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Resolution improvement in STED super resolution microscopy at low power using a phasor plot approach

Abstract

Stimulated emission depletion (STED) microscopy is a powerful super resolution microscopy technique that has achieved a series of significant results in breaking the resolution limit and relevant applications. In principle, STED super resolution is obtained by means of 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; this laser power is harmful for live biological specimens due to its high phototoxicity and photo-bleaching effects. Therefore, further improving STED resolution at a lower level of depletion power has recently attracted the increasing interest of researchers from various fields. In this work, a phasor plot approach combined with fluorescence lifetime imaging microscopy (FLIM) was used to resolve this problem based on a long- and short-lifetime criterion. First, the time-resolved data obtained by STED-FLIM was converted to the frequency domain by a phasor approach. Next, partial data were extracted according to the information on the phase and amplitude for resolution improvement. Then, fluorescent microspheres (100 nm in diameter) were observed under different depletion powers, resulting in a series of improved resolution by phasor plots. Finally, the method was applied to image human Nup153 in fixed HeLa cells, providing a resolution of 86 nm higher than that in traditional STED imaging at the depletion power of 20 mW.

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Publication details

The article was received on 02 May 2018, accepted on 07 Aug 2018 and first published on 08 Aug 2018


Article type: Paper
DOI: 10.1039/C8NR03584A
Citation: Nanoscale, 2018, Accepted Manuscript
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    Resolution improvement in STED super resolution microscopy at low power using a phasor plot approach

    L. Wang, B. Chen, W. Yan, Z. yang, X. peng, D. Lin, X. weng, T. Ye and J. Qu, Nanoscale, 2018, Accepted Manuscript , DOI: 10.1039/C8NR03584A

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