Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing

Abstract Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and reti...

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Autores principales:	Chiara Stringari, Lamiae Abdeladim, Guy Malkinson, Pierre Mahou, Xavier Solinas, Isabelle Lamarre, Sébastien Brizion, Jean-Baptiste Galey, Willy Supatto, Renaud Legouis, Ana-Maria Pena, Emmanuel Beaurepaire
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/428881b7060047e0a490e4d6be92d52a
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spelling	oai:doaj.org-article:428881b7060047e0a490e4d6be92d52a2021-12-02T11:40:20ZMulticolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing10.1038/s41598-017-03359-82045-2322https://doaj.org/article/428881b7060047e0a490e4d6be92d52a2017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03359-8https://doaj.org/toc/2045-2322Abstract Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.Chiara StringariLamiae AbdeladimGuy MalkinsonPierre MahouXavier SolinasIsabelle LamarreSébastien BrizionJean-Baptiste GaleyWilly SupattoRenaud LegouisAna-Maria PenaEmmanuel BeaurepaireNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Chiara Stringari Lamiae Abdeladim Guy Malkinson Pierre Mahou Xavier Solinas Isabelle Lamarre Sébastien Brizion Jean-Baptiste Galey Willy Supatto Renaud Legouis Ana-Maria Pena Emmanuel Beaurepaire Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
description	Abstract Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.
format	article
author	Chiara Stringari Lamiae Abdeladim Guy Malkinson Pierre Mahou Xavier Solinas Isabelle Lamarre Sébastien Brizion Jean-Baptiste Galey Willy Supatto Renaud Legouis Ana-Maria Pena Emmanuel Beaurepaire
author_facet	Chiara Stringari Lamiae Abdeladim Guy Malkinson Pierre Mahou Xavier Solinas Isabelle Lamarre Sébastien Brizion Jean-Baptiste Galey Willy Supatto Renaud Legouis Ana-Maria Pena Emmanuel Beaurepaire
author_sort	Chiara Stringari
title	Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
title_short	Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
title_full	Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
title_fullStr	Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
title_full_unstemmed	Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
title_sort	multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/428881b7060047e0a490e4d6be92d52a
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Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing

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