JP3727494B2

JP3727494B2 - Optical measurement method and apparatus

Info

Publication number: JP3727494B2
Application number: JP23909599A
Authority: JP
Inventors: 和男袴田
Original assignee: Fuji Photo Film Co Ltd
Current assignee: Fujifilm Holdings Corp
Priority date: 1999-08-26
Filing date: 1999-08-26
Publication date: 2005-12-14
Anticipated expiration: 2019-08-26
Also published as: JP2001066254A

Description

【０００１】
【発明の属する技術分野】
本発明は、照明光や外光等の背景光の存在下においても高い精度で被検出光の測定を行うことができる光計測方法および装置に関するものである。
【０００２】
【従来の技術】
従来より、照明光や外光等の背景光によるノイズを除去する光計測方法および装置が各種研究されている。例えば生体等の試料に励起光を照射し、試料から発生する蛍光等の再放射光を測定する光計測装置においては、微弱な再放射光を測定するときに室内の照明および戸外の光等の背景光がノイズとして混入し測定品質を劣化させる。
【０００３】
そこで、励起光の照射時と非照射時のそれぞれにおいて同一時間に亘って測定を行い、励起光の照射により再放射光が発生しているときに測定した測定値から励起光が照射されていないときに測定した再放射光を含まない測定値を減算することにより背景光の影響を除去したり、周期的に背景光の強度が変化する場合には背景光の強度変化、例えば室内照明の１００Ｈｚまたは１２０Ｈｚ等の強度変化の周期に同期させて、励起光の照射時と非照射時のそれぞれにおいて同一の時間に亘って測定を行い、前者の測定値から後者の測定値を減算することにより背景光の影響を除去したりする方式等が研究されている。
【０００４】
【発明が解決しようとする課題】
しかしながら、強度変化の周期が異なる２種類以上の背景光が存在する場合には、従来研究されていた１種類の背景光の強度変化の周期に注目して背景光を除去する方式では他の背景光の影響を除去することはできず、測定の品質が劣化し、精度の高い測定値を得ることが困難となる。
【０００５】
本発明は、上記の事情に鑑みてなされたものであり、異なる周期を備えた２種類以上の背景光が存在する環境下であっても、背景光の影響を除去した精度の高い測定値を得ることができる光計測方法および装置を提供することを目的とする。
【０００６】
【課題を解決するための手段】
本発明の第１の光計測方法は、互いに異なる周期を備えた２種類以上の背景光の存在下で、被検出光を含む光を測定する検出測定と、被検出光を含まない光を測定する参照測定とを行い、検出測定で取得した値から参照測定で取得した値を減算することにより検出測定で取得した値から背景光の影響を除去する光計測方法であって、検出測定と参照測定との時間間隔を、各背景光がそれぞれ備える周期の最小公倍数の整数倍としたことを特徴とするものである。
【０００７】
本発明の第２の光計測方法は、互いに異なる周期を備えた２種類以上の背景光の存在下で、被検出光を含む光を測定する検出測定と、被検出光を含まない光を測定する参照測定とを行い、検出測定で取得した値から参照測定で取得した値を減算することにより検出測定で取得した値から背景光の影響を除去する光計測方法であって、検出測定の時間および参照測定の時間を、各背景光がそれぞれ備える周期の最小公倍数の整数倍としたことを特徴とするものである。
【０００８】
本発明の第３の光計測方法は、互いに異なる周期を備えた２種類以上の背景光の存在下で、被検出光を含む光を測定する検出測定と、被検出光を含まない光を測定する参照測定とを行い、検出測定で取得した値から参照測定で取得した値を減算することにより検出測定で取得した値から背景光の影響を除去する光計測方法であって、検出測定と参照測定との時間間隔を、背景光の中から選択された背景光がそれぞれ備える周期の最小公倍数の整数倍とし、かつ検出測定および参照測定の時間を、前記選択された背景光以外の背景光がそれぞれ備える周期の最小公倍数の整数倍としたことを特徴とするものである。
【０００９】
本発明の第１の光計測装置は、互いに異なる周期を備えた２種類以上の背景光の存在下で被検出光を測定する測定部と、被検出光を含む光を測定部によって測定する検出測定と被検出光を含まない光を測定部によって測定する参照測定との時間間隔、および検出測定と参照測定それぞれの測定時間を制御する制御部と、検出測定で取得した値から参照測定で取得した値を減算する演算部とを備えた光計測装置であって、検出測定と参照測定との時間間隔が、各背景光がそれぞれ備える周期の最小公倍数の整数倍であることを特徴とするものである。
【００１０】
本発明の第２の光計測装置は、互いに異なる周期を備えた２種類以上の背景光の存在下で被検出光を測定する測定部と、被検出光を含む光を測定部によって測定する検出測定と被検出光を含まない光を測定部によって測定する参照測定との時間間隔、および検出測定と参照測定それぞれの測定時間を制御する制御部と、検出測定で取得した値から参照測定で取得した値を減算する演算部とを備えた光計測装置であって、検出測定の時間および参照測定の時間が、各背景光がそれぞれ備える周期の最小公倍数の整数倍であることを特徴とするものである。
【００１１】
本発明の第３の光計測装置は、互いに異なる周期を備えた２種類以上の背景光の存在下で被検出光を測定する測定部と、被検出光を含む光を測定部によって測定する検出測定と被検出光を含まない光を測定部によって測定する参照測定との時間間隔、および検出測定と参照測定それぞれの測定時間を制御する制御部と、検出測定で取得した値から参照測定で取得した値を減算する演算部とを備えた光計測装置であって、検出測定と参照測定との時間間隔が、背景光の中から選択された背景光がそれぞれ備える周期の最小公倍数の整数倍であり、かつ検出測定および参照測定の測定時間が、前記選択された背景光以外の背景光がそれぞれ備える周期の最小公倍数の整数倍であることを特徴とするものである。
【００１２】
前記第１〜第３の方法および装置においては、前記検出測定および参照測定は複数回行うことができ、また、前記複数回行う検出測定および参照測定の各測定は、前記選択された背景光の強度変化の最小値近傍で行うことができ、さらに、前記選択した１種類の背景光は、全ての背景光の中で周期が最小のものとすることができる。
【００１３】
なお、前記検出測定と参照測定とは、れぞれ同じ時間に亘って測定が行われる。
【００１４】
また、「２種類以上の背景光」とは、除去対象である背景光を意味する。
【００１５】
また、前記背景光の中から選択された背景光もしくは前記選択された背景光以外の背景光が１つの場合、前記「背景光がそれぞれ備える周期の最小公倍数」とは前記１つの背景光の周期そのものとなる。
【００１６】
また、前記「選択された背景光以外の１つもしくは複数の背景光」とは、選択された背景光を除く残りの全ての背景光を意味している。
【００１７】
【発明の効果】
本発明の光計測方法および装置によれば、互いに異なる周期を備えた２種類以上の背景光の存在下で、検出測定と参照測定との時間間隔を、各背景光がそれぞれ備える周期の最小公倍数の整数倍としたり（第１の方法および装置）、検出測定の時間および参照測定の時間を、各背景光がそれぞれ備える周期の最小公倍数の整数倍としたり（第２の方法および装置）、検出測定と参照測定との時間間隔を、背景光の中から選択された背景光がそれぞれ備える周期の最小公倍数の整数倍とし、かつ検出測定および参照測定の測定時間を、前記選択された背景光以外の背景光がそれぞれ備える周期の最小公倍数の整数倍としたり（第３の方法および装置）することにより、被検出光を含む検出測定によって取得される値に含まれる除去対象となる上記２種類以上の背景光の値と、被検出光を含まない参照測定によって取得される値に含まれる除去対象となる上記２種類以上の背景光の値とが等しい値となるように検出測定と参照測定の測定時間あるいは測定間隔が設定される。そして、この設定に従い測定を行い検出測定で得られた値から参照測定で得られた値を減算することにより、除去対象となる上記２種類以上の背景光の値を消去することができ、異なる周期を備えた２種類以上の背景光が存在する環境下であっても、背景光の影響が除去された精度の高い測定結果を得ることができる。
【００１８】
【発明の実施の形態】
以下、本発明の具体的な実施の形態について図面を参照して説明する。
【００１９】
図１は本発明の光計測方法を実施する光計測装置の第１の実施形態として蛍光スペクトルを測定するスペクトル測定装置の概略を示す図である。
【００２０】
本実施の形態のスペクトル測定装置１００は、試料１０に波長４１０ｎｍ近傍の励起光Ｌｅを照射する励起光源２０と、前記励起光Ｌａの照射により試料１０から発生する蛍光Ｋｅ等を含む複数の光のスペクトル強度を測定し、それらの値を出力する測定部３０と、外部から入力される除去対象となる背景光の強度変化の周期に基づき測定のタイミングを制御し、前記測定により測定部３０から出力される複数の光のスペクトル強度の値を演算処理して背景光を除去した結果を出力する制御部４０と、制御部４０から出力される演算結果を表示する表示器５０とから構成されている。
【００２１】
測定部３０には、励起光Ｌａを遮断し蛍光Ｋｅを透過させる励起光カットフィルタ３３を備え、試料１０から発生する蛍光Ｋｅを集光する光学系３２および光学系３２により集光された蛍光Ｋｅを分光および測光し、そのスペクトル強度の値を制御部に出力する分光測光器３１が、測定部３０の光軸に沿って配設されている。
【００２２】
制御部４０には、外部から入力される背景光の強度変化の周期に基づき測定条件を設定する測定条件設定部４２と、この測定条件設定部４２によって設定された測定タイミングに基づき励起光源２０に励起光Ｌｅの照射の開始および終了の命令を出力し、分光測光器３１に測定の開始および終了の命令を出力するタイミングコントローラ４３と、分光測光器３１から入力された複数のスペクトル強度の値を演算処理してその結果を表示器５０に出力する演算処理部４４とを備えている。
【００２３】
次に、このスペクトル測定装置の作用について説明する。
【００２４】
まず始めに、コネクタ４１を介して外部から除去対象となる背景光の強度変化の周期データＤａが測定条件設定部４２に入力される。この周期データＤａは、スペクトル測定装置１００の背景光として存在する除去対象となる室内照明光と試料１０を観察するために必要な他の照明光の２種類の背景光の強度変化の周期の値からなり、これらの周期の値はあらかじめ別の測定装置等により測定され得られたものである。
【００２５】
測定条件設定部４２では、入力された周期データＤａに基づき測定時間、測定の時間間隔および励起光の照射時間が定められ、これらの値に基づいて、励起光Ｌｅの照射、励起光Ｌｅの試料１０への照射により発生した蛍光の測定を行う検出測定、および起光Ｌｅを照射せず試料１０から蛍光が発生しない状態で測定を行う参照測定の測定タイミングが求められ、その測定タイミングはタイミングコントローラ４３に出力される。
【００２６】
測定タイミングを入力したタイミングコントローラ４３は、その設定値に従って、励起光の照射、検出測定および参照測定それぞれの開始および終了の命令を測定部３０および励起光源２０に対して出力してスペクトル強度の測定が行われる。
【００２７】
ここで、検出測定が行われるときには、励起光Ｌｅの照射により発生した蛍光および前記２種類の背景光等が光学系３２を介して分光測光器３１に入射され、該分光測光器３１によって検出スペクトル強度が測定され、一方、参照測定が行われるときには、前記蛍光は発生させず２種類の背景光等が光学系３２を介して分光測光器３１に入射し、該分光測光器３１によって参照スペクトル強度が測定される。
【００２８】
分光測光器３１で測定された検出スペクトル強度および参照スペクトル強度は演算処理部４４に入力され減算処理が施され背景光が除去される。
【００２９】
ここで、上記測定および減算処理に関する作用について具体的に説明する。除去対象となる２種類の背景光は、図２のタイミングチャートに示すように、一方は室内照明光Ｆａ（ｔ）でその強度変化の周期ｆａは０．０２秒（５０Ｈｚ）であり、他方は試料１０をカラー画像として撮像するための面順次のＲ．Ｇ．Ｂ．の照明光（以下ＲＧＢ照明光Ｆｂ（ｔ）と呼ぶ）でその強度変化の周期ｆｂは０．０５秒（２０Ｈｚ）である。
【００３０】
上記室内照明光Ｆａ（ｔ）の周期ｆａ＝０．０２秒とＲＧＢ照明光の周期ｆｂ＝０．０５秒が周期データＤａとして測定条件設定部４２に入力されると該設定部４２では以下のように測定時間、測定間隔および励起光の照射時間が定められ、これらに基づいて測定タイミングが求められる。
【００３１】
検出測定と参照測定との測定間隔Ｔｋは、２種類の背景光がそれぞれ備える周期ｆａおよびｆｂの最小公倍数の整数倍とされ、周期ｆａ＝０．０２秒と周期ｆｂ＝０．０５秒との最小公倍数である０．１秒の２倍（整数倍）である０．２秒に定められる。また、測定時間Ｔｊは測定間隔Ｔｋより短時間の０．０７秒に、励起光Ｌｅの照射時間Ｔｒは測定時間Ｔｊより短時間の０．０２秒に定められる。そしてこれらの値を基にして検出測定の測定タイミングｔ１、ｔ２、ｔ３、ｔ４と参照測定の測定タイミングｔ５、ｔ６とが求められる。これらの測定タイミングは、測定条件設定部４２からタイミングコントローラ４３に出力され、タイミングコントローラ４３によって動作を制御する信号に変換され測定部３０および励起光源２０に出力され以下のようなタイミングで測定が行われる。
【００３２】
時刻ｔ１において検出測定が開始されると光学系３２を介して分光測光器３１に入射した光は、該測光器３１により分光され各波長に対応する光量が測定されて検出スペクトル強度ＳＰｃが求められる。検出測定は時刻ｔ１からｔ４までの測定時間Ｔｊ＝０．０７秒の間継続されるが、その中の時刻ｔ２からｔ３までの照射時間Ｔｒ＝０．０２秒の間励起光が試料１に照射され、試料から蛍光Ｋｅが発生し、この蛍光Ｋｅは光学系３２を介して分光測光器３１に入射され測定される。このとき試料１０等で反射され光学系３２に向う励起光Ｌｅは励起光カットフィルタ３３によって遮断されるので励起光Ｌｅが分光測光器３１によって検出されることはない。
【００３３】
時刻ｔ４で検出測定が終了すると、時刻ｔ１から測定間隔Ｔｋ＝０．２秒後の時刻ｔ５に参照測定が開始され、光学系３２を介して分光測光器３１に入射する光は、分光測光器３１によって分光され各波長に対応する光量が測定されて参照スペクトル強度ＳＰｓが求められる。参照測定は検出測定と同様に時刻ｔ５からｔ６までの測定時間Ｔｊ＝０．０７秒の間継続されるが、この間に励起光Ｌｅは試料１０に照射されないので蛍光Ｋｅが検出されることはない。
【００３４】
次に、検出測定で測定された検出スペクトル強度ＳＰｃおよび参照測定で測定された参照スペクトル強度ＳＰｓは分光測光器３１から演算処理部４４に出力され、該処理部４４において検出スペクトル強度ＳＰｃから参照スペクトル強度ＳＰｓを減算することにより背景光の影響を除去した蛍光スペクトル強度Ｓｐｋが求められ、その結果は表示器５０に出力され表示される。
【００３５】
ここで、２種類の背景光が上記減算処理によって除去される作用について説明する。測定間隔Ｔｋは、周期ｆａと周期ｆｂとの最小公倍数の２倍となっているので、その間隔Ｔｋは周期ｆａの１０周期分、かつ周期ｆｂの４周期分となり、検出測定が開始される時刻ｔ１における室内照明光Ｆａ（ｔ１）の位相Ｐａ１は参照測定が開始される時刻ｔ５における室内照明光Ｆａ（ｔ５）の位相Ｐａ２と一致し、同様に検出測定が開始される時刻ｔ１におけるＲＧＢ照明光Ｆｂ（ｔ１）の位相Ｐｂ１は、参照測定が開始される時刻ｔ５におけるＲＧＢ照明光Ｆｂ（ｔ５）の位相Ｐｂ２と一致する。さらに、検出測定および参照測定共に等しい測定時間Ｔｊ＝０．０７秒の間測定が行われるので、検出測定において測定される室内照明光の測定光量Ａ１と参照測定において測定される室内照明光の測定光量Ａ２とは等しい値となり、測定光量Ａ１を分光して求められる検出室内照明スペクトル強度Ｓｐａ１と測定光量Ａ２を分光して求められる参照室内照明スペクトル強度Ｓｐａ２とは等しくなる。
【００３６】
同様に検出測定および参照測定において測定されるＲＧＢ照明光の測定光量Ｂ１およびＢ２も等しく、これらの光量を分光して求められる検出ＲＧＢ照明スペクトル強度Ｓｐｂ１および参照ＲＧＢ照明スペクトル強度Ｓｐｂ２も等しくなる。
【００３７】
一方、励起光Ｌｅは検出測定の測定時間Ｔｊ内に０．０２秒間照射され、この照射により発生した蛍光から得られる光量Ｃは分光されて蛍光スペクトル強度Ｓｐｋとして測定されるが、この値は検出測定において測定される検出室内照明スペクトル強度Ｓｐａ１および検出ＲＧＢ照明スペクトル強度Ｓｐｂ１に加算されて検出される。
【００３８】
従って、検出測定において求められた検出スペクトル強度ＳＰｃ、すなわち蛍光スペクトル強度Ｓｐｋ、検出室内照明スペクトル強度Ｓｐａ１および検出ＲＧＢ照明スペクトル強度Ｓｐｂ１の和、から参照測定において求められた参照スペクトル強度ＳＰｓ、すなわち参照室内照明スペクトル強度Ｓｐａ２および参照ＲＧＢ照明スペクトル強度Ｓｐｂ２の和を減算すると、背景光の影響が除去された蛍光スペクトル強度Ｓｐｋを求めることができる。すなわち、

