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Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)
Author(s): Iván Coto Hernández; Luca Lanzano; Marco Castello; Nate Jowett; Giorgio Tortarolo; Alberto Diaspro; Giuseppe Vicidomini
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Paper Abstract

Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.

Paper Details

Date Published: 23 February 2018
PDF: 7 pages
Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104982U (23 February 2018); doi: 10.1117/12.2286912
Show Author Affiliations
Iván Coto Hernández, Harvard Medical School (United States)
Luca Lanzano, Istituto Italiano di Tecnologia (Italy)
Marco Castello, Istituto Italiano di Tecnologia (Italy)
Nate Jowett, Harvard Medical School (United States)
Giorgio Tortarolo, Istituto Italiano di Tecnologia (Italy)
Alberto Diaspro, Istituto Italiano di Tecnologia (Italy)
Giuseppe Vicidomini, Istituto Italiano di Tecnologia (Italy)

Published in SPIE Proceedings Vol. 10498:
Multiphoton Microscopy in the Biomedical Sciences XVIII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)

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