Department of Pharmaceutical Sciences, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA.
Opt Lett. 2011 Aug 15;36(16):3185-7. doi: 10.1364/OL.36.003185.
Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.
双光子荧光（TPF）是生物成像最重要的发现之一。尽管连续波（cw）激光被认为可以激发 TPF，但由于激发效率低，其在 TPF 成像中的应用受到了很大限制。在这里，我们直接用用于光镊的红外连续波激光激发荧光团，实现了单分子荧光灵敏度：观察到增强型绿色荧光蛋白的离散逐步光漂白。单分子荧光强度分析和 ON 时间分布强烈表明，连续波激光可以产生可在单分子水平检测到的 TPF，从而为使用连续波激光进行单分子 TPF 成像开辟了道路。
