Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.
Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.
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DOI：
10.1038/srep14766
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发表时间：
2015-10-01
影响因子：
4.6
通讯作者：
Stefani E
中科院分区：
文献类型：
--
作者：
Wu Y;Wu X;Lu R;Zhang J;Toro L;Stefani E
光漂白是超分辨受激耗尽发射（STED）显微术的一个主要限制。快速扫描一直被认为是一种有效的手段，以减少在荧光显微镜的光漂白，但仔细定量研究这个问题是失踪。在本文中，我们表明，在STED显微镜的光漂白率可以减慢和荧光产率被提高，通过高速扫描，使使用定制的共振扫描STED显微镜的大视场。扫描速度对光漂白和荧光产率的影响在较高水平的耗尽激光辐照度下更为显著，并且在传统的共聚焦显微镜中几乎消失。在≥6 GW/cm−2的耗尽辐照度下，我们能够将Atto 647 N和Abberior星星635 P的荧光团存活时间延长约80%，视野扩大8倍。我们证实，STED光漂白主要是由耗尽光作用于激发的荧光团引起的。实验数据与理论模型一致。我们的研究结果鼓励进一步提高线性扫描速度，减少在STED显微镜的光漂白。
Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm−2 depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.