the A / D converter 32 is 5.4 × 10 6.^-12It corresponds to the amount of W light. Therefore, the light quantity that can be measured in the second electron irradiation mode is 1.1 × 10^-9~ 2.2 × 10^-8The resolution in the W range can be improved.
[0059]
In the optical measuring device 10 according to the above embodiment, the APD 28b is used as the semiconductor detector, but this may be a photodiode or the like.
[0060]
Moreover, although the optical measuring device concerning the said embodiment measured the fluorescence emitted from the sample which is a to-be-measured object, this may measure chemiluminescence and other lights.
[0061]
【The invention's effect】
The optical measurement device and the optical measurement method of the present invention have a first operation state and a second operation state, and the dynamic range can be widened by switching between the operation states. Further, based on the output signal when the reference light is incident on the photocathode in the first operation state and the output signal when the reference light is incident on the photocathode in the second operation state, the first operation is performed. By correcting the output signal in the state or the output signal in the second operation state, the difference in sensitivity of the semiconductor detector between the first operation state and the second operation state can be compensated, and high-precision measurement can be performed. It becomes possible.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of an optical measurement device.
FIG. 2 is a flowchart showing a procedure of optical measurement.
FIG. 3 is a flowchart showing a procedure for obtaining a coefficient C2.
FIG. 4 is a flowchart showing a procedure for obtaining a coefficient G1.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 10 ... Optical measuring device, 12 ... Light source chamber, 14 ... Sample chamber, 16 ... Measurement chamber, 18 ... Sample, 20 ... Light source, 22 ... Vial, 24 ... Reflection mirror, 26 ... Condensing mirror, 28 ... HPD, 28a ... Photocathode, 28b ... APD, 30 ... TZ amplifier, 32 ... A / D converter, 34 ... Voltage switching unit, 36 ... correction unit, 38 ... memory, 40 ... display, 42 ... shutter

Claims

In the first operating state, which has a photocathode and a semiconductor detector, and the first voltage is applied between the photocathode and the semiconductor detector, the light enters the photocathode. The photoelectron emitted from the photocathode is detected by the semiconductor detector and an output signal corresponding to the number of photoelectrons emitted is output, and the first voltage is applied between the photocathode and the semiconductor detector. In a second operating state in which a second voltage having a smaller absolute value is applied, a light beam transmitted through the photocathode out of light incident on the photocathode is detected by the semiconductor detector and the photocathode. An output signal corresponding to the amount of the light beam passing through the first detector is output , and the absolute value is smaller than the first voltage and larger than the second voltage between the photocathode and the semiconductor detector. The third applied with a voltage of 3 In work state, the light detecting means for outputting an output signal corresponding to the number of emitted photoelectrons with the photoelectrons emitted from the photocathode in accordance with the incidence of light to the photocathode for detecting by said semiconductor detector When,
Switching means for switching the operation state of the light detection means based on an output signal output from the light detection means;
An output signal when is incident reference light to the photocathode in the first operating state, the output signal when is incident the reference light to the photocathode in the second operating state, the third An output signal when reference light is incident on the photocathode in the operation state, an output signal when light is not incident on the photocathode in the third operation state, and the photoelectric signal in the second operation state. Based on the output signal when no light is incident on the surface, the current multiplication factor of the light detection means in the first operation state, and the current multiplication factor of the light detection means in the third operation state , A compensation for correcting the output signal in the first operation state or the output signal in the second operation state so as to compensate for the difference in sensitivity of the semiconductor detector between the first operation state and the second operation state. Optical measuring apparatus characterized by comprising a means.

The correction means includes
An output signal Is3 when reference light is incident on the photocathode in the third operating state, an output signal Id3 when light is not incident on the photocathode in the third operating state, and the second An output signal Is2 when reference light is incident on the photocathode in the operating state, an output signal Id2 when light is not incident on the photocathode in the second operating state, and the output signal Id2 in the first operating state Using the current multiplication factor G1 of the light detection means and the current multiplication factor G3 of the light detection means in the third operating state,
C3 = G · (K1-1) +1
G = G1 / G3
K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C3 represented by the following equation, or multiplying the output signal in the second operation state by the coefficient C3, the output signal in the first operation state or The optical measurement apparatus according to claim 1 , wherein an output signal in the second operation state is corrected.

