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ライトシート読み出しモード | sCMOSカメラ
ライトシート読み出しモードとは
ライトシート読み出しモードは、ライトシート顕微鏡のS/Nを改善するsCMOSカメラの読み出し方法です。ライトシート顕微鏡は、シート状の励起光を試料の側面から照射することで光学断面像を得る蛍光顕微鏡で、この手法は、低褪色、低光毒性、高速性、深部観察能といった、生物個体や組織の生体イメージングに大変有利な特徴を持っています。近年、生体観察用の顕微鏡として非常に注目を集めており、発展をしています。
ライトシート読み出しモードでは、カメラの読み出しのタイミングを励起光の動きに同期して調整できるため、散乱の影響を受けないS/Nの高い画像取得が可能になります。
撮像例
ライトシート読み出しモードの原理と特長
ローリングシャッタ
CMOSイメージセンサの露光、読み出し方式は、大きく2つに分類されます。ローリングシャッタとグローバルシャッタです。
ローリングシャッタは、1画素または1ラインを1単位として露光・読み出しを順次行います。この為、1画面中で露光タイミングが異なります。一方、グローバルシャッタは、全画素同時に露光・読み出しを行うため、1画面中の露光タイミングは同時です。（図1参照）
浜松ホトニクスはsCMOSカメラの特長であるローリングシャッタを応用し、ライトシート顕微鏡での撮像に最適なライトシート読み出しモードを開発しました。
図1 ローリングシャッタとグローバルシャッタの読み出しタイミング
ライトシート読み出しモードの読み出し方式
通常モードの読み出し方式では、センサ中央の水平2ラインから同時に読み出しを開始し、上下にそれぞれ1ラインずつ読み出しを行ないます。（図2参照）
ライトシート読み出しモードでは、センサの最上位ラインより順番に下方に読み出しをしていきます。（図3参照）
図2 通常モード
図3 ライトシート読み出しモード
また、センサの最上部の水平1ラインからセンサの下方向に向かって読み出すフォワード読み出しとセンサ最下部の水平ラインから上方に向かって読み出すバックワード読み出しの２つのモードがあり、切り替えることができます。
図4 フォワード読み出し
図5 バックワード読み出し
特長1：露光リセット
ローリングシャッタを応用したライトシート読み出しモードの読み出し方式では、それぞれの水平1ラインは次の露光を開始する前に電荷をリセットすることができるため、レーザビームが照射されている領域からの光のみを効率良く検出することができます。それに対しグローバルシャッタでは、同時に全てのラインが露光するため、レーザビームが照射されている領域以外からボケの要因となる散乱光も検出してしまいます。（図6参照）
ローリングシャッタを応用したライトシート読み出しモードの読み出し方式では、実際にグローバルシャッタ方式に比べて優れた画質の画像を得る事ができます。（図7参照）
図6 露光リセット
図7 露光リセットの効果
“The gentle illumination schemes of light-sheet microscopes allow for imaging thick samples with unprecedented speed and resolution. However scattering within the sample can impair image quality. Electronic confocal detection in modern sCMOS cameras allows for rejection of scattered photons, resulting in better image quality."
Lars Hufnagel, PhD
特長2：露光時間と読み出し時間がフレキシブルに調整可能
読み出し時間や読み出し方向、そしてレーザの照射幅や照射スピードに合わせた露光時間の調整など、フレキシブルな調整が可能です。読み出し時間の設定は、水平1ラインごとに10 us～100 msの間で調整できるため、容易に光源との同期を取ることができます。フレキシブルな露光時間の調整ができるため、ライトシート読み出しモードは様々な照射幅の照明にも対応が可能です。（図8参照）
図8 露光時間と読み出し時間がフレキシブルに調整可能
特長3：外部トリガ出力
ライトシート読み出しモードでは、水平1ラインごとや毎フレームごとに外部トリガを出力することが可能です。このトリガ信号を利用して、カメラをマスタとしたステージ制御や光源の制御が可能です。（図9参照）
図9 外部トリガ出力
ライトシート読み出しモード 特許リスト（2021年12月現在）
| 国 | 取得状況 | 特許番号 |
|---|---|---|
| 日本 | 登録 | JP06475307、JP05770958、JP06240056、JP05639670 |
| イギリス | 登録 | GB2544913、GB2544666、GB2544667、GB2535367、GB2532906、GB2531200、GB2524183、GB2523012、GB2523263、GB2522793 |
| ドイツ | 登録 | DE112014006152、DE112014000240、DE112014000195.3 |
| アメリカ | 登録 | US10142566、US9423601、US10362245、US10602087 |
| 中国 | 登録 | ZL201480002237.1、ZL201810113454.8、ZL201810113455.2 |
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