なぜなら
Ｓｐａ１＝Ｓｐａ２、Ｓｐｂ１＝Ｓｐｂ２
なお、上記各スペクトル強度は図３に示すように各波長に対する強度分布で表される値であるが、簡易的に上記減算の式の形で示した。
【００３９】
上記演算が演算処理部４４において施された後、その結果は表示器５０に出力され表示される。
【００４０】
図４は本発明の光計測方法を実施するスペクトル測定装置の第２の実施形態のタイミングチャート図であり、第２の実施形態のスペクトル測定装置の概略構成図は図１を兼用し、記号等についても第１の実施形態で用いられた記号を兼用する。
【００４１】
第２の実施形態は、検出測定および参照測定の測定時間を、除去対象の２つの背景光がそれぞれ備える周期の最小公倍数の整数倍に設定し背景光を除去する方式である。すなわち、図４のタイミングチャートに示すように、除去対象の背景光である室内照明光の強度変化Ｆａ（ｔ）の周期ｆａを０．０２秒（５０Ｈｚ）とし、もう一つの除去対象の背景光であるＲＧＢ照明光の強度変化Ｆｂ（ｔ）の周期ｆｂを０．０５秒（２０Ｈｚ）とすると、これらの周期は、周期データＤａとして測定条件設定部４２に入力され、該設定部４２では以下のように測定時間、測定間隔および励起光の照射時間が算定されて各タイミングの設定値が求められる。
【００４２】
検出測定および参照測定の測定時間Ｔｊは、２種類の背景光がそれぞれ備える周期ｆａおよびｆｂの最小公倍数の整数倍とされ、周期ｆａ＝０．０２秒と周期ｆｂ＝０．０５秒との最小公倍数である０．１秒の１倍（整数倍）である０．１秒に定められる。また、測定間隔Ｔｋは測定時間Ｔｊより長時間の０．１７秒に、励起光Ｌｅの照射時間Ｔｒは測定時間Ｔｊより短時間の０．０２秒に定められる。そしてこれらの値を基にして検出測定の測定タイミングｔ１、ｔ２、ｔ３、ｔ４と参照測定の測定タイミングｔ５、ｔ６とが設定される。この測定タイミングは、測定条件設定部４２からタイミングコントローラ４３に出力され、タイミングコントローラ４３によって動作を制御する信号に変換され測定部３０および励起光源２０に出力され以下のようなタイミングで測定が行われる。
【００４３】
時刻ｔ１において検出測定が開始されると光学系３２を介して分光測光器３１に入射した光は、該測光器３１により分光され各波長に対応する光量が測定されて検出スペクトル強度ＳＰｃが求められる。検出測定は時刻ｔ１からｔ４までの測定時間Ｔｊ＝０．１秒の間継続されるが、その中の時刻ｔ２からｔ３までのＴｒ＝０．０２秒の間励起光が試料１に照射され、試料から蛍光Ｋｅが発生し、この蛍光Ｋｅは光学系３２を介して分光測光器３１に入射され測定される。
【００４４】
時刻ｔ４で検出測定が終了すると、時刻ｔ１から測定間隔Ｔｋ＝０．１７秒後の時刻ｔ５に参照測定が開始され、光学系３２を介して分光測光器３１に入射する光は、分光測光器３１によって分光され各波長に対応する光量が測定されて参照スペクトル強度ＳＰｓが求められる。参照測定は検出測定と同様に時刻ｔ５からｔ６までの測定時間Ｔｊ＝０．１秒の間継続されるが、この間に励起光Ｌｅは試料１０に照射されないので蛍光Ｋｅが測定されることはない。
【００４５】
次に、検出測定で測定された検出スペクトル強度ＳＰｃおよび参照測定で測定された参照スペクトル強度ＳＰｓは演算処理部４４に出力され、該処理部４４において検出スペクトル強度ＳＰｃから参照スペクトル強度ＳＰｓを減算することにより背景光の影響を除去した蛍光スペクトル強度Ｓｐｋが求められ、その結果は表示器５０に出力され表示される。
【００４６】
ここで、２種類の背景光が上記減算処理によって除去される作用について説明する。測定時間Ｔｊは、周期ｆａと周期ｆｂとの最小公倍数の１倍となっているので、その時間Ｔｊは周期ｆａの５周期分、かつ周期ｆｂの２周期分となる。すなわち、検出測定開始時と参照測定開始時のそれぞれの背景光の位相に無関係に、室内照明光に関しては、検出測定および参照測定共に５周期分の測定光量Ａ１およびＡ２が測定され、一方ＲＧＢ照明光に関しては、検出測定および参照測定共に２周期分の測定光量Ｂ１およびＢ２が測定される。ここで、室内照明光の検出測定開始時と参照測定開始時の位相が異なっていても５周期分を測定するので測定される光量は等しくＡ１＝Ａ２となり、これらの光量を分光して求められる検出室内照明スペクトル強度Ｓｐａ１も参照室内照明スペクトル強度Ｓｐａ２と等しくなる。また、同様にＲＧＢ照明光の検出測定開始時と参照測定開始時の位相が異なっていても２周期分を測定するので測定される光量は等しくＢ１＝Ｂ２となり、これらの光量を分光して求められる検出ＲＧＢ照明スペクトル強度Ｓｐｂ１も参照ＲＧＢ照明スペクトル強度Ｓｐｂ２と等しくなる。
【００４７】
一方、励起光Ｌｅは検出測定の測定時間Ｔｊ内に０．０２秒間照射され、この照射により発生した蛍光から得られる光量Ｃは分光されて蛍光スペクトル強度Ｓｐｋとして測定されるが、この値は検出測定において測定される検出室内照明スペクトル強度Ｓｐａ１および検出ＲＧＢ照明スペクトル強度Ｓｐｂ１に加算されて検出される。
【００４８】
従って、検出測定において求められた検出スペクトル強度ＳＰｃ、すなわち蛍光スペクトル強度Ｓｐｋ、検出室内照明スペクトル強度Ｓｐａ１および検出ＲＧＢ照明スペクトル強度Ｓｐｂ１の和、から参照測定において求められた参照スペクトル強度ＳＰｓ、すなわち参照室内照明スペクトル強度Ｓｐａ２および参照ＲＧＢ照明スペクトル強度Ｓｐｂ２の和を減算すると、背景光の影響が除去された蛍光スペクトル強度Ｓｐｋを求めることができる。すなわち、

