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SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-...

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Autores principales: Antolovic, Ivan Michel, Burri, Samuel, Bruschini, Claudio, Hoebe, Ron A., Charbon, Edoardo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347095/
https://www.ncbi.nlm.nih.gov/pubmed/28287122
http://dx.doi.org/10.1038/srep44108
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author Antolovic, Ivan Michel
Burri, Samuel
Bruschini, Claudio
Hoebe, Ron A.
Charbon, Edoardo
author_facet Antolovic, Ivan Michel
Burri, Samuel
Bruschini, Claudio
Hoebe, Ron A.
Charbon, Edoardo
author_sort Antolovic, Ivan Michel
collection PubMed
description sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm.
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spelling pubmed-53470952017-03-14 SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking Antolovic, Ivan Michel Burri, Samuel Bruschini, Claudio Hoebe, Ron A. Charbon, Edoardo Sci Rep Article sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm. Nature Publishing Group 2017-03-13 /pmc/articles/PMC5347095/ /pubmed/28287122 http://dx.doi.org/10.1038/srep44108 Text en Copyright © 2017, The Author(s) 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/
spellingShingle Article
Antolovic, Ivan Michel
Burri, Samuel
Bruschini, Claudio
Hoebe, Ron A.
Charbon, Edoardo
SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
title SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
title_full SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
title_fullStr SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
title_full_unstemmed SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
title_short SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
title_sort spad imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347095/
https://www.ncbi.nlm.nih.gov/pubmed/28287122
http://dx.doi.org/10.1038/srep44108
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