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Linear Approaches to Intramolecular Förster Resonance Energy Transfer Probe Measurements for Quantitative Modeling
Date Issued
2011-11-16
Date Available
2013-11-29T09:21:50Z
Abstract
Numerous unimolecular, genetically-encoded Förster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted Ralt) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on Ralt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.
Other Sponsorship
Science Foundation Ireland Grant No. 06/CE/B1129.Marie Curie International Incoming Fellowship (236758) and an European Molecular Biology Organization long-term fellowship (ALTF 81)
Type of Material
Journal Article
Publisher
Public Library of Science
Journal
PLoS ONE
Volume
6
Issue
11
Copyright (Published Version)
2011 Public Library of Science
Language
English
Status of Item
Peer reviewed
This item is made available under a Creative Commons License
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