なぜなら
Ｓｐａ１＝Ｓｐａ２、Ｓｐｂ１＝Ｓｐｂ２
上記演算が施された後、その結果は表示器５０に出力され表示される。その他の構成および作用は第１の実施形態と同様である。
【００４９】
図５は本発明の光計測方法を実施するスペクトル測定装置の第３の実施形態のタイミングチャート図であり、第３の実施形態のスペクトル測定装置の概略構成図は図１を兼用し、記号等についても第１の実施形態で用られた記号を兼用する。
【００５０】
第３の実施形態は、検出測定と参照測定との時間間隔を、除去対象の２つの背景光の中の一方の背景光が備える周期の整数倍とし、かつ検出測定の時間および参照測定の時間を、他方の背景光が備える周期の整数倍に設定し背景光を除去する方式である。
【００５１】
すなわち、図５のタイミングチャートに示すように、除去対象の背景光である室内照明光の強度変化Ｆａ（ｔ）の周期ｆａを０．０２秒（５０Ｈｚ）とし、もう一つの除去対象の背景光であるＲＧＢ照明光の強度変化Ｆｂ（ｔ）の周期ｆｂを０．０５秒（２０Ｈｚ）とすると、これらの周期は、周期データＤａとして測定条件設定部４２に入力され、該設定部４２では以下のように測定時間、測定間隔および励起光の照射時間が定められて、測定タイミングが求められる。
【００５２】
測定間隔Ｔｋは周期ｆｂの３倍（整数倍）としてＴｋ＝０．１５秒に、検出測定および参照測定の測定時間Ｔｊは周期ｆａの４倍（整数倍）としてＴｊ＝０．０８秒に定められる。また、励起光Ｌｅの照射時間Ｔｒは測定時間Ｔｊより短時間の０．０２秒に定められる。
【００５３】
そしてこれらの値を基にして検出測定の測定タイミングｔ１、ｔ２、ｔ３、ｔ４と参照測定の測定タイミングｔ５、ｔ６とが設定される。これらの測定タイミングは、測定条件設定部４２からタイミングコントローラ４３に出力され、タイミングコントローラ４３によって動作を制御する信号に変換され測定部３０および励起光源２０に出力され以下のようなタイミングで測定が行われる。
【００５４】
時刻ｔ１において検出測定が開始されると光学系３２を介して分光測光器３１に入射した光は、該測光器３１により分光され各波長に対応する光量が測定されて検出スペクトル強度ＳＰｃが求められる。検出測定は時刻ｔ１からｔ４までの測定時間Ｔｊ＝０．０８秒の間継続されるが、その中の時刻ｔ２からｔ３までのＴｒ＝０．０２秒の間励起光が試料１に照射され、試料から蛍光Ｋｅが発生し、この蛍光Ｋｅは光学系３２を介して分光測光器３１に入射され測定される。
【００５５】
時刻ｔ４で検出測定が終了すると、時刻ｔ１から測定間隔Ｔｋ＝０．１５秒後の時刻ｔ５に参照測定が開始され、光学系３２を介して分光測光器３１に入射する光は、分光測光器３１によって分光され各波長に対応する光量が測定されて参照スペクトル強度ＳＰｓが求められる。参照測定は検出測定と同様に時刻ｔ５からｔ６までの測定時間Ｔｊ＝０．０８秒の間継続されるが、この間に励起光Ｌｅは試料１０に照射されないので蛍光Ｋｅが測定されることはない。
【００５６】
次に、検出測定で測定された検出スペクトル強度ＳＰｃおよび参照測定で測定された参照スペクトル強度ＳＰｓは演算処理部４４に出力され、該処理部４４において検出スペクトル強度ＳＰｃから参照スペクトル強度ＳＰｓを減算することにより背景光の影響を除去した蛍光スペクトル強度Ｓｐｋが求められ、その結果は表示器５０に出力され表示される。
【００５７】
ここで、２種類の背景光が上記減算処理によって除去される作用について説明する。室内照明光Ｆａ（ｔ）に関しては、検出測定および参照測定における測定時間Ｔｊはその周期ｆａの４倍であり、測定が開始される位相に関係なく、検出測定および参照測定共に４周期分の等しい光量Ａ１およびＡ２が測定され、これらの光量を分光して求められる検出室内照明スペクトル強度Ｓｐａ１と参照室内照明スペクトル強度Ｓｐａ２とは等しくなる。
【００５８】
ＲＧＢ照明光Ｆａ（ｔ）に関しては、測定間隔Ｔｋはその周期ｆｂの３倍であり検出測定および参照測定において測定を開始する位相Ｐｂ１とＰｂ２とが常に同一位相から始まり、かつそれぞれの測定は同じ時間に亘って行われるので、等しい光量Ｂ１およびＢ２が測定され、これらの光量を分光して求められる検出ＲＧＢ照明スペクトル強度Ｓｐｂ１と参照ＲＧＢ照明スペクトル強度Ｓｐｂ２とは等しくなる。
【００５９】
一方、励起光Ｌｅは検出測定の測定時間Ｔｊ内に０．０２秒間照射され、この照射により発生した蛍光から得られる光量Ｃは分光されて蛍光スペクトル強度Ｓｐｋとして測定されるが、この値は検出測定において測定される検出室内照明スペクトル強度Ｓｐａ１および検出ＲＧＢ照明スペクトル強度Ｓｐｂ１に加算されて検出される。
【００６０】
従って、検出測定において求められた検出スペクトル強度ＳＰｃ、すなわち蛍光スペクトル強度Ｓｐｋ、検出室内照明スペクトル強度Ｓｐａ１および検出ＲＧＢ照明スペクトル強度Ｓｐｂ１の和、から参照測定において求められた参照スペクトル強度ＳＰｓ、すなわち参照室内照明スペクトル強度Ｓｐａ２および参照ＲＧＢ照明スペクトル強度Ｓｐｂ２の和を減算すると、背景光の影響が除去された蛍光スペクトル強度Ｓｐｋを求めることができる。すなわち、

