JP7057926B1 - 蛍光測定装置 - Google Patents
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
Description
2 連続光源
3 励起光照射部
4 励起光強度検出部
5 蛍光検出部
6 矩形波変調回路
7 タイミング回路
8 ゲートカウンタ回路
9 物理パラメータ情報取得部
10 濃度演算器
Claims (7)
- 測定対象物質の励起光を発生する連続光源と、
試料を導入し、前記連続光源からの励起光を照射する励起光照射部と、
励起光強度と比例する光量を検出し、デジタル信号に変換する励起光強度検出部と、
測定対象物質が発する蛍光をフォトンカウンティング方式で検出する蛍光検出部と、
前記連続光源を点灯及び消灯状態に対応する矩形波で変調する矩形波変調回路と、
同一のクロックにより、前記矩形波変調回路に供給する矩形波パルス及び信号処理用のゲートパルスを生成するタイミング回路と
前記蛍光検出部からの1光子に対応するパルス信号を、所定時間内において前記ゲートパルスの期間に計数するゲートカウンタ回路と、
前記測定対象物質の濃度の演算において必要とされる、現在時点の物理パラメータ値を取得する物理パラメータ情報取得部と、
前記ゲートカウンタ回路で得られた現在時点の光子計数値から、現在時点のバックグラウンド光子計数換算値を差し引きして、バックグラウンドの影響を除いた蛍光光子計数値を求め、得られた前記蛍光光子計数値を、前記励起光強度検出部で測定された現在時点の励起光強度計測値と、濃度校正時に測定された励起光強度基準値との、比として表される励起光強度補正係数で除することにより、励起光強度補正蛍光光子計数値を算出し、さらに前記励起光強度補正蛍光光子計数値を、前記濃度校正時に求めた濃度当りの蛍光光子計数値として表される前記測定対象物質の濃度係数で除することにより、前記測定対象物質の濃度をデジタル演算する濃度演算器とを、備え、
前記濃度演算器における演算は、
前記濃度校正時における前記測定対象物質を含まない試料で測定した基準バックグラウンド光子計数値が、前記励起光強度基準値に依存しない定数項と、前記励起光強度補正係数に比例する比例項の基準値との、総和で構成されるとして各項の値を保管し、
前記現在時点のバックグラウンド光子計数換算値が、保管された前記定数項と、前記励起光強度補正係数に比例する比例項との、総和で構成されるとして、
前記現在時点のバックグラウンド光子計数換算値の比例項は、保管された前期比例項の基準値並びに前記基準物理パラメータ値、前記現在時点の物理パラメータ値、及び前記励起光強度補正係数を用いて計算されることを特徴とする蛍光測定装置。 - 前記連続光源はLEDであることを特徴とする請求項1に記載の蛍光測定装置。
- 前記濃度演算器における演算は、
前記基準バックグラウンド光子計数値の比例項の基準値が、
前記励起光照射部での反射並びに屈折による基準励起光散乱寄与項と、試料媒質の主要成分による基準レーリー散乱寄与項との、総和で構成されるとして、各項の値を保管し、
前記現在時点のバックグラウンド光子計数換算値の比例項が、
現在時点の励起光散乱寄与項と、現在時点のレーリー散乱寄与項との、総和で構成されるとして、
前記現在時点の励起光散乱寄与項は、保管された前記基準励起光散乱寄与項並びに前記基準物理パラメータ値、前記現在時点の物理パラメータ値、及び前記励起光強度補正係数を用いて計算され、
前記現在時点のレーリー散乱寄与項は、保管された前記基準レーリー散乱寄与項並びに前記基準物理パラメータ値、前記現在時点の物理パラメータ値、及び前記励起光強度補正係数を用いて計算されることを特徴とする請求項1又は2に記載の蛍光測定装置。 - 前記濃度演算器における演算は、
前記基準バックグラウンド光子計数値の比例項の基準値が、
前記基準励起光散乱寄与項と、前記基準レーリー散乱寄与項に加え、試料中のラマン活性を有する成分による基準自然ラマン散乱寄与項との、総和で構成されるとして、各項の値を保管し、
前記現在時点のバックグラウンド光子計数換算値の比例項が、
前記現在時点の励起光散乱寄与項と、前記現在時点のレーリー散乱寄与項に加え、現在時点の自然ラマン散乱寄与項との、総和で構成されるとして、
前記現在時点の自然ラマン散乱寄与項は、保管された前記基準自然ラマン散乱寄与項並びに前記基準物理パラメータ値、前記現在時点の物理パラメータ値、及び前記励起光強度補正係数を用いて計算されることを特徴とする請求項1又は2に記載の蛍光測定装置。 - 前記濃度演算器における演算は、
前記基準バックグラウンド光子計数値の比例項の基準値が、
前記基準励起光散乱寄与項と、前記基準レーリー散乱寄与項と、前記基準自然ラマン散乱寄与項に加え、試料による基準自家蛍光寄与項との、総和で構成されるとして、各項の値を保管し、
前記現在時点のバックグラウンド光子計数換算値の比例項が、
前記現在時点の励起光散乱寄与項と、前記現在時点のレーリー散乱寄与項と、前記現在時点の自然ラマン散乱寄与項に加え、現在時点の自家蛍光寄与項との、総和で構成されるとして、
前記現在時点の自家蛍光寄与項は、保管された前記基準自家蛍光寄与項並びに前記基準物理パラメータ値、前記現在時点の物理パラメータ値、及び前記励起光強度補正係数を用いて計算されることを特徴とする請求項1又は2に記載の蛍光測定装置。 - 前記濃度演算器における前記測定対象物質の濃度の計算は、
前記励起光強度補正蛍光光子計数値を、
さらに消光物質の非存在下における無消光励起光強度補正蛍光光子計数値と、前記消光物質の濃度と、消光速度定数及び前記消光物質の非存在下の蛍光寿命の積であるStern-Volmer定数との間の、Stern-Volmerの関係式より求まる前記無消光励起光強度補正蛍光光子計数値に、
置換えて演算することを特徴とする請求項1~5のいずれか一項に記載の蛍光測定装置。 - 前記濃度演算器における前記測定対象物質の濃度の計算は、
複数の前記励起光強度補正蛍光光子計数値、もしくは複数の前記励起光強度補正蛍光光子計数値が複数の前記無消光励起光強度補正蛍光光子計数値に置換えられた場合にあっては、前期複数の無消光励起光強度補正蛍光光子計数値が、同数の前記測定対象物質の濃度と同数の前記濃度係数とによる線形結合であるものとして、
連立方程式の求解により各前記測定対象物質の濃度を、演算することを特徴とする請求項1~6のいずれか一項に記載の蛍光測定装置。
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