23 February 2018 Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)
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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.
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Iván Coto Hernández, Iván Coto Hernández, Luca Lanzano, Luca Lanzano, Marco Castello, Marco Castello, Nate Jowett, Nate Jowett, Giorgio Tortarolo, Giorgio Tortarolo, Alberto Diaspro, Alberto Diaspro, Giuseppe Vicidomini, 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); doi: 10.1117/12.2286912; https://doi.org/10.1117/12.2286912

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