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Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo

RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a...

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Autores principales: Dreier, Jes, Castello, Marco, Coceano, Giovanna, Cáceres, Rodrigo, Plastino, Julie, Vicidomini, Giuseppe, Testa, Ilaria
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358637/
https://www.ncbi.nlm.nih.gov/pubmed/30710076
http://dx.doi.org/10.1038/s41467-019-08442-4
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author Dreier, Jes
Castello, Marco
Coceano, Giovanna
Cáceres, Rodrigo
Plastino, Julie
Vicidomini, Giuseppe
Testa, Ilaria
author_facet Dreier, Jes
Castello, Marco
Coceano, Giovanna
Cáceres, Rodrigo
Plastino, Julie
Vicidomini, Giuseppe
Testa, Ilaria
author_sort Dreier, Jes
collection PubMed
description RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70–90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20–30 µm inside a living Caenorhabditis elegans worm.
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spelling pubmed-63586372019-02-04 Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo Dreier, Jes Castello, Marco Coceano, Giovanna Cáceres, Rodrigo Plastino, Julie Vicidomini, Giuseppe Testa, Ilaria Nat Commun Article RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70–90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20–30 µm inside a living Caenorhabditis elegans worm. Nature Publishing Group UK 2019-02-01 /pmc/articles/PMC6358637/ /pubmed/30710076 http://dx.doi.org/10.1038/s41467-019-08442-4 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Dreier, Jes
Castello, Marco
Coceano, Giovanna
Cáceres, Rodrigo
Plastino, Julie
Vicidomini, Giuseppe
Testa, Ilaria
Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
title Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
title_full Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
title_fullStr Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
title_full_unstemmed Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
title_short Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo
title_sort smart scanning for low-illumination and fast resolft nanoscopy in vivo
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358637/
https://www.ncbi.nlm.nih.gov/pubmed/30710076
http://dx.doi.org/10.1038/s41467-019-08442-4
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