Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides inf...

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Autores principales:	Valentin Dunsing, Annett Petrich, Salvatore Chiantia
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	fluorescence optical microscopy virus assembly protein-protein interactions diffusion Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/7f29b1f383df4e06bca7cd732370dc2e
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spelling	oai:doaj.org-article:7f29b1f383df4e06bca7cd732370dc2e2021-11-30T09:57:55ZMulticolor fluorescence fluctuation spectroscopy in living cells via spectral detection10.7554/eLife.696872050-084Xe69687https://doaj.org/article/7f29b1f383df4e06bca7cd732370dc2e2021-09-01T00:00:00Zhttps://elifesciences.org/articles/69687https://doaj.org/toc/2050-084XSignaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.Valentin DunsingAnnett PetrichSalvatore ChiantiaeLife Sciences Publications Ltdarticlefluorescenceoptical microscopyvirus assemblyprotein-protein interactionsdiffusionMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	fluorescence optical microscopy virus assembly protein-protein interactions diffusion Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	fluorescence optical microscopy virus assembly protein-protein interactions diffusion Medicine R Science Q Biology (General) QH301-705.5 Valentin Dunsing Annett Petrich Salvatore Chiantia Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
description	Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.
format	article
author	Valentin Dunsing Annett Petrich Salvatore Chiantia
author_facet	Valentin Dunsing Annett Petrich Salvatore Chiantia
author_sort	Valentin Dunsing
title	Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
title_short	Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
title_full	Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
title_fullStr	Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
title_full_unstemmed	Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
title_sort	multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/7f29b1f383df4e06bca7cd732370dc2e
work_keys_str_mv	AT valentindunsing multicolorfluorescencefluctuationspectroscopyinlivingcellsviaspectraldetection AT annettpetrich multicolorfluorescencefluctuationspectroscopyinlivingcellsviaspectraldetection AT salvatorechiantia multicolorfluorescencefluctuationspectroscopyinlivingcellsviaspectraldetection
_version_	1718406692560961536

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection

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