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生体組織中における二光子励起の4次元時空間制御 (本文)

石川, 智啓 慶應義塾大学

2022.07.06

概要

集光されたパルスの伝播とともにパルス幅が劇的に変化する時空間集光(TF)技術の発明によって,生命現象を解明するために有用な新しい多光子励起技術が開発されてきた.広視野TF顕微鏡は,走査型多光子顕微鏡のように集光スポットを走査することなく断層像が得られる高速なイメージング技術であるため,多細胞間相互作用の観察へ応用されている.また,TF技術をホログラフィック技術と組み合わせたパターン照明顕微鏡は,細胞の活動を光で操作するオプトジェネティクスに応用され,離れて存在する複数の細胞の活動を同時に励起することが可能となっている.しかし,TF技術にはまだ,いくつかの課題がある.例えば,厚みのある試料内部において歪んだTFパルスの補償が困難であること,走査型多光子顕微鏡より光軸方向の分解能が低いこと,パターン照明において隣り合うマルチスポット間で生じる干渉縞がスポットの均一性を劣化させることなどである.これらの問題を解決するために,本研究では,生体組織において,TFパルスの歪み補償を行うための時空間ロックイン検出技術,走査型多光子顕微鏡と同等の光軸方向の分解能が得られる時間多重化(TM)マルチライン(ML)TF技術,均一なパターン照明が可能なマルチフォーカス(MF)TM-TF技術の開発に取り組んだ.

 第1章では,背景および研究目的について述べた.

 第2章では,本研究の基礎となる理論や技術として,非線形光学,超短パルスレーザ技術,イメージング技術について説明した.

 第3章では,広視野TF顕微鏡において広い視野で同時に多光子励起するために,本研究で構築したYbファイバレーザの構成および出力特性について述べた.平均出力3.9W,ピーク波長1059nm,繰り返し710kHz,パルス幅110fsを達成した.

 第4章では,広視野TF顕微鏡の補償光学技術として開発した時空間ロックイン検出技術について述べた.従来は,TF顕微鏡において厚みのある試料を用いると,パルスの時間特性を最適化することは困難であったが,本検出技術により厚みのある試料でもパルスの時間特性の最適化ができることを実証した.

 第5章では,TM-TF顕微鏡において,視野と光軸方向の分解能の両立を可能とするためマルチライン化を組み合わせたTM-ML-TF顕微鏡を提案した.実際に,従来のTM-TF顕微鏡で達成されている視野を5倍に拡大し,TF顕微鏡に対して光軸方向の分解能を2.5倍向上することに成功した.

 第6章では,均一なパターン照明が可能な技術として開発したMF-TM-TF技術について述べた.マルチスポットを近接して配置しても,フリンジ・スペックルフリーを実現できるパターン照明技術を確立した.開発した技術をTM-TF顕微鏡の視野拡大にも応用し,視野を12倍にまで拡大することに成功した.

 第7章は,結論であり,本博士論文で開発してきた技術に関する知見をまとめ,開発した技術の生物学・医科学応用への展望について述べた.

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