Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging.

Details

Serval ID
serval:BIB_85110E93ED03
Type
Article: article from journal or magazin.
Collection
Publications
Title
Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging.
Journal
Journal of Biomedical Optics
Author(s)
Pelