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Proceedings Paper

A novel pulsed STED microscopy method using FastFLIM and the phasor plots
Author(s): Yuansheng Sun; Giorgio Tortarolo; Kai-Wen Teng; Yuji Ishitsuka; Ulas C. Coskun; Shih-Chu Jeff Liao; Alberto Diaspro; Giuseppe Vicidomini; Paul R. Selvin; Beniamino Barbieri
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Paper Abstract

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.

Paper Details

Date Published: 21 February 2017
PDF: 16 pages
Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100691C (21 February 2017); doi: 10.1117/12.2267880
Show Author Affiliations
Yuansheng Sun, ISS, Inc. (United States)
Giorgio Tortarolo, Istituto Italiano di Tecnologia (Italy)
Kai-Wen Teng, Univ. of Illinois (United States)
Yuji Ishitsuka, Univ. of Illinois at Urbana-Champaign (United States)
Ulas C. Coskun, ISS, Inc. (United States)
Shih-Chu Jeff Liao, ISS, Inc. (United States)
Alberto Diaspro, Istituto Italiano di Tecnologia (Italy)
Giuseppe Vicidomini, Istituto Italiano di Tecnologia (Italy)
Paul R. Selvin, Univ. of Illinois at Urbana-Champaign (United States)
Beniamino Barbieri, ISS, Inc. (United States)


Published in SPIE Proceedings Vol. 10069:
Multiphoton Microscopy in the Biomedical Sciences XVII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)

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