Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/83631
Citations
Scopus Web of Science® Altmetric
?
?
Full metadata record
DC FieldValueLanguage
dc.contributor.authorMustafa, S.en
dc.contributor.authorHannagan, J.en
dc.contributor.authorRigby, P.en
dc.contributor.authorPfleger, K.en
dc.contributor.authorCorry, B.en
dc.date.issued2013en
dc.identifier.citationJournal of Biomedical Optics, 2013; 18(2):026024-1-026024-14en
dc.identifier.issn1083-3668en
dc.identifier.issn1560-2281en
dc.identifier.urihttp://hdl.handle.net/2440/83631-
dc.description.abstractAccurate quantification of Förster resonance energy transfer (FRET) using intensity-based methods is difficult due to the overlap of fluorophore excitation and emission spectra. Consequently, mechanisms are required to remove bleedthrough of the donor emission into the acceptor channel and direct excitation of the acceptor when aiming to excite only the donor fluorophores. Methods to circumvent donor bleedthrough using the unmixing of emission spectra have been reported, but these require additional corrections to account for direct excitation of the acceptor. Here we present an alternative method for robust quantification of FRET efficiencies based upon the simultaneous spectral unmixing of both excitation and emission spectra. This has the benefit over existing methodologies in circumventing the issue of donor bleedthrough and acceptor cross excitation without the need for additional corrections. Furthermore, we show that it is applicable with as few as two excitation wavelengths and so can be used for quantifying FRET efficiency in microscope images as easily as for data collected on a spectrofluorometer. We demonstrate the accuracy of the approach by reproducing efficiency values in well characterized FRET standards: HEK cells expressing a variety of linked cerulean and venus fluorescent proteins. Finally we describe simple ImageJ plugins that can be used to calculate and create images of FRET efficiencies from microscope images.en
dc.description.statementofresponsibilitySanam Mustafa, John Hannagan, Paul Rigby, Kevin Pfleger, and Ben Corryen
dc.language.isoenen
dc.publisherSPIE-Int Society Optical Engineeringen
dc.rights© The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.18.2.026024]en
dc.subjectresonance energy transfer; ImageJ; FRET efficiency; fluorescent protein; spectral unmixing; confocal; spectral imaging; spectral bleedthroughen
dc.titleQuantitative Föster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectraen
dc.title.alternativeQuantitative Foster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectraen
dc.typeJournal articleen
dc.identifier.rmid0020138123en
dc.identifier.doi10.1117/1.JBO.18.2.026024en
dc.relation.granthttp://purl.org/au-research/grants/arc/FT100100271en
dc.identifier.pubid14482-
pubs.library.collectionMedical Sciences publicationsen
pubs.verification-statusVerifieden
pubs.publication-statusPublisheden
dc.identifier.orcidMustafa, S. [0000-0002-8677-5151]en
Appears in Collections:Medical Sciences publications

Files in This Item:
File Description SizeFormat 
hdl_83631.pdfPublished version6.05 MBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.