Show simple item record

dc.contributor.authorBirtwistle, Marc R.
dc.contributor.authorvon Kriegsheim, Alexander
dc.contributor.authorKida, Katarzyna
dc.contributor.authorSchwarz, Juliane P.
dc.contributor.authorAnderson, Kurt I.
dc.contributor.authorKolch, Walter
dc.date.accessioned2012-10-26T20:30:41Z
dc.date.available2012-10-26T20:30:41Z
dc.date.issued2011-11-16en_US
dc.identifier.citationPLoS One. 2011 Nov 16; 6(11):e27823en_US
dc.identifier.issn1932-6203en_US
dc.identifier.pmid22114702en_US
dc.identifier.doi10.1371/journal.pone.0027823en_US
dc.identifier.urihttp://hdl.handle.net/10675.2/768
dc.description.abstractNumerous unimolecular, genetically-encoded Forster 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.
dc.rightsThis is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.en_US
dc.subjectResearch Articleen_US
dc.subjectBiologyen_US
dc.subjectBiochemistryen_US
dc.subjectEnzymesen_US
dc.subjectEnzyme Kineticsen_US
dc.subjectComputational Biologyen_US
dc.subjectBiochemical Simulationsen_US
dc.subjectSignaling Networksen_US
dc.subjectSystems Biologyen_US
dc.subjectDevelopmental Biologyen_US
dc.subjectMolecular Developmenten_US
dc.subjectSignalingen_US
dc.subjectMolecular Cell Biologyen_US
dc.subjectSignal Transductionen_US
dc.subjectSignaling Cascadesen_US
dc.subjectSignaling in Cellular Processesen_US
dc.subjectSignaling in Selected Disciplinesen_US
dc.subjectSignaling Pathwaysen_US
dc.subjectNeuroscienceen_US
dc.subjectMolecular Neuroscienceen_US
dc.subjectSignaling Pathwaysen_US
dc.subjectSystems Biologyen_US
dc.subjectChemistryen_US
dc.subjectPhysical Chemistryen_US
dc.subjectEnergy Transferen_US
dc.subjectEngineeringen_US
dc.subjectBioengineeringen_US
dc.subjectBiological Systems Engineeringen_US
dc.titleLinear Approaches to Intramolecular Forster Resonance Energy Transfer Probe Measurements for Quantitative Modelingen_US
dc.typeArticleen_US
dc.identifier.pmcidPMC3218046en_US
dc.contributor.corporatenameGHSU Cancer Center
refterms.dateFOA2019-04-10T00:48:50Z
html.description.abstractNumerous unimolecular, genetically-encoded Forster 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.


Files in this item

Thumbnail
Name:
pone.0027823.pdf
Size:
395.0Kb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record