Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach
Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach
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使用相量图方法提高低功率 STED 超分辨率显微镜的分辨率
DOI：
10.1039/c8nr03584a
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发表时间：
2018-09-14
期刊：
影响因子：
6.7
通讯作者：
Qu, Junle
中科院分区：
文献类型：
--
作者：
Wang, Luwei;Chen, Bingling;Qu, Junle
受激发射耗尽（STED）显微镜是一种强大的超分辨率显微镜技术，在突破分辨率限制和相关应用方面取得了重大成果。原则上，STED超分辨率是通过受激发射部分抑制衍射受限区域外围的自发发射而获得的。然而，为了提高成像分辨率，通常需要非常高的耗尽激光功率，由于其高光毒性和光漂白效应，对活体生物标本有害。因此，进一步提高低损耗功率水平下的STED分辨率，近年来引起了各领域研究人员越来越多的兴趣。在这项工作中，相量图方法结合荧光寿命成像显微镜（FLIM）来解决基于长寿命和短寿命标准的上述问题。首先，通过相量法将STED-FLIM获得的时间分辨数据转换到频域；其次，根据相位和幅值信息提取部分数据，提高分辨率。然后，在不同的耗尽功率下观察荧光微球（直径100 nm），通过相量图获得一系列提高的分辨率。最后，将该方法应用于固定HeLa细胞中人类Nup153的成像，在20 mW的耗尽功率下，提供了比传统STED成像高86 nm的分辨率。
Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications. In principle, STED super resolution is obtained by stimulated emission partially inhibiting the spontaneous emission in the periphery of a diffraction-limited area. However, very high depletion laser power is generally necessary for the enhancement of imaging resolution, which is harmful to live biological specimens due to its high phototoxicity and photo-bleaching effects. Therefore, further improving the STED resolution at a lower depletion power level has recently attracted increasing interest from researchers in various fields. In this work, a phasor plot approach combined with fluorescence lifetime imaging microscopy (FLIM) is used to resolve the abovementioned problem based on a long- and short-lifetime criterion. Firstly, the time-resolved data obtained by STED-FLIM is converted to the frequency domain via a phasor approach. Next, partial data is extracted according to the information on the phase and amplitude for resolution improvement. Then, fluorescent microspheres (100 nm in diameter) are observed under different depletion powers, resulting in a series of improved resolution through phasor plots. Finally, this method is applied to image human Nup153 in fixed HeLa cells, providing a 86 nm higher resolution than that in traditional STED imaging at a depletion power of 20 mW.