Long-lived bright red emitting azaoxa-triangulenium fluorophores.

The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measure...

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Autores principales:	Badri P Maliwal, Rafal Fudala, Sangram Raut, Rutika Kokate, Thomas J Sørensen, Bo W Laursen, Zygmunt Gryczynski, Ignacy Gryczynski
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7e16835107f64f7ea4ef417c3e35cc61
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spelling	oai:doaj.org-article:7e16835107f64f7ea4ef417c3e35cc612021-11-18T07:46:36ZLong-lived bright red emitting azaoxa-triangulenium fluorophores.1932-620310.1371/journal.pone.0063043https://doaj.org/article/7e16835107f64f7ea4ef417c3e35cc612013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23667570/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.Badri P MaliwalRafal FudalaSangram RautRutika KokateThomas J SørensenBo W LaursenZygmunt GryczynskiIgnacy GryczynskiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e63043 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Badri P Maliwal Rafal Fudala Sangram Raut Rutika Kokate Thomas J Sørensen Bo W Laursen Zygmunt Gryczynski Ignacy Gryczynski Long-lived bright red emitting azaoxa-triangulenium fluorophores.
description	The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.
format	article
author	Badri P Maliwal Rafal Fudala Sangram Raut Rutika Kokate Thomas J Sørensen Bo W Laursen Zygmunt Gryczynski Ignacy Gryczynski
author_facet	Badri P Maliwal Rafal Fudala Sangram Raut Rutika Kokate Thomas J Sørensen Bo W Laursen Zygmunt Gryczynski Ignacy Gryczynski
author_sort	Badri P Maliwal
title	Long-lived bright red emitting azaoxa-triangulenium fluorophores.
title_short	Long-lived bright red emitting azaoxa-triangulenium fluorophores.
title_full	Long-lived bright red emitting azaoxa-triangulenium fluorophores.
title_fullStr	Long-lived bright red emitting azaoxa-triangulenium fluorophores.
title_full_unstemmed	Long-lived bright red emitting azaoxa-triangulenium fluorophores.
title_sort	long-lived bright red emitting azaoxa-triangulenium fluorophores.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/7e16835107f64f7ea4ef417c3e35cc61
work_keys_str_mv	AT badripmaliwal longlivedbrightredemittingazaoxatrianguleniumfluorophores AT rafalfudala longlivedbrightredemittingazaoxatrianguleniumfluorophores AT sangramraut longlivedbrightredemittingazaoxatrianguleniumfluorophores AT rutikakokate longlivedbrightredemittingazaoxatrianguleniumfluorophores AT thomasjsørensen longlivedbrightredemittingazaoxatrianguleniumfluorophores AT bowlaursen longlivedbrightredemittingazaoxatrianguleniumfluorophores AT zygmuntgryczynski longlivedbrightredemittingazaoxatrianguleniumfluorophores AT ignacygryczynski longlivedbrightredemittingazaoxatrianguleniumfluorophores
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Long-lived bright red emitting azaoxa-triangulenium fluorophores.

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