カルシウムイメージングとは、Ca2+プローブを用いて、細胞内のカルシウムイオンの濃度や流動を観察する手法のことです。Ca2+プローブは細胞内のカルシウムイオン濃度に応じて蛍光輝度が変化するという特長を持っています。カルシウムイオンは、神経伝達物質やホルモンなどの分泌、筋肉の収縮など生命や健康の維持に関与しているため、カルシウムイオンの蛍光輝度の変化をイメージングすることで、細胞内へのカルシウムイオンの流動や活動状態を観察することができます。
カルシウムイメージングには、Fluo-3やFluo-4、G-CaMPといったカルシウムイオンの濃度変化に応じ蛍光輝度が変化するプローブと、Fura-2やIndo-1といったカルシウムイオンの濃度変化に応じて、励起光や蛍光のスペクトルが変化するプローブの大きく2種類が存在します。前者は1波長の蛍光輝度を測定するのに対し、後者は2波長のレシオを測定するという点が異なります。
図2 : フィルタホイールを用いた落射蛍光顕微鏡の模式図
フィルタホイール方式は、励起フィルタや蛍光(吸収)フィルタのホルダ部をホイール型のホルダにし、複数のフィルタを入れたホイールを回すことでさまざまな波長に対応できるようにする方式です。撮影時にフィルタホイールの位置を変えながら撮影することで、多波長撮影が可能になります。
図3 : W-VIEW GEMINIを用いた落射蛍光顕微鏡の模式図
2波長同時計測方式とは、W-VIEW GEMINIというイメージスプリッティング光学系を使用し、カメラの上下(または左右)で波長ごとに像を分けて結像させることで、2波長を同時に撮影できるようにする方式のことです。ダイクロイックミラー、バンドパスフィルタを変えることでさまざまな波長の組み合わせに対応できます。
図4に示すように、フィルタの切り替えには通常200 ms程度の時間を要します。例えば、膜電位変化のような高速な生命現象はフィルタ切り替えの間にも進行してしまうため、フィルタホイール方式を用いて観察時に起こりうる生命現象を正確に計測することはできません。このような高速な生命現象を捉えるには、2つの波長を同時に計測する必要があります。
図4 : フィルタホイール方式と2波長同時計測方式の撮像タイミング比較
カルシウムイメージングでは、単純に計測したCa2+プローブの蛍光輝度の変化を、そのまま目的の現象や標的分子の動態と捉えると誤った計測になりかねません。Ca2+プローブから検出される蛍光輝度は、蛍光プローブの褪色、局在濃度のばらつき、サンプルの移動などさまざまな要因で変化するためです。
「1波長イメージングと2波長レシオイメージングの違い」のページでは1波長イメージングと2波長レシオイメージングの違い、2波長レシオイメージングのメリットなどについて解説しています。
心筋細胞の拍動に伴うカルシウムイメージング
共焦点高速カルシウムイメージング
hiPS-CMの細胞内におけるCa2+の蛍光輝度変化と位相差像の同時測定
FDSS-GX カイネティクスプレートイメージャは、創薬スクリーニング分野で培った技術を最大限に活かしたハイスループットスクリーニング(HTS)向けのカイネティクスプレートイメ―ジャです。蛍光/発光測定における高い定量性が特長であるFDSS専用qCMOSセンサと、正確性かつ再現性のよい微量分注を可能にする全ウエル独立ピストン内蔵の分注ヘッド(1536 ch チップ式/384 ch チップ式/96 ch チップ式)を用いることで、薬剤のプレート一括分注・同時測定及び解析を実現しました。各種Ca2+アッセイに対応しています。
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