なぜなら
Ｓｐａ１＝Ｓｐａ２、Ｓｐｂ１＝Ｓｐｂ２
上記演算が施された後、その結果は表示器５０に出力され表示される。その他の構成および作用は第１の実施形態と同様である。
【００６１】
また、上記検出測定と参照測定とを２組行うこともできる。すなわち、図６のタイミングチャートに示すように、除去対象の背景光である室内照明光の強度変化Ｆａ（ｔ）の周期ｆａを０．０２秒（５０Ｈｚ）とし、もう一つの除去対象の背景光であるＲＧＢ照明光の強度変化Ｆｂ（ｔ）の周期ｆｂを０．０５秒（２０Ｈｚ）とすると、これらの周期は、周期データＤａとして測定条件設定部４２に入力され、該設定部４２では以下のように測定時間、測定間隔および励起光の照射時間が定められてこれらの値に基づき測定タイミングが求められる。
【００６２】
検出測定と参照測定との測定間隔Ｔｋは周期ｆｂの３倍（整数倍）のＴｋ＝０．１５秒に設定され、検出測定および参照測定の測定時間Ｔｊは周期ｆａの１倍（整数倍）としてＴｊ＝０．０２秒に定められる。また、励起光Ｌｅの照射時間Ｔｒは測定時間Ｔｊより短時間の時間０．０１秒に定められる。そしてこれらの値を基にして、１組目の検出測定の測定タイミングｔ１、ｔ２、ｔ３、ｔ４と参照測定の測定タイミングｔ５、ｔ６、および２組目の検出測定の測定タイミングｔ１’、ｔ２’、ｔ３’、ｔ４’と参照測定の測定タイミングｔ５’、ｔ６’とが求められる。これらの測定タイミングは、測定条件設定部４２からタイミングコントローラ４３に出力され、タイミングコントローラ４３によって動作を制御する信号に変換され測定部３０および励起光源２０に出力され以下のようなタイミングで測定が行われる。
【００６３】
１組目の測定は検出測定を時刻ｔ１からｔ４までの時間Ｔｊに亘って行なわれ、その中で励起光Ｌｅを時刻ｔ２からｔ３までの時間Ｔｒの間照射し蛍光Ｋｅを発生させ、蛍光の光量Ｃ２および種類の背景光の光量Ａ１、Ｂ１を得、これらの光量の和が分光されて検出スペクトル強度ＳＰｃが求められ、一方、参照測定を時刻ｔ１から測定間隔Ｔｋが経過した時刻ｔ５からｔ６までの測定時間Ｔｊに亘って行い、２種類の背景光の光量Ａ２およびＢ２を得、これらの光量の和が分光されて参照スペクトル強度ＳＰｓが求められる。
【００６４】
２組目の測定は、検出測定を時刻ｔ１’からｔ４’までの時間Ｔｊに亘って行なわれ、その中で励起光Ｌｅを時刻ｔ２’からｔ３’までの時間Ｔｒの間照射し蛍光Ｋｅを発生させ、蛍光の光量Ｃ’および２種類の背景光の光量Ａ１’、Ｂ１’を得、これらの光量の和が分光されて検出スペクトル強度ＳＰｃ’が求められ、一方、参照測定を時刻ｔ１’から測定間隔Ｔｋが経過した時刻ｔ５’からｔ６’までの測定時間Ｔｊに亘って行い、２種類の背景光の光量Ａ２’およびＢ２’を得、これらの光量の和が分光されて参照スペクトル強度ＳＰｓ’が求められる。
【００６５】
ここで、１組目の検出スペクトル強度ＳＰｃから参照スペクトル強度ＳＰｓを、もしくは２組目の検出スペクトル強度ＳＰｃ’から参照スペクトル強度ＳＰｓ’を減算することにより、上記に説明した内容と同様に背景光を除去することができ、蛍光光量Ｃもしくは蛍光光量Ｃ’に対応した蛍光スペクトル強度Ｓｐｋもしくは蛍光スペクトル強度Ｓｐｋ’が求められる。
【００６６】
従って、背景光を除去した蛍光スペクトル強度は以下の式によって求めることができる。
【００６７】
Ｓｐｋ＋Ｓｐｋ’＝（ＳＰｃ＋ＳＰｃ’）−（ＳＰｓ＋ＳＰｓ’）
なお、上記検出測定と参照測定とを３組以上行い同様の方式により背景光の影響を除去した蛍光スペクトル強度を得ることもできる。
【００６８】
また、第１の実施の形態において検出測定と参照測定とを２組行うこともできる。すなわち、図７のタイミングチャートに示すように、除去対象の背景光である室内照明光の強度変化Ｆａ（ｔ）の周期ｆａを０．０２秒（５０Ｈｚ）とし、もう一つの除去対象の背景光であるＲＧＢ照明光の強度変化Ｆｂ（ｔ）の周期ｆｂを０．０５秒（２０Ｈｚ）とすると、これらの周期は、周期データＤａとして測定条件設定部４２に入力され、該設定部４２では以下のように測定時間、測定間隔および励起光の照射時間が定められて、これらに基づき測定タイミングが求められる。
【００６９】
検出測定と参照測定との測定間隔は、２種類の背景光がそれぞれ備える周期ｆａおよびｆｂの最小公倍数の整数倍に定められるので、測定間隔Ｔｋは周期ｆａ＝０．０２秒と周期ｆｂ＝０．０５秒との最小公倍数である０．１秒の２倍（整数倍）である０．２秒に定められ。また、測定時間Ｔｊは測定間隔Ｔｋより短時間の０．０１秒に、励起光Ｌｅの照射時間Ｔｒは測定時間Ｔｊより短時間の０．００５秒に定められる。そしてこれらの値を基にして、１組目の検出測定の測定タイミングｔ１、ｔ２、ｔ３、ｔ４と参照測定の測定タイミングｔ５、ｔ６および２組目の検出測定の測定タイミングｔ１’、ｔ２’、ｔ３’、ｔ４’と参照測定の測定タイミングｔ５’、ｔ６’とが求められる。これらの測定タイミングは、測定条件設定部４２からタイミングコントローラ４３に出力され、タイミングコントローラ４３によって動作を制御する信号に変換され測定部３０および励起光源２０に出力され以下のようなタイミングで測定が行われる。
【００７０】
１組目の測定は検出測定を時刻ｔ１からｔ４までの時間Ｔｊに亘って行いその中で、励起光Ｌｅを時刻ｔ２からｔ３までの時間Ｔｒの間照射し蛍光Ｋｅを発生させ、蛍光の光量Ｃ２および種類の背景光の光量Ａ１、Ｂ１を得、これらの光量の和が分光されて検出スペクトル強度ＳＰｃが求められ、一方、参照測定は時刻ｔ１から測定間隔Ｔｋ＝０．０２秒後の時刻ｔ５からｔ６までの測定時間Ｔｊに亘って行い、２種類の背景光の光量Ａ２およびＢ２を得、これらの光量の和が分光されて参照スペクトル強度ＳＰｓが求められる。
【００７１】
２組目の測定は、検出測定を時刻ｔ１’からｔ４’までの時間Ｔｊに亘って行いその中で、励起光Ｌｅを時刻ｔ２からｔ３までの時間Ｔｒの間照射し蛍光Ｋｅを発生させ、蛍光の光量Ｃ’および２種類の背景光の光量Ａ１’、Ｂ１’を得、これらの光量の和が分光されて２組目の検出スペクトル強度ＳＰｃ’が求められ、一方、参照測定は時刻ｔ１’から測定間隔Ｔｋ＝０．０２秒後の時刻ｔ５’からｔ６’までの測定時間Ｔｊに亘って行い、２種類の背景光の光量Ａ２’およびＢ２’を得、これらの光量の和が分光されて２組目の参照スペクトル強度ＳＰｓ’が求められる。
【００７２】
ここで、１組目の検出スペクトル強度ＳＰｃから参照スペクトル強度ＳＰｓを、あるいは２組目の検出スペクトル強度ＳＰｃ’から参照スペクトル強度ＳＰｓ’を減算することにより、第１の実施形態で説明した内容と同様に背景光を除去することができ、蛍光スペクトル強度ＳｐｋもしくはＳｐｋ’が求められる。
【００７３】
従って、背景光を除去した蛍光スペクトル強度は以下の式によって求めることができる。
【００７４】
Ｓｐｋ＋Ｓｐｋ’＝（ＳＰｃ＋ＳＰｃ’）−（ＳＰｓ＋ＳＰｓ’）
なお、図７のタイミングチャートに示すように、このとき検出測定を行う時刻ｔ１からｔ４および参照測定を行う時刻ｔ５からｔ６、あるいは検出測定を行う時刻ｔ１’からｔ４’および参照測定を行う時刻ｔ５’からｔ６’は、各背景光の中で周期が最小の背景光、すなわち最小の周期ｆａを備えた室内照明光Ｆａ（ｔ）に同期させかつ各周期の中で強度が最小となる位相の近傍に検出測定および参照測定の測定タイミングを設定することにより背景光の影響をさらに低減することができる。
【００７５】
また、上記検出測定と参照測定とを３組以上行い同様の方式により背景光の影響を除去した蛍光スペクトル強度を得ることもできる。
【００７６】
なお、上記全ての実施形態においては、励起光の照射は検出測定の中で１回行われる設定としたが励起光の照射は同一検出測定の中で複数回行ってもよい。
【００７７】
また、上記全ての実施形態においては背景光は２種類としたが、背景光が３種類以上ある場合、例えば背景光がＦ１（ｔ）、Ｆ２（ｔ）、Ｆ３（ｔ）、Ｆ４（ｔ）、Ｆ５（ｔ）、Ｆ６（ｔ）およびＦ７（ｔ）の７種類ある場合にはＦ１（ｔ）、Ｆ２（ｔ）およびＦ３（ｔ）を合成したものを１つの背景光Ｆｘ（ｔ）と考え、その強度変化の周期ｆｘを求め、前記以外のＦ４（ｔ）、Ｆ５（ｔ）、Ｆ６（ｔ）およびＦ７（ｔ）を合成したものを、もう１つの背景光Ｆｙ（ｔ）と考え、その強度変化の周期ｆｙを求め、合成された２種類の背景光の強度変化の周期ｆｘとｆｙと用いて背景光の影響を除去する上記方式を適用することができる。
【００７８】
また、本発明の光計測方法および光計測装置は、蛍光、吸収、燐光などを測定対象とするスペクトル測定装置、定量計測装置、画像計測装置等にも適用することができる。
【００７９】
以上のように本発明の光計測方法および光計測装置によれば、異なる周期を備えた２種類以上の背景光が存在する環境下であっても、背景光の影響を除去した精度の高い測定値を得ることができる。
【図面の簡単な説明】
【図１】本発明の光計測方法および装置をスペクトル測定装置に適用した概略構成図
【図２】本発明の第１の実施形態のスペクトル測定方式のタイミングチャート図
【図３】スペクトル強度の図
【図４】本発明の第２の実施形態のスペクトル測定方式のタイミングチャート図
【図５】本発明の第３の実施形態のスペクトル測定方式のタイミングチャート図
【図６】本発明の第３の実施形態において複数回の測定を繰り返し行うスペクトル測定方式のタイミングチャート図
【図７】本発明の第１の実施形態において複数回の測定を繰り返し行うスペクトル測定方式のタイミングチャート図
【符号の説明】
１０試料
２０励起光源
３０測定部
４０制御部
５０表示器
１００スペクトル測定装置
Ｌａ励起光
Ｋｅ蛍光[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an optical measurement method and apparatus capable of measuring light to be detected with high accuracy even in the presence of background light such as illumination light and external light.
[0002]
[Prior art]
Conventionally, various researches have been made on optical measurement methods and apparatuses for removing noise caused by background light such as illumination light and external light. For example, in an optical measurement device that irradiates a sample such as a living body with excitation light and measures re-emitted light such as fluorescence generated from the sample, when measuring weak re-emitted light, such as indoor lighting and outdoor light Background light is mixed in as noise and degrades measurement quality.
[0003]
Therefore, the measurement is performed over the same time during excitation light irradiation and during non-irradiation, and no excitation light is irradiated from the measured value when re-radiated light is generated by irradiation with excitation light. In some cases, the influence of the background light is removed by subtracting the measured value not including the re-radiated light, or when the intensity of the background light periodically changes, for example, 100 Hz of the indoor lighting Alternatively, in synchronization with the period of intensity change such as 120 Hz, the measurement is performed over the same time in each of the excitation light irradiation and non-irradiation, and the latter measurement value is subtracted from the former measurement value. A method for removing the influence of light has been studied.
[0004]
[Problems to be solved by the invention]
However, when there are two or more types of background light having different intensity change periods, the method of removing background light by paying attention to the intensity change period of one type of background light, which has been studied in the past, is used for other background lights. The influence of light cannot be removed, the quality of measurement deteriorates, and it becomes difficult to obtain highly accurate measurement values.
[0005]
The present invention has been made in view of the above circumstances, and provides highly accurate measurement values that eliminate the influence of background light even in an environment where two or more types of background light having different periods exist. An object is to provide an optical measurement method and apparatus that can be obtained.
[0006]
[Means for Solving the Problems]
In the first optical measurement method of the present invention, in the presence of two or more types of background light having different periods, detection measurement for measuring light including the detected light and measurement of light not including the detected light are performed. This is an optical measurement method that removes the influence of background light from the value obtained by detection measurement by subtracting the value obtained by reference measurement from the value obtained by detection measurement. The time interval from the measurement is an integer multiple of the least common multiple of the period of each background light.
[0007]
In the second optical measurement method of the present invention, in the presence of two or more types of background light having different periods, detection measurement for measuring light including the detected light and measurement of light not including the detected light are performed. A measurement method that eliminates the influence of background light from the value obtained by detection measurement by subtracting the value obtained by reference measurement from the value obtained by detection measurement. The reference measurement time is set to an integer multiple of the least common multiple of the period of each background light.
[0008]
In the third optical measurement method of the present invention, in the presence of two or more types of background light having different periods, detection measurement for measuring light including the detected light and measurement of light not including the detected light are performed. This is an optical measurement method that removes the influence of background light from the value obtained by detection measurement by subtracting the value obtained by reference measurement from the value obtained by detection measurement. The time interval with the measurement is set to an integer multiple of the least common multiple of the periods of the background light selected from the background light, and the detection measurement and reference measurement times are set to the background light other than the selected background light. It is characterized by being an integral multiple of the least common multiple of the periods provided.
[0009]
A first optical measurement apparatus according to the present invention includes a measurement unit that measures detected light in the presence of two or more types of background light having different periods, and a detection that measures light including the detected light by the measurement unit. Obtained by reference measurement from the time interval between the measurement and the reference measurement in which light that does not contain the detected light is measured by the measurement unit, and the control unit that controls the measurement time of each of the detection measurement and reference measurement, and the value obtained by the detection measurement An optical measurement device including a calculation unit that subtracts the measured value, wherein the time interval between the detection measurement and the reference measurement is an integral multiple of the least common multiple of the period of each background light. It is.
[0010]
The second optical measurement apparatus of the present invention includes a measurement unit that measures the detected light in the presence of two or more types of background light having different periods, and a detection that measures the light including the detected light by the measurement unit. Obtained by reference measurement from the time interval between the measurement and the reference measurement in which the light not including the detected light is measured by the measurement unit, and the control unit that controls the measurement time of each of the detection measurement and reference measurement, and the value obtained by the detection measurement An optical measurement device including a calculation unit that subtracts the measured value, wherein the time of detection measurement and the time of reference measurement are integer multiples of the least common multiple of the period of each background light. It is.
[0011]
The third optical measurement apparatus of the present invention includes a measuring unit that measures the detected light in the presence of two or more types of background light having different periods, and a detection that measures the light including the detected light by the measuring unit. Obtained by reference measurement from the time interval between the measurement and the reference measurement in which the light not including the detected light is measured by the measurement unit, and the control unit that controls the measurement time of each of the detection measurement and reference measurement, and the value obtained by the detection measurement And a time interval between the detection measurement and the reference measurement is an integer multiple of the least common multiple of the periods of the background light selected from the background light. And the measurement time of the detection measurement and the reference measurement is an integral multiple of the least common multiple of the periods of the background light other than the selected background light.
[0012]
In the first to third methods and apparatuses, the detection measurement and the reference measurement can be performed a plurality of times, and the detection measurement and the reference measurement performed the plurality of times are performed on the selected background light. It can be performed in the vicinity of the minimum value of the intensity change, and the selected one type of background light can have the minimum period among all the background lights.
[0013]
Note that the detection measurement and the reference measurement are performed over the same time.
[0014]
“Two or more types of background light” means background light to be removed.
[0015]
In addition, when there is one background light selected from the background light or a background light other than the selected background light, the “least common multiple of the period of each background light” is the period of the one background light It becomes itself.
[0016]
The “one or more background lights other than the selected background light” means all the remaining background lights except the selected background light.
[0017]
【The invention's effect】
According to the optical measurement method and apparatus of the present invention, in the presence of two or more types of background light having different periods, the time interval between the detection measurement and the reference measurement is the least common multiple of the periods each background light has. Or an integer multiple of the least common multiple of the period of each background light (second method and apparatus) The time interval between the measurement and the reference measurement is an integer multiple of the least common multiple of the periods of the background light selected from the background light, and the measurement time for the detection measurement and the reference measurement is other than the selected background light. By using an integer multiple of the least common multiple of the periods included in each of the background light (third method and apparatus), it becomes a removal target included in the value acquired by the detection measurement including the detected light. The detection measurement is performed such that two or more types of background light values are equal to the two or more types of background light values to be removed included in a value acquired by reference measurement that does not include detected light. The measurement time or measurement interval of the reference measurement is set. Then, by performing the measurement according to this setting and subtracting the value obtained by the reference measurement from the value obtained by the detection measurement, the values of the two or more types of background light to be removed can be erased. Even in an environment where two or more types of background light having a period exist, it is possible to obtain a highly accurate measurement result from which the influence of the background light has been removed.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, specific embodiments of the present invention will be described with reference to the drawings.
[0019]
FIG. 1 is a diagram showing an outline of a spectrum measuring apparatus that measures a fluorescence spectrum as a first embodiment of an optical measuring apparatus that performs an optical measuring method of the present invention.
[0020]
The spectrum measuring apparatus 100 according to the present embodiment includes an excitation light source 20 that irradiates a sample 10 with excitation light Le having a wavelength of about 410 nm, and a plurality of lights including fluorescence Ke generated from the sample 10 by irradiation with the excitation light La. The measurement unit 30 that measures the spectrum intensity and outputs those values, and the measurement timing is controlled based on the period of the intensity change of the background light to be removed input from the outside, and is output from the measurement unit 30 by the measurement. The control unit 40 outputs the result of removing the background light by calculating the values of the spectral intensities of the plurality of lights, and the display 50 displaying the calculation result output from the control unit 40. .
[0021]
The measurement unit 30 includes an excitation light cut filter 33 that blocks the excitation light La and transmits the fluorescence Ke. The measurement system 30 collects the fluorescence Ke generated from the sample 10 and the fluorescence Ke condensed by the optical system 32. A spectrophotometer 31 is provided along the optical axis of the measuring unit 30 for spectrally and photometrically measuring and outputting the value of the spectral intensity to the control unit.
[0022]
The control unit 40 includes a measurement condition setting unit 42 that sets measurement conditions based on a period of intensity change of background light input from the outside, and an excitation light source 20 based on the measurement timing set by the measurement condition setting unit 42. A timing controller 43 that outputs an instruction to start and end the irradiation of the excitation light Le and outputs an instruction to start and end the measurement to the spectrophotometer 31, and a plurality of spectral intensity values input from the spectrophotometer 31. And an arithmetic processing unit 44 that performs arithmetic processing and outputs the result to the display 50.
[0023]
Next, the operation of this spectrum measuring apparatus will be described.
[0024]
First, period data Da of intensity change of background light to be removed is input to the measurement condition setting unit 42 from the outside via the connector 41. This period data Da is the value of the period of intensity change of two types of background light, that is, the room illumination light to be removed that exists as background light of the spectrum measuring apparatus 100 and the other illumination light necessary for observing the sample 10. These period values are obtained in advance by another measuring device or the like.
[0025]
In the measurement condition setting unit 42, the measurement time, the measurement time interval, and the excitation light irradiation time are determined based on the input period data Da. Based on these values, the excitation light Le irradiation and the excitation light Le sample are determined. The measurement timing of the detection measurement for measuring the fluorescence generated by the irradiation of the light source 10 and the reference measurement for performing the measurement in the state where the fluorescence is not generated from the sample 10 without irradiating the electromotive light Le is obtained. 43 is output.
[0026]
The timing controller 43 to which the measurement timing is input outputs the commands for starting and ending excitation light irradiation, detection measurement, and reference measurement to the measurement unit 30 and the excitation light source 20 according to the set value, and measures the spectral intensity. Is done.
[0027]
Here, when detection measurement is performed, the fluorescence generated by the irradiation of the excitation light Le, the two types of background light, and the like are incident on the spectrophotometer 31 via the optical system 32, and the spectrophotometer 31 detects the detection spectrum. When the intensity is measured and the reference measurement is performed, the fluorescence is not generated, and two kinds of background light and the like enter the spectrophotometer 31 through the optical system 32, and the spectrophotometer 31 uses the reference spectrum intensity. Is measured.
[0028]
The detected spectrum intensity and the reference spectrum intensity measured by the spectrophotometer 31 are input to the arithmetic processing unit 44 and subjected to a subtraction process to remove background light.
[0029]
Here, the effect | action regarding the said measurement and subtraction process is demonstrated concretely. As shown in the timing chart of FIG. 2, one of the two types of background light to be removed is room illumination light Fa (t), and its intensity change period fa is 0.02 seconds (50 Hz), and the other is A frame sequential R.D. for picking up the sample 10 as a color image. G. B. Of the illumination light (hereinafter referred to as RGB illumination light Fb (t)), the intensity change period fb is 0.05 seconds (20 Hz).
[0030]
When the cycle fa = 0.02 seconds of the indoor illumination light Fa (t) and the cycle fb = 0.05 seconds of the RGB illumination light are input to the measurement condition setting unit 42 as the cycle data Da, the setting unit 42 Thus, the measurement time, the measurement interval, and the excitation light irradiation time are determined, and the measurement timing is obtained based on these.
[0031]
The measurement interval Tk between the detection measurement and the reference measurement is an integer multiple of the least common multiple of the periods fa and fb included in each of the two types of background light, and the period fa = 0.02 seconds and the period fb = 0.05 seconds. It is set to 0.2 seconds that is twice (integer multiple) 0.1 seconds which is the least common multiple. The measurement time Tj is set to 0.07 seconds, which is shorter than the measurement interval Tk, and the irradiation time Tr of the excitation light Le is set to 0.02 seconds, which is shorter than the measurement time Tj. Based on these values, the measurement timings t1, t2, t3, and t4 of the detection measurement and the measurement timings t5 and t6 of the reference measurement are obtained. These measurement timings are output from the measurement condition setting unit 42 to the timing controller 43, converted into signals for controlling the operation by the timing controller 43, and output to the measurement unit 30 and the excitation light source 20, and are measured at the following timings. Is called.
[0032]
When detection measurement is started at time t1, the light incident on the spectrophotometer 31 via the optical system 32 is split by the photometer 31 and the amount of light corresponding to each wavelength is measured to obtain the detected spectral intensity SPc. . The detection measurement is continued for the measurement time Tj = 0.07 seconds from the time t1 to t4, and the sample 1 is irradiated with the excitation light for the irradiation time Tr = 0.02 seconds from the time t2 to t3. Then, fluorescence Ke is generated from the sample, and this fluorescence Ke enters the spectrophotometer 31 through the optical system 32 and is measured. At this time, the excitation light Le reflected by the sample 10 or the like and directed to the optical system 32 is blocked by the excitation light cut filter 33, so that the excitation light Le is not detected by the spectrophotometer 31.
[0033]
When the detection measurement is completed at time t4, the reference measurement is started at time t5 after the measurement interval Tk = 0.2 seconds from time t1, and the light incident on the spectrophotometer 31 via the optical system 32 is transmitted to the spectrophotometer. The amount of light corresponding to each wavelength is measured by 31 and the reference spectrum intensity SPs is obtained. The reference measurement is continued for the measurement time Tj = 0.07 seconds from the time t5 to t6 as in the detection measurement. However, since the excitation light Le is not irradiated on the sample 10 during this time, the fluorescence Ke is not detected. .
[0034]
Next, the detected spectrum intensity SPc measured by the detection measurement and the reference spectrum intensity SPs measured by the reference measurement are output from the spectrophotometer 31 to the arithmetic processing unit 44, and the processing unit 44 uses the detected spectrum intensity SPc to the reference spectrum. By subtracting the intensity SPs, the fluorescence spectrum intensity Spk from which the influence of background light is removed is obtained, and the result is output to the display 50 and displayed.
[0035]
Here, an operation in which two types of background light are removed by the subtraction process will be described. Since the measurement interval Tk is twice the least common multiple of the cycle fa and the cycle fb, the interval Tk is 10 cycles of the cycle fa and 4 cycles of the cycle fb, and the time when detection measurement is started The phase Pa1 of the room illumination light Fa (t1) at t1 coincides with the phase Pa2 of the room illumination light Fa (t5) at time t5 when the reference measurement is started, and similarly, RGB illumination light at time t1 when detection measurement is started. The phase Pb1 of Fb (t1) matches the phase Pb2 of the RGB illumination light Fb (t5) at the time t5 when the reference measurement is started. Further, since the detection measurement and the reference measurement are performed for the same measurement time Tj = 0.07 seconds, the measurement light quantity A1 of the room illumination light measured in the detection measurement and the measurement of the room illumination light measured in the reference measurement. The detected light amount A2 is equal to the detected indoor illumination spectrum intensity Spa1 obtained by dispersing the measured light amount A1, and the reference indoor illumination spectrum intensity Spa2 obtained by dispersing the measured light amount A2.
[0036]
Similarly, the measured light amounts B1 and B2 of the RGB illumination light measured in the detection measurement and the reference measurement are also equal, and the detected RGB illumination spectrum intensity Spb1 and the reference RGB illumination spectrum intensity Spb2 obtained by dispersing these light quantities are also equal.
[0037]
On the other hand, the excitation light Le is irradiated for 0.02 seconds within the measurement time Tj of the detection measurement, and the light amount C obtained from the fluorescence generated by this irradiation is dispersed and measured as the fluorescence spectrum intensity Spk, but this value is detected. It is added to the detected indoor illumination spectrum intensity Spa1 and the detected RGB illumination spectrum intensity Spb1 measured in the measurement.
[0038]
Accordingly, the reference spectrum intensity SPs obtained in the reference measurement from the detected spectrum intensity SPc obtained in the detection measurement, that is, the sum of the fluorescence spectrum intensity Spk, the detected indoor illumination spectrum intensity Spa1, and the detected RGB illumination spectrum intensity Spb1, that is, the reference room. By subtracting the sum of the illumination spectrum intensity Spa2 and the reference RGB illumination spectrum intensity Spb2, it is possible to obtain the fluorescence spectrum intensity Spk from which the influence of background light has been removed. That is,

Because
Spa1 = Spa2, Spb1 = Spb2
In addition, although each said spectrum intensity is a value represented by the intensity distribution with respect to each wavelength, as shown in FIG. 3, it showed with the form of the said subtraction type | formula simply.
[0039]
After the calculation is performed in the calculation processing unit 44, the result is output to the display 50 and displayed.
[0040]
FIG. 4 is a timing chart of the second embodiment of the spectrum measuring apparatus for carrying out the optical measuring method of the present invention. The schematic configuration diagram of the spectrum measuring apparatus of the second embodiment is also used in FIG. The symbol used in the first embodiment is also used.
[0041]
In the second embodiment, the measurement time of detection measurement and reference measurement is set to an integral multiple of the least common multiple of the periods of the two background lights to be removed, and the background light is removed. That is, as shown in the timing chart of FIG. 4, the period fa of the intensity change Fa (t) of the indoor illumination light that is the background light to be removed is set to 0.02 seconds (50 Hz), and another background light to be removed. Assuming that the period fb of the intensity change Fb (t) of the RGB illumination light is 0.05 seconds (20 Hz), these periods are input to the measurement condition setting unit 42 as the period data Da. As described above, the measurement time, the measurement interval, and the irradiation time of the excitation light are calculated, and the set value of each timing is obtained.
[0042]
The measurement time Tj of the detection measurement and the reference measurement is an integral multiple of the least common multiple of the periods fa and fb included in each of the two types of background light, and is the minimum of the period fa = 0.02 seconds and the period fb = 0.05 seconds. The common multiple is set to 0.1 second, which is 1 time (integer multiple) of 0.1 seconds. The measurement interval Tk is set to 0.17 seconds, which is longer than the measurement time Tj, and the irradiation time Tr of the excitation light Le is set to 0.02 seconds, which is shorter than the measurement time Tj. Based on these values, measurement timings t1, t2, t3, and t4 for detection measurement and measurement timings t5 and t6 for reference measurement are set. This measurement timing is output from the measurement condition setting unit 42 to the timing controller 43, converted into a signal for controlling the operation by the timing controller 43, output to the measurement unit 30 and the excitation light source 20, and measured at the following timing. .
[0043]
When detection measurement is started at time t1, the light incident on the spectrophotometer 31 via the optical system 32 is split by the photometer 31 and the amount of light corresponding to each wavelength is measured to obtain the detected spectral intensity SPc. . The detection measurement is continued for a measurement time Tj = 0.1 seconds from time t1 to t4, and during that period, Tr = 0.02 seconds from time t2 to t3, the sample 1 is irradiated with excitation light, Fluorescence Ke is generated from the sample, and this fluorescence Ke enters the spectrophotometer 31 through the optical system 32 and is measured.
[0044]
When the detection measurement is completed at time t4, the reference measurement is started at time t5 after the measurement interval Tk = 0.17 seconds from time t1, and the light incident on the spectrophotometer 31 via the optical system 32 is transmitted to the spectrophotometer. The amount of light corresponding to each wavelength is measured by 31 and the reference spectrum intensity SPs is obtained. The reference measurement is continued for a measurement time Tj = 0.1 seconds from time t5 to t6 as in the detection measurement, but during this time, the excitation light Le is not irradiated onto the sample 10, and thus the fluorescence Ke is not measured. .
[0045]
Next, the detected spectrum intensity SPc measured by the detection measurement and the reference spectrum intensity SPs measured by the reference measurement are output to the arithmetic processing unit 44, and the processing unit 44 subtracts the reference spectrum intensity SPs from the detected spectrum intensity SPc. Thus, the fluorescence spectrum intensity Spk from which the influence of the background light is removed is obtained, and the result is output to the display 50 and displayed.
[0046]
Here, an operation in which two types of background light are removed by the subtraction process will be described. Since the measurement time Tj is 1 times the least common multiple of the cycle fa and the cycle fb, the time Tj is 5 cycles of the cycle fa and 2 cycles of the cycle fb. That is, regardless of the phase of the background light at the start of detection measurement and reference measurement, for the room illumination light, the measurement light amounts A1 and A2 for five periods are measured for both detection measurement and reference measurement, while RGB illumination Regarding light, measurement light amounts B1 and B2 for two periods are measured for both detection measurement and reference measurement. Here, even if the phase at the start of detection and measurement of the indoor illumination light is different from the phase at the start of the reference measurement, the light quantity to be measured is equal to A1 = A2 because it is measured for five periods. The detected room illumination spectrum intensity Spa1 is also equal to the reference room illumination spectrum intensity Spa2. Similarly, even if the phase at the start of detection and detection of RGB illumination light is different from that at the start of reference measurement, two periods are measured, so the measured light amounts are equal to B1 = B2, and these light amounts are obtained by spectroscopy. The detected RGB illumination spectrum intensity Spb1 is also equal to the reference RGB illumination spectrum intensity Spb2.
[0047]
On the other hand, the excitation light Le is irradiated for 0.02 seconds within the measurement time Tj of the detection measurement, and the light amount C obtained from the fluorescence generated by this irradiation is dispersed and measured as the fluorescence spectrum intensity Spk, but this value is detected. It is added to the detected indoor illumination spectrum intensity Spa1 and the detected RGB illumination spectrum intensity Spb1 measured in the measurement.
[0048]
Accordingly, the reference spectrum intensity SPs obtained in the reference measurement from the detected spectrum intensity SPc obtained in the detection measurement, that is, the sum of the fluorescence spectrum intensity Spk, the detected indoor illumination spectrum intensity Spa1, and the detected RGB illumination spectrum intensity Spb1, that is, the reference room. By subtracting the sum of the illumination spectrum intensity Spa2 and the reference RGB illumination spectrum intensity Spb2, it is possible to obtain the fluorescence spectrum intensity Spk from which the influence of background light has been removed. That is,

