Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis

We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested i...

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Main Authors:	Bianchini, Paolo, Cardarelli, Francesco, Luca, Mariagrazia Di, Diaspro, Alberto, Bizzarri, Ranieri
Format:	Online
Language:	English
Published:	Public Library of Science 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072630/

id	pubmed-4072630
recordtype	oai_dc
spelling	pubmed-40726302014-07-02 Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis Bianchini, Paolo Cardarelli, Francesco Luca, Mariagrazia Di Diaspro, Alberto Bizzarri, Ranieri Research Article We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500–700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode. Public Library of Science 2014-06-26 /pmc/articles/PMC4072630/ /pubmed/24967681 http://dx.doi.org/10.1371/journal.pone.0099619 Text en © 2014 Bianchini et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
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	Bianchini, Paolo Cardarelli, Francesco Luca, Mariagrazia Di Diaspro, Alberto Bizzarri, Ranieri
spellingShingle	Bianchini, Paolo Cardarelli, Francesco Luca, Mariagrazia Di Diaspro, Alberto Bizzarri, Ranieri Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis
author_facet	Bianchini, Paolo Cardarelli, Francesco Luca, Mariagrazia Di Diaspro, Alberto Bizzarri, Ranieri
author_sort	Bianchini, Paolo
title	Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis
title_short	Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis
title_full	Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis
title_fullStr	Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis
title_full_unstemmed	Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis
title_sort	nanoscale protein diffusion by sted-based pair correlation analysis
description	We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500–700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode.
publisher	Public Library of Science
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072630/
_version_	1612106804060225536

Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis

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