Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)

Iván Coto Hernández; Luca Lanzano; Marco Castello; Nate Jowett; Giorgio Tortarolo; Alberto Diaspro; Giuseppe Vicidomini

doi:10.1117/12.2286912

23 February 2018 Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)

Iván Coto Hernández, Luca Lanzano, Marco Castello, Nate Jowett, Giorgio Tortarolo, Alberto Diaspro, Giuseppe Vicidomini

Proceedings Volume 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII; 104982U (2018) https://doi.org/10.1117/12.2286912
Event: SPIE BiOS, 2018, San Francisco, California, United States

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.

Citation Download Citation

Iván Coto Hernández, Luca Lanzano, Marco Castello, Nate Jowett, Giorgio Tortarolo, Alberto Diaspro, and Giuseppe Vicidomini "Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)", Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104982U (23 February 2018); https://doi.org/10.1117/12.2286912

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