Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb;18(2):26024.
doi: 10.1117/1.JBO.18.2.026024.

Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

Sanam Mustafa  1 , John Hannagan, Paul Rigby, Kevin Pfleger, Ben Corry
Affiliations
Free article

Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

Sanam Mustafa et al. J Biomed Opt. 2013 Feb.
Free article

Abstract

Accurate 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.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

Publication types

MeSH terms