The correction means includes
An output signal Is3 when reference light is incident on the photocathode in the third operating state, an output signal Id3 when light is not incident on the photocathode in the third operating state, and the second An output signal Is2 when reference light is incident on the photocathode in the operating state, an output signal Id2 when light is not incident on the photocathode in the second operating state, and the output signal Id2 in the first operating state Using the current multiplication factor G1 of the light detection means and the current multiplication factor G3 of the light detection means in the third operating state,
C4 = G ・ K1
G = G1 / G3
K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C4 represented by the following equation, or multiplying the output signal in the second operation state by the coefficient C4, the output signal in the first operation state or The optical measurement apparatus according to claim 1 , wherein an output signal in the second operation state is corrected.

In the first operating state, which has a photocathode and a semiconductor detector, and the first voltage is applied between the photocathode and the semiconductor detector, the light enters the photocathode. The photoelectron emitted from the photocathode is detected by the semiconductor detector and an output signal corresponding to the number of photoelectrons emitted is output, and the first voltage is applied between the photocathode and the semiconductor detector. In a second operating state in which a second voltage having a smaller absolute value is applied, a light beam transmitted through the photocathode out of light incident on the photocathode is detected by the semiconductor detector and the photocathode. An output signal corresponding to the amount of the light beam passing through the first detector is output , and the absolute value is smaller than the first voltage and larger than the second voltage between the photocathode and the semiconductor detector. The third applied with a voltage of 3 In work state, the optical measurement device for outputting an output signal corresponding to the number of emitted photoelectrons with the photoelectrons emitted from the photocathode in accordance with the incidence of light detected by the semiconductor detector to the photocathode An optical measurement method using
A switching step of switching the operating state based on the output signal;
An output signal when is incident reference light to the photocathode in the first operating state, the output signal when is incident the reference light to the photocathode in the second operating state, the third An output signal when reference light is incident on the photocathode in the operation state, an output signal when light is not incident on the photocathode in the third operation state, and the photoelectric signal in the second operation state. Based on the output signal when no light is incident on the surface, the current multiplication factor of the light detection means in the first operation state, and the current multiplication factor of the light detection means in the third operation state , A compensation for correcting the output signal in the first operation state or the output signal in the second operation state so as to compensate for the difference in sensitivity of the semiconductor detector between the first operation state and the second operation state. Optical measurement method characterized by comprising the step.

The correction step includes
An output signal Is3 when reference light is incident on the photocathode in the third operating state, an output signal Id3 when light is not incident on the photocathode in the third operating state, and the second An output signal Is2 when reference light is incident on the photocathode in the operating state, an output signal Id2 when light is not incident on the photocathode in the second operating state, and the output signal Id2 in the first operating state Using the current multiplication factor G1 of the light detection means and the current multiplication factor G3 of the light detection means in the third operating state,
C3 = G · (K1-1) +1
G = G1 / G3
K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C3 represented by the following equation, or multiplying the output signal in the second operation state by the coefficient C3, the output signal in the first operation state or The optical measurement method according to claim 4 , wherein an output signal in the second operation state is corrected.

The correction step includes
An output signal Is3 when reference light is incident on the photocathode in the third operating state, an output signal Id3 when light is not incident on the photocathode in the third operating state, and the second An output signal Is2 when reference light is incident on the photocathode in the operating state, an output signal Id2 when light is not incident on the photocathode in the second operating state, and the output signal Id2 in the first operating state Using the current multiplication factor G1 of the light detection means and the current multiplication factor G3 of the light detection means in the third operating state,
C4 = G ・ K1
G = G1 / G3
K1 = (Is3-Id3) / (Is2-Id2)
By dividing the output signal in the first operation state by the coefficient C4 represented by the following equation, or multiplying the output signal in the second operation state by the coefficient C4, the output signal in the first operation state or The optical measurement method according to claim 4 , wherein an output signal in the second operation state is corrected.