Two-Photon Excitation STED Microscopy with Time-Gated Detection

We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing...

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Main Authors:	Coto Hernández, Iván, Castello, Marco, Lanzanò, Luca, d’Amora, Marta, Bianchini, Paolo, Diaspro, Alberto, Vicidomini, Giuseppe
Format:	Online
Language:	English
Published:	Nature Publishing Group 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725939/

id	pubmed-4725939
recordtype	oai_dc
spelling	pubmed-47259392016-01-28 Two-Photon Excitation STED Microscopy with Time-Gated Detection Coto Hernández, Iván Castello, Marco Lanzanò, Luca d’Amora, Marta Bianchini, Paolo Diaspro, Alberto Vicidomini, Giuseppe Article We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture’s complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source. Nature Publishing Group 2016-01-13 /pmc/articles/PMC4725939/ /pubmed/26757892 http://dx.doi.org/10.1038/srep19419 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Coto Hernández, Iván Castello, Marco Lanzanò, Luca d’Amora, Marta Bianchini, Paolo Diaspro, Alberto Vicidomini, Giuseppe
spellingShingle	Coto Hernández, Iván Castello, Marco Lanzanò, Luca d’Amora, Marta Bianchini, Paolo Diaspro, Alberto Vicidomini, Giuseppe Two-Photon Excitation STED Microscopy with Time-Gated Detection
author_facet	Coto Hernández, Iván Castello, Marco Lanzanò, Luca d’Amora, Marta Bianchini, Paolo Diaspro, Alberto Vicidomini, Giuseppe
author_sort	Coto Hernández, Iván
title	Two-Photon Excitation STED Microscopy with Time-Gated Detection
title_short	Two-Photon Excitation STED Microscopy with Time-Gated Detection
title_full	Two-Photon Excitation STED Microscopy with Time-Gated Detection
title_fullStr	Two-Photon Excitation STED Microscopy with Time-Gated Detection
title_full_unstemmed	Two-Photon Excitation STED Microscopy with Time-Gated Detection
title_sort	two-photon excitation sted microscopy with time-gated detection
description	We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture’s complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source.
publisher	Nature Publishing Group
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725939/
_version_	1613527993857081344

Two-Photon Excitation STED Microscopy with Time-Gated Detection

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