Because
Spa1 = Spa2, Spb1 = Spb2
After the above calculation is performed, the result is output to the display 50 and displayed. Other configurations and operations are the same as those of the first embodiment.
[0049]
FIG. 5 is a timing chart of the third embodiment of the spectrum measuring apparatus for carrying out the optical measuring method of the present invention. The schematic configuration diagram of the spectrum measuring apparatus of the third embodiment is the same as FIG. The symbol used in the first embodiment is also used.
[0050]
In the third embodiment, the time interval between the detection measurement and the reference measurement is an integer multiple of the period of one of the two background lights to be removed, and the detection measurement time and the reference measurement time Is set to an integral multiple of the period of the other background light, and the background light is removed.
[0051]
That is, as shown in the timing chart of FIG. 5, the period fa of the intensity change Fa (t) of the indoor illumination light that is the background light to be removed is set to 0.02 seconds (50 Hz), and another background light to be removed. Assuming that the period fb of the intensity change Fb (t) of the RGB illumination light is 0.05 seconds (20 Hz), these periods are input to the measurement condition setting unit 42 as the period data Da. As described above, the measurement time, the measurement interval, and the irradiation time of the excitation light are determined, and the measurement timing is obtained.
[0052]
The measurement interval Tk is set to Tk = 0.15 seconds as 3 times (integer multiple) of the period fb, and the measurement time Tj for detection measurement and reference measurement is set to Tj = 0.08 seconds as 4 times (integer multiple) of the period fa. It is done. The irradiation time Tr of the excitation light Le is set to 0.02 seconds, which is shorter than the measurement time Tj.
[0053]
Based on these values, measurement timings t1, t2, t3, and t4 for detection measurement and measurement timings t5 and t6 for reference measurement are set. These measurement timings are output from the measurement condition setting unit 42 to the timing controller 43, converted into signals for controlling the operation by the timing controller 43, and output to the measurement unit 30 and the excitation light source 20, and are measured at the following timings. Is called.
[0054]
When detection measurement is started at time t1, the light incident on the spectrophotometer 31 via the optical system 32 is split by the photometer 31 and the amount of light corresponding to each wavelength is measured to obtain the detected spectral intensity SPc. . The detection measurement is continued for a measurement time Tj = 0.08 seconds from time t1 to t4, and excitation light is irradiated on the sample 1 for Tr = 0.02 seconds from time t2 to t3, Fluorescence Ke is generated from the sample, and this fluorescence Ke enters the spectrophotometer 31 through the optical system 32 and is measured.
[0055]
When the detection measurement is completed at time t4, the reference measurement is started at time t5 after measurement interval Tk = 0.15 seconds from time t1, and the light incident on the spectrophotometer 31 via the optical system 32 is transmitted to the spectrophotometer. The amount of light corresponding to each wavelength is measured by 31 and the reference spectrum intensity SPs is obtained. The reference measurement is continued for the measurement time Tj = 0.08 seconds from the time t5 to t6 as in the detection measurement. However, since the excitation light Le is not irradiated on the sample 10 during this time, the fluorescence Ke is not measured. .
[0056]
Next, the detected spectrum intensity SPc measured by the detection measurement and the reference spectrum intensity SPs measured by the reference measurement are output to the arithmetic processing unit 44, and the processing unit 44 subtracts the reference spectrum intensity SPs from the detected spectrum intensity SPc. Thus, the fluorescence spectrum intensity Spk from which the influence of the background light is removed is obtained, and the result is output to the display 50 and displayed.
[0057]
Here, an operation in which two types of background light are removed by the subtraction process will be described. With respect to the indoor illumination light Fa (t), the measurement time Tj in the detection measurement and the reference measurement is four times the period fa, and the detection measurement and the reference measurement are equal for four periods regardless of the phase at which the measurement is started. The light quantities A1 and A2 are measured, and the detected indoor illumination spectrum intensity Spa1 and the reference indoor illumination spectrum intensity Spa2 obtained by spectrally dividing these light quantities are equal.
[0058]
For the RGB illumination light Fa (t), the measurement interval Tk is three times the period fb, and the phases Pb1 and Pb2 that start measurement in the detection measurement and the reference measurement always start from the same phase, and each measurement is the same Since it is performed over time, the equal amounts of light B1 and B2 are measured, and the detected RGB illumination spectrum intensity Spb1 and the reference RGB illumination spectrum intensity Spb2 obtained by spectrally dividing these amounts of light are equal.
[0059]
On the other hand, the excitation light Le is irradiated for 0.02 seconds within the measurement time Tj of the detection measurement, and the light amount C obtained from the fluorescence generated by this irradiation is dispersed and measured as the fluorescence spectrum intensity Spk, but this value is detected. It is added to the detected indoor illumination spectrum intensity Spa1 and the detected RGB illumination spectrum intensity Spb1 measured in the measurement.
[0060]
Accordingly, the reference spectrum intensity SPs obtained in the reference measurement from the detected spectrum intensity SPc obtained in the detection measurement, that is, the sum of the fluorescence spectrum intensity Spk, the detected indoor illumination spectrum intensity Spa1, and the detected RGB illumination spectrum intensity Spb1, that is, the reference room. By subtracting the sum of the illumination spectrum intensity Spa2 and the reference RGB illumination spectrum intensity Spb2, it is possible to obtain the fluorescence spectrum intensity Spk from which the influence of background light has been removed. That is,

