Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules

We developed two new FRET imaging measures for intramolecular FRET biosensors, called linearly proportional (LP) and highly contrasting (HC) measures, which can be easily calculated by the fluorescence intensities of donor and acceptor as a ratio between their weighted sums. As an alternative to the...

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Main Authors:	Yamao, Masataka, Aoki, Kazuhiro, Yukinawa, Naoto, Ishii, Shin, Matsuda, Michiyuki, Naoki, Honda
Format:	Online
Language:	English
Published:	Public Library of Science 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079603/

id	pubmed-5079603
recordtype	oai_dc
spelling	pubmed-50796032016-11-04 Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules Yamao, Masataka Aoki, Kazuhiro Yukinawa, Naoto Ishii, Shin Matsuda, Michiyuki Naoki, Honda Research Article We developed two new FRET imaging measures for intramolecular FRET biosensors, called linearly proportional (LP) and highly contrasting (HC) measures, which can be easily calculated by the fluorescence intensities of donor and acceptor as a ratio between their weighted sums. As an alternative to the conventional ratiometric measure, which non-linearly depends on the fraction of active molecule, we first developed the LP measure, which is linearly proportional to the fraction of active molecules. The LP measure inherently unmixes bleed-through signals and is robust against fluorescence noise. By extending the LP measure, we furthermore designed the HC measure, which provides highly contrasting images of the molecular activity, more than the ratiometric measure. In addition to their advantages, these measures are insensitive to the biosensor expression level, which is a fundamental property of the ratiometric measure. Using artificial data and FRET imaging data, we showed that the LP measure effectively represents the fraction of active molecules and that the HC measure improves visual interpretability by providing high contrast images of molecular activity. Therefore, the LP and HC measures allow us to gain more quantitative and qualitative insights from FRET imaging than the ratiometric measure. Public Library of Science 2016-10-25 /pmc/articles/PMC5079603/ /pubmed/27780260 http://dx.doi.org/10.1371/journal.pone.0164254 Text en © 2016 Yamao 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 (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are 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	Yamao, Masataka Aoki, Kazuhiro Yukinawa, Naoto Ishii, Shin Matsuda, Michiyuki Naoki, Honda
spellingShingle	Yamao, Masataka Aoki, Kazuhiro Yukinawa, Naoto Ishii, Shin Matsuda, Michiyuki Naoki, Honda Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules
author_facet	Yamao, Masataka Aoki, Kazuhiro Yukinawa, Naoto Ishii, Shin Matsuda, Michiyuki Naoki, Honda
author_sort	Yamao, Masataka
title	Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules
title_short	Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules
title_full	Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules
title_fullStr	Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules
title_full_unstemmed	Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules
title_sort	two new fret imaging measures: linearly proportional to and highly contrasting the fraction of active molecules
description	We developed two new FRET imaging measures for intramolecular FRET biosensors, called linearly proportional (LP) and highly contrasting (HC) measures, which can be easily calculated by the fluorescence intensities of donor and acceptor as a ratio between their weighted sums. As an alternative to the conventional ratiometric measure, which non-linearly depends on the fraction of active molecule, we first developed the LP measure, which is linearly proportional to the fraction of active molecules. The LP measure inherently unmixes bleed-through signals and is robust against fluorescence noise. By extending the LP measure, we furthermore designed the HC measure, which provides highly contrasting images of the molecular activity, more than the ratiometric measure. In addition to their advantages, these measures are insensitive to the biosensor expression level, which is a fundamental property of the ratiometric measure. Using artificial data and FRET imaging data, we showed that the LP measure effectively represents the fraction of active molecules and that the HC measure improves visual interpretability by providing high contrast images of molecular activity. Therefore, the LP and HC measures allow us to gain more quantitative and qualitative insights from FRET imaging than the ratiometric measure.
publisher	Public Library of Science
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079603/
_version_	1613697601489600512

Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules

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