Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitatio...

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Autores principales:	Yusuke Niino, Kohji Hotta, Kotaro Oka
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/02669813f9744f4384fe59fe263dc4e8
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spelling	oai:doaj.org-article:02669813f9744f4384fe59fe263dc4e82021-11-25T06:21:56ZSimultaneous live cell imaging using dual FRET sensors with a single excitation light.1932-620310.1371/journal.pone.0006036https://doaj.org/article/02669813f9744f4384fe59fe263dc4e82009-06-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19551140/?tool=EBIhttps://doaj.org/toc/1932-6203Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+) and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.Yusuke NiinoKohji HottaKotaro OkaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 6, p e6036 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yusuke Niino Kohji Hotta Kotaro Oka Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
description	Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+) and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.
format	article
author	Yusuke Niino Kohji Hotta Kotaro Oka
author_facet	Yusuke Niino Kohji Hotta Kotaro Oka
author_sort	Yusuke Niino
title	Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
title_short	Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
title_full	Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
title_fullStr	Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
title_full_unstemmed	Simultaneous live cell imaging using dual FRET sensors with a single excitation light.
title_sort	simultaneous live cell imaging using dual fret sensors with a single excitation light.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/02669813f9744f4384fe59fe263dc4e8
work_keys_str_mv	AT yusukeniino simultaneouslivecellimagingusingdualfretsensorswithasingleexcitationlight AT kohjihotta simultaneouslivecellimagingusingdualfretsensorswithasingleexcitationlight AT kotarooka simultaneouslivecellimagingusingdualfretsensorswithasingleexcitationlight
_version_	1718413782297870336

Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

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