The importance of the photon arrival times in STED microscopy

Marco Castello; Luca L. Lanzanò; Iván Coto Hernández; Christian Eggeling; Alberto Diaspro; Giuseppe Vicidomini

doi:10.1117/12.2081553

9 March 2015 The importance of the photon arrival times in STED microscopy

Marco Castello, Luca L. Lanzanò, Iván Coto Hernández, Christian Eggeling, Alberto Diaspro, Giuseppe Vicidomini

Proceedings Volume 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII; 93310X (2015) https://doi.org/10.1117/12.2081553
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

In a stimulated emission depletion (STED) microscope the region from which a fluorophore can spontaneously emit shrinks with the continued STED beam action after the excitation event. This fact has been recently used to implement a versatile, simple and cheap STED microscope that uses a pulsed excitation beam, a STED beam running in continuous-wave (CW) and a time-gated detection: By collecting only the delayed (with respect to the excitation events) fluorescence, the STED beam intensity needed for obtaining a certain spatial resolution strongly reduces, which is fundamental to increase live cell imaging compatibility. This new STED microscopy implementation, namely gated CW-STED, is in essence limited (only) by the reduction of the signal associated with the time-gated detection. Here we show the recent advances in gated CW-STED microscopy and related methods. We show that the time-gated detection can be substituted by more efficient computational methods when the arrival-times of all fluorescence photons are provided.

Citation Download Citation

Marco Castello, Luca L. Lanzanò, Iván Coto Hernández, Christian Eggeling, Alberto Diaspro, and Giuseppe Vicidomini "The importance of the photon arrival times in STED microscopy", Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 93310X (9 March 2015); https://doi.org/10.1117/12.2081553

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