Because
Spa1 = Spa2, Spb1 = Spb2
After the above calculation is performed, the result is output to the display 50 and displayed. Other configurations and operations are the same as those of the first embodiment.
[0061]
Two sets of the detection measurement and the reference measurement can be performed. That is, as shown in the timing chart of FIG. 6, the period fa of the intensity change Fa (t) of the indoor illumination light that is the background light to be removed is set to 0.02 seconds (50 Hz), and another background light to be removed. Assuming that the period fb of the intensity change Fb (t) of the RGB illumination light is 0.05 seconds (20 Hz), these periods are input to the measurement condition setting unit 42 as the period data Da. As described above, the measurement time, the measurement interval, and the irradiation time of the excitation light are determined, and the measurement timing is obtained based on these values.
[0062]
The measurement interval Tk between the detection measurement and the reference measurement is set to Tk = 0.15 seconds, which is 3 times (integer multiple) of the period fb, and the measurement time Tj for detection measurement and reference measurement is 1 time (integer multiple) of the period fa. As Tj = 0.02 seconds. Further, the irradiation time Tr of the excitation light Le is set to 0.01 seconds, which is shorter than the measurement time Tj. Based on these values, the measurement timing t1, t2, t3, t4 of the first set of detection measurements, the measurement timing t5, t6 of the reference measurement, and the measurement timings t1 ′, t2 ′ of the second set of detection measurements , T3 ′, t4 ′ and reference measurement timings t5 ′, t6 ′. These measurement timings are output from the measurement condition setting unit 42 to the timing controller 43, converted into signals for controlling the operation by the timing controller 43, and output to the measurement unit 30 and the excitation light source 20, and are measured at the following timings. Is called.
[0063]
In the first set of measurements, detection measurement is performed over a time Tj from time t1 to t4, in which excitation light Le is irradiated for a time Tr from time t2 to t3 to generate fluorescence Ke, The light amount C2 and the light amounts A1 and B1 of the types of background light are obtained, and the sum of these light amounts is dispersed to obtain the detected spectrum intensity SPc, while the reference measurement is performed from the time t5 to the time t5 when the measurement interval Tk has elapsed The measurement is performed over the measurement time Tj until two types of light amounts A2 and B2 of the background light are obtained, and the sum of these light amounts is dispersed to obtain the reference spectrum intensity SPs.
[0064]
In the second set of measurements, the detection measurement is performed over a time Tj from time t1 ′ to t4 ′, in which excitation light Le is emitted for a time Tr from time t2 ′ to t3 ′, and fluorescence Ke is emitted. The amount of fluorescence C ′ and the amounts of light A1 ′ and B1 ′ of two types of background light are obtained, and the sum of these amounts of light is dispersed to obtain the detected spectrum intensity SPc ′, while the reference measurement is performed at time t1 ′. Over the measurement time Tj from the time t5 ′ to the time t6 ′ when the measurement interval Tk has passed, the two types of light amounts A2 ′ and B2 ′ of the background light are obtained, and the sum of these light amounts is dispersed and the reference spectrum intensity SPs ′ is determined.
[0065]
Here, by subtracting the reference spectrum intensity SPs from the first set of detected spectrum intensity SPc, or by subtracting the reference spectrum intensity SPs ′ from the second set of detected spectrum intensity SPc ′, the background light in the same manner as described above. The fluorescence spectrum intensity Spk or the fluorescence spectrum intensity Spk ′ corresponding to the fluorescence light amount C or the fluorescence light amount C ′ is obtained.
[0066]
Therefore, the fluorescence spectrum intensity from which the background light is removed can be obtained by the following equation.
[0067]
Spk + Spk ′ = (SPc + SPc ′) − (SPs + SPs ′)
It is also possible to obtain three or more sets of detection measurements and reference measurements and obtain a fluorescence spectrum intensity from which the influence of background light has been removed by the same method.
[0068]
In the first embodiment, two sets of detection measurement and reference measurement can be performed. That is, as shown in the timing chart of FIG. 7, the period fa of the intensity change Fa (t) of the indoor illumination light that is the background light to be removed is set to 0.02 seconds (50 Hz), and another background light to be removed. Assuming that the period fb of the intensity change Fb (t) of the RGB illumination light is 0.05 seconds (20 Hz), these periods are input to the measurement condition setting unit 42 as the period data Da. Thus, the measurement time, the measurement interval, and the irradiation time of the excitation light are determined, and the measurement timing is obtained based on these.
[0069]
Since the measurement interval between the detection measurement and the reference measurement is determined to be an integral multiple of the least common multiple of the periods fa and fb included in each of the two types of background light, the measurement interval Tk is the period fa = 0.02 seconds and the period fb = 0. .05 seconds is set to 0.2 seconds which is twice (integer multiple) of 0.1 seconds which is the least common multiple of 05 seconds. The measurement time Tj is set to 0.01 seconds, which is shorter than the measurement interval Tk, and the irradiation time Tr of the excitation light Le is set to 0.005 seconds, which is shorter than the measurement time Tj. Based on these values, the measurement timing t1, t2, t3, t4 of the first set of detection measurements, the measurement timing t5, t6 of the reference measurement, and the measurement timings t1 ′, t2 ′ of the second set of detection measurements, t3 ′ and t4 ′ and measurement timings t5 ′ and t6 ′ of the reference measurement are obtained. These measurement timings are output from the measurement condition setting unit 42 to the timing controller 43, converted into signals for controlling the operation by the timing controller 43, and output to the measurement unit 30 and the excitation light source 20, and are measured at the following timings. Is called.
[0070]
In the first set of measurements, detection measurement is performed over time Tj from time t1 to t4, and during that time, excitation light Le is emitted for time Tr from time t2 to t3 to generate fluorescence Ke, and the amount of fluorescence C2 and types of background light amounts A1 and B1 are obtained, and the sum of these light amounts is dispersed to obtain the detected spectrum intensity SPc. On the other hand, the reference measurement is the time after the measurement interval Tk = 0.02 seconds from the time t1. Over the measurement time Tj from t5 to t6, two types of light amounts A2 and B2 of background light are obtained, and the sum of these light amounts is dispersed to obtain the reference spectral intensity SPs.
[0071]
In the second measurement, detection measurement is performed over time Tj from time t1 ′ to time t4 ′. Among them, excitation light Le is irradiated for time Tr from time t2 to time t3 to generate fluorescence Ke, The light quantity C ′ of fluorescence and the light quantities A1 ′ and B1 ′ of two types of background light are obtained, and the sum of these light quantities is dispersed to obtain the second set of detected spectrum intensity SPc ′, while the reference measurement is performed at time t1. Is measured over a measurement time Tj from 't5' to t6 'after a measurement interval Tk = 0.02 seconds, and two types of light amounts A2' and B2 'of background light are obtained. Thus, the second set of reference spectral intensity SPs ′ is obtained.
[0072]
Here, by subtracting the reference spectrum intensity SPs from the first set of detected spectrum intensity SPc or subtracting the reference spectrum intensity SPs ′ from the second set of detected spectrum intensity SPc ′, the contents described in the first embodiment can be obtained. Similarly, background light can be removed, and the fluorescence spectrum intensity Spk or Spk ′ is obtained.
[0073]
Therefore, the fluorescence spectrum intensity from which the background light is removed can be obtained by the following equation.
[0074]
Spk + Spk ′ = (SPc + SPc ′) − (SPs + SPs ′)
As shown in the timing chart of FIG. 7, detection times t1 to t4 and reference measurement times t5 to t6, or detection measurement times t1 ′ to t4 ′ and reference measurement time t5 are performed. 'To t6' has a phase that is synchronized with the background light having the minimum period among the background lights, that is, the indoor illumination light Fa (t) having the minimum period fa and has the minimum intensity in each period. By setting the measurement timing of detection measurement and reference measurement in the vicinity, the influence of background light can be further reduced.
[0075]
It is also possible to obtain three or more sets of detection measurements and reference measurements and obtain a fluorescence spectrum intensity from which the influence of background light has been removed by the same method.
[0076]
In all the embodiments described above, the excitation light irradiation is set to be performed once in the detection measurement, but the excitation light irradiation may be performed a plurality of times in the same detection measurement.
[0077]
In all the embodiments described above, two types of background light are used. However, when there are three or more types of background light, for example, the background light is F1 (t), F2 (t), F3 (t), F4 (t). , F5 (t), F6 (t) and F7 (t), there are seven types of F1 (t), F2 (t) and F3 (t) combined as one background light Fx (t). Then, the period fx of the intensity change is obtained, and the combination of F4 (t), F5 (t), F6 (t) and F7 (t) other than the above is considered as another background light Fy (t). The above-described method for obtaining the intensity change period fy and using the synthesized two kinds of intensity change periods fx and fy of the background light to remove the influence of the background light can be applied.
[0078]
The optical measurement method and optical measurement device of the present invention can also be applied to a spectrum measurement device, a quantitative measurement device, an image measurement device, and the like that measure fluorescence, absorption, phosphorescence, and the like.
[0079]
As described above, according to the optical measurement method and the optical measurement device of the present invention, even in an environment where two or more types of background light having different periods exist, highly accurate measurement in which the influence of the background light is removed. A value can be obtained.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram of an optical measurement method and apparatus according to the present invention applied to a spectrum measurement apparatus.
FIG. 2 is a timing chart of the spectrum measurement method according to the first embodiment of the present invention.
Fig. 3 Spectral intensity diagram
FIG. 4 is a timing chart of a spectrum measurement method according to a second embodiment of the present invention.
FIG. 5 is a timing chart of the spectrum measurement method according to the third embodiment of the present invention.
FIG. 6 is a timing chart of a spectrum measurement method in which measurement is repeated a plurality of times in the third embodiment of the present invention.
FIG. 7 is a timing chart of a spectrum measurement method in which measurement is repeated a plurality of times in the first embodiment of the present invention.
[Explanation of symbols]
10 samples
20 Excitation light source
30 Measuring unit
40 Control unit
50 indicator
100 Spectrum measuring device
La excitation light
Ke fluorescence

