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Proceedings Paper

Two-color CW STED nanoscopy
Author(s): Xuanze Chen; Yujia Liu; Xusan Yang; Tingting Wang; Eric Alonas; Philip J. Santangelo; Qiushi Ren; Peng Xi
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Paper Abstract

Fluorescent microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile even the most advanced confocal microscopy can only yield optical resolution approaching Abbe diffraction limit of ~200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade.

In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction.

In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.

Paper Details

Date Published: 22 February 2013
PDF: 7 pages
Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 859017 (22 February 2013); doi: 10.1117/12.2001178
Show Author Affiliations
Xuanze Chen, Peking Univ. (China)
Yujia Liu, Peking Univ. (China)
Shanghai Jiao Tong Univ. (China)
Xusan Yang, Peking Univ. (China)
Tingting Wang, Peking Univ. (China)
Eric Alonas, Georgia Institute of Technology (United States)
Emory Univ. (United States)
Philip J. Santangelo, Georgia Institute of Technology (United States)
Emory Univ. (United States)
Qiushi Ren, Peking Univ. (China)
Shanghai Jiao Tong Univ. (China)
Peng Xi, Peking Univ. (China)

Published in SPIE Proceedings Vol. 8590:
Single Molecule Spectroscopy and Superresolution Imaging VI
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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