Linear Approaches to Intramolecular Förster Resonance Energy Transfer Probe Measurements for Quantitative Modeling

Files in This Item:
File Description SizeFormat 
Paper143.pdf395.05 kBAdobe PDFDownload
Title: Linear Approaches to Intramolecular Förster Resonance Energy Transfer Probe Measurements for Quantitative Modeling
Authors: Birtwistle, Marc R.
Kriegsheim, Alexander von
Kida, Katarzyna
et al.
Permanent link: http://hdl.handle.net/10197/5031
Date: 16-Nov-2011
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.
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
Keywords: Förster Resonance Energy Transfer (FRET)biochemical activityfluorescence lifetime imaging (FLIM)ratiometric methods
DOI: 10.1371/journal.pone.0027823
Language: en
Status of Item: Peer reviewed
Appears in Collections:SBI Research Collection

Show full item record

SCOPUSTM   
Citations 50

7
Last Week
0
Last month
checked on Sep 17, 2018

Page view(s) 50

44
checked on May 25, 2018

Download(s) 50

22
checked on May 25, 2018

Google ScholarTM

Check

Altmetric


This item is available under the Attribution-NonCommercial-NoDerivs 3.0 Ireland. No item may be reproduced for commercial purposes. For other possible restrictions on use please refer to the publisher's URL where this is made available, or to notes contained in the item itself. Other terms may apply.