Claims

In the presence of two or more types of background light having different periods, detection measurement for measuring light including the detected light and reference measurement for measuring light not including the detected light are performed. An optical measurement method for removing the influence of background light from a value acquired by the detection measurement by subtracting a value acquired by the reference measurement from the acquired value,
The optical measurement method characterized in that the time interval between the detection measurement and the reference measurement is set to an integer multiple of the least common multiple of the period of each background light.

In the presence of two or more types of background light having different periods, detection measurement for measuring light including the detected light and reference measurement for measuring light not including the detected light are performed. An optical measurement method for removing the influence of background light from a value acquired by the detection measurement by subtracting a value acquired by the reference measurement from the acquired value,
The optical measurement method characterized in that the detection measurement time and the reference measurement time are set to integer multiples of the least common multiple of the periods of each background light.

In the presence of two or more types of background light having different periods, detection measurement for measuring light including the detected light and reference measurement for measuring light not including the detected light are performed. An optical measurement method for removing the influence of background light from a value acquired by the detection measurement by subtracting a value acquired by the reference measurement from the acquired value,
The time interval between the detection measurement and the reference measurement is set to an integer multiple of the least common multiple of the period of each of the one or more background lights selected from the background light, and the detection measurement and reference measurement times are An optical measurement method characterized in that an integer multiple of a least common multiple of periods of one or more background lights other than the selected background light is provided.

The optical measurement method according to claim 1, wherein the detection measurement and the reference measurement are performed a plurality of times.

The detection measurement and the reference measurement are performed a plurality of times, and each of the detection measurement and the reference measurement is performed in synchronization with a period of one type of background light selected from the background light. Item 3. The optical measurement method according to Item 1.

6. The optical measurement method according to claim 5, wherein each of the detection measurement and the reference measurement is performed in the vicinity of the minimum value of the intensity change of the selected background light.

7. The optical measurement method according to claim 5, wherein a background light having a minimum period among all the background lights is selected as the one type of background light.

A measurement unit that measures the detected light in the presence of two or more types of background light having different periods, a detection measurement that measures the light including the detected light by the measurement unit, and the detected light is not included The control unit that controls the time interval between the reference measurement in which the light is measured by the measurement unit and the measurement time of each of the detection measurement and the reference measurement, and the value acquired in the reference measurement is subtracted from the value acquired in the detection measurement An optical measuring device comprising a computing unit for performing
The time interval between the detection measurement and the reference measurement is an integer multiple of the least common multiple of the period of each background light.

A measurement unit that measures the detected light in the presence of two or more types of background light having different periods, a detection measurement that measures the light including the detected light by the measurement unit, and the detected light is not included The control unit that controls the time interval between the reference measurement in which the light is measured by the measurement unit and the measurement time of each of the detection measurement and the reference measurement, and the value acquired in the reference measurement is subtracted from the value acquired in the detection measurement An optical measuring device comprising a computing unit for performing
The optical measurement device characterized in that the time of the detection measurement and the time of the reference measurement are integer multiples of the least common multiple of the periods of each of the background lights.

A measurement unit that measures the detected light in the presence of two or more types of background light having different periods, a detection measurement that measures the light including the detected light by the measurement unit, and the detected light is not included The control unit that controls the time interval between the reference measurement in which the light is measured by the measurement unit and the measurement time of each of the detection measurement and the reference measurement, and the value acquired in the reference measurement is subtracted from the value acquired in the detection measurement An optical measuring device comprising a computing unit for performing
The time interval between the detection measurement and the reference measurement is an integer multiple of the least common multiple of the periods of one or more background lights selected from the background light, and the measurement of the detection measurement and the reference measurement An optical measuring device characterized in that the time is an integer multiple of the least common multiple of the periods of one or more background lights other than the selected background light.

The optical measurement apparatus according to claim 8, wherein the detection measurement and the reference measurement are performed a plurality of times.

The detection measurement and the reference measurement are performed a plurality of times, and each of the detection measurement and the reference measurement performed a plurality of times is performed in synchronization with a period of one type of background light selected from the background light. The optical measuring device according to claim 8.

13. The optical measurement apparatus according to claim 12, wherein each of the detection measurement and the reference measurement performed a plurality of times is performed in the vicinity of the minimum value of the intensity change of the selected background light.

14. The optical measurement apparatus according to claim 12, wherein the selected one type of background light has a minimum period among all the background light.