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Journal of Biomedical Optics

Comparing the quantification of Förster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging
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Paper Abstract

The measurement of Förster resonance energy transfer (FRET) in microscopes can be realized by different imaging modalities. In the present work, reference FRET constructs are developed to allow the comparison of FRET microscopy measurements using intensity, spectral, and lifetime imaging. Complimentary DNA strands are respectively labeled with Oregon Green 488 (OG488) or tetramethylrhodamine (TMR). The OG488 dye is fixed at the 5′ end of one strand, and the TMR label position is allowed to vary along the complimentary strand. Since OG488 and TMR are FRET pairs, the FRET efficiency can be determined theoretically from the distance separating the two dyes of the double-stranded DNA molecules. Microscopic images are formed by imaging microcapillaries containing various mixtures of oligonucleotides labeled with the FRET fluorophore pair, only the donor, or only acceptor. Traditional two-channel intensity measurements are compared with spectrally resolved imaging and fluorescence lifetime imaging by calculating a FRET index. The latter proves to be the best method to quantify FRET efficiency in the image. More importantly, the intensity fraction of molecules undergoing FRET can be quantitatively measured in each pixel of the image.

Paper Details

Date Published: 1 May 2006
PDF: 11 pages
J. Biomed. Opt. 11(3) 034017 doi: 10.1117/1.2203664
Published in: Journal of Biomedical Optics Volume 11, Issue 3
Show Author Affiliations
Serge Blaise Pelet, Massachusetts Institute of Technology (United States)
Michael John Robert Previte, Massachusetts Institute of Technology (United States)
Peter T. C. So, Massachusetts Institute of Technology (United States)

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