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ORCA®-Questを用いたゼブラフィッシュの神経活動のin vivo定量計測
ほとんど一瞬で変化が起こるような生体を高画質で撮影することは、多くの課題が存在する複雑な観測となります。
現在、ドイツのマックス・プランク研究所（Max Planck Institute）で研究を行っているDrew Robson博士は、光子数識別が行える定量的なカメラ、ORCA-Questを用いて、ゼブラフィッシュの幼生が自然に振舞っている際の脳機能を観察するためのさまざまな手法を開発しています。
ゼブラフィッシュの脳からヒトの脳へ
水族館のゼブラフィッシュ
ゼブラフィッシュは、脳の活動を研究するための新たなモデル生物です。実際に、ゼブラフィッシュとヒトには多くの共通点があり、私たちの脳機能、健康、疾病を理解する上で理想的な生物といえます。Harvard大学で博士号を取得したDrew Robson博士は現在、このゼブラフィッシュの幼生期を研究しています。幼生期のゼブラフィッシュは透明で、成長中の体内の細胞を直接観察しやすくなっています。
幼生が泳いでいる状態を観察するために、Robson博士はClosed-loopシステムを考案し、脳を顕微鏡の視野内に維持することで、幼生の位置を追跡・補正しています。幼生の自然な振舞いを調べる実験作業はとても複雑で、適切な手法を選定する必要があります。
顕微鏡観察における課題
幼生の脳で起きている一瞬の変化を捉えるためには、細胞サイズ以下の分解能で空間方向、時間方向も含めた高解像度イメージングを行う必要があります。しかし、生物を生きたまま撮影することは容易ではなく、多くの課題を抱えています。この実験では、収差やノイズの影響を最小限に抑えるため、ライトシート蛍光顕微鏡をもとに、優れた光学設計と検出能力をもつカスタマイズされたセットアップが必要でした。
Rahul Trivedi博士：マックス・プランク研究所の博士研究員
ゼブラフィッシュの脳内の信号源は、蛍光タンパク質型カルシウムセンサーです。蛍光シグナルそのものは、励起光エネルギー量が大きいほど、より良い信号対雑音比（SNR：Signal-to-noise ratio）が得られます。その一方で、励起光エネルギー量の増大は、正常な脳機能を損傷・阻害し、蛍光タンパク質の褪色リスクを生じさせる可能性があるため、長時間の観察には限界があります。
さらに、神経細胞のシグナル伝達は短い時間スケールで行われるため、露光時間を短くする必要があり、上に述べた励起光エネルギー量とSNRのジレンマが顕著になります。つまり、最良の結果を得るためには、励起光エネルギー量とSNRのバランスを実験ごとに注意深く調整する必要があるのです。
神経活動の定量計測
Volume render of pan-neuronal H2B-GCaMP6s (green) and ReaChR in Islet2b (ORCA-Questで撮影)
Drew Robson博士は、この絶妙なバランスに対応したいという要求を世界で初めて満たせるカメラとして、光子数識別カメラを入手しました。qCMOSⓇ (Quantitative CMOS) カメラとして知られるORCA-Questは、非常に優れた読み出しノイズ性能や高速なフレームレート等により、励起光エネルギー量とSNRの間に生じるジレンマを大きく緩和することに成功しました。結果として、ORCA-Questによりゼブラフィッシュの脳を長時間にわたって3次元で観察することが可能になりました。
ORCA-Questは、光子のショットノイズが顕在化する低光量領域においても、神経細胞の活動を定量化するためのリニアな結果を提供します。また、高画素数のセンサフォーマットにより、ゼブラフィッシュの脳全体を細胞サイズ以下の分解能で2チャンネル観察することができます。
Maximum intensity projection of pan-neuronal H2B-GCaMP6s (green) and ReaChR in Islet2b neurons (red) (ORCA-Questで撮影)
Drew Robson博士について
Princeton大学在籍時、Olga TroyanskayaとEric Wieschausの研究室にて計算生物学と生物物理学の研究に従事し、数学の学士号を取得。Harvard大学在籍時は、SchierとEngertの研究室にて熱感知行動と脳全体の神経イメージングの研究に従事し、博士号を取得。2014年から2019年までHarvard大学のRowland研究所にてJennifer Liとシステム神経科学と神経工学の共同研究室を率いた後、2019年にRowland研究所からMax Planck生物サイバネティックス研究所に自身の研究室を移した。
この事例で使用された製品
※現在は後継機種のORCA-Quest 2 qCMOSカメラ C15550-22UPを販売しています。
その他のお客様導入事例
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