title: Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules.
abstract: 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.
authors: Graduate School of Information Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
authors: Masataka
authors: M
authors: Yamao
authors: National Institute for Basic Biology, Okazaki, Aichi, Japan.
authors: Kazuhiro
authors: K
authors: Aoki
authors: Okinawa Institute of Science and Technology Graduate University, Kunigami, Okinawa, Japan.
authors: Naoto
authors: N
authors: Yukinawa
authors: Imaging Platform for Spatio-temporal Information, Kyoto University, Sakyo, Kyoto, Japan.|Graduate School of Informatics, Kyoto University, Sakyo, Kyoto, Japan.
authors: Shin
authors: S
authors: Ishii
authors: Imaging Platform for Spatio-temporal Information, Kyoto University, Sakyo, Kyoto, Japan.|Graduate School of Medicine, Kyoto University, Sakyo, Kyoto, Japan.
authors: Michiyuki
authors: M
authors: Matsuda
authors: Imaging Platform for Spatio-temporal Information, Kyoto University, Sakyo, Kyoto, Japan.|Graduate School of Medicine, Kyoto University, Sakyo, Kyoto, Japan.
authors: Honda
authors: H
authors: Naoki
mesh_terms: D000465:Algorithms; D015374:Biosensing Techniques / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*