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2波長同時レシオイメージング
2．実測データで見るレシオイメージング
生命現象の変化をレシオイメージングで捉える方法について、新薬候補化合物の心毒性評価に用いる細胞として注目されているiPS細胞由来心筋細胞の膜電位変化を、膜電位感受性色素di-4-ANEPPSを用いて計測したデータから説明します。
計測した心筋細胞は、自発的な拍動に伴う厚みの変化や位置の変動が起きやすく、これらの理由で蛍光輝度は変動します。膜電位に由来する蛍光輝度の変動を効果的に抜き出さないと正確な計測は出来ません。レシオイメージングにて拍動する心筋から、膜電位に由来した成分がどの様に抜き出されているかを説明します。
動画1において左側の2つの画像は、計測した2波長各々の蛍光画像です。拍動に伴い試料が大きく移動するため、観察領域のdi-4-ANEPPSの存在量そのものも大きく変動していることが想定されます。蛍光輝度変化には膜電位の変化と、色素の存在量の変化の両方の情報が混ざっています。一方、右側の画像は2波長蛍光のレシオ値を疑似カラーにして重ねたものです。レシオ値は試料移動の影響を受けず、心筋細胞が拍動する瞬間に先立って膜電位が変化する様子がはっきりと捉えられています。
レシオイメージングでどの様に試料移動や退色の影響が除かれ、本来計測したい信号が取り出されるのか、計測した蛍光輝度の経時変化グラフから見ていきましょう。特定の観察領域における2波長の蛍光各々の経時変化をグラフ化すると、以下の波形を示します。
図7：蛍光膜電位観察におけるCh1、Ch2の蛍光輝度の経時変化
図7のグラフを見ると鋭いピークを持つ波形や、ゆるやかな変化を示す波形が混在しており、この波形だけから膜電位変化のタイミングや大きさを導き出すには困難が多いと言わざるを得ません。さらにグラフ全体が右肩下がりであることから、蛍光プローブの退色が起きており輝度の絶対値を活動の指標にする事も困難であると言えます。
次に、Ch1とCh２のレシオ値を算出しグラフに加えると、以下の様になります。
図8：蛍光膜電位観察におけるレシオ値の経時変化と波形比較
図8において緑色のレシオグラフでは、明瞭なピークを持った波形が得られています。青い領域で示す様にレシオグラフがピークを示す範囲で、Ch1、Ch2はそれぞれ逆方向に輝度変化しており、このピークが膜電位変化に伴う蛍光輝度の変化であることが判別できます。
図9：試料移動による蛍光輝度の変動とレシオ演算による補正効果
また図9の赤い領域で示す様にCh1、Ch2が向きを揃えて輝度増減する部分は、心筋の移動や厚みの変化による観察領域のプローブ存在量変化のためと考えられます。この部分はレシオグラフでは波形の変化が見られず、目的としている膜電位以外の要因による変動が抑えられていることが分かります。
この様に、レシオグラフは試料移動の様な単純な輝度変化に影響されず、膜電位の変化に応じて変動すると言えます。動画2において試料移動と膜電位変化の様子を画像と連動させ確認してみましょう。
動画2は試料の動きを見やすくするため、動画1の一部を切り出して示したものです。計測時間3 secから3.5 secの間に弛緩によって試料が動いている事が左の動画から分かります。右のグラフをみると、その間はCh1、Ch2観察領域の蛍光輝度が試料移動に伴い変化しています。しかし一方でレシオ値は変化していません。
次に計測時間3.57 secにレシオ値が大きく変化する瞬間があります。この瞬間は正に膜電位が上昇する瞬間ですが、この時レシオ値の急激な変化に比べ、試料移動は殆ど無いことが画像から分かります。この瞬間のCh1、Ch2の輝度変化が画像の位置ズレ等の計測上の不具合の為でない事が確認できます。これは後述するフィルタホイールを用いた計測でしばしば問題となります。
図10：退色による蛍光輝度の変化とレシオ演算による補正効果
図10の破線は各グラフのベースラインを示したものです。Ch1、Ch2共にベースラインに経時的な減少が見られ、6 secの間に退色していることが分かります。一方レシオグラフではベースラインは水平に保たれ、退色に影響されないグラフを得られます。
ここまで見てきた様に、心筋細胞の膜電位変化を単純な蛍光輝度変化で捉えようとすると、様々な困難があることが分かります。そしてレシオイメージングでは膜電位変化を、より確実に計測出来ることを示しました。
ここまでは、iPS細胞由来心筋細胞の膜電位変化の実測データを示しながら、生命現象の変化をレシオイメージングで捉える方法について説明してきました。次のページでは、高速な生命現象をレシオイメージングで確実に捉える方法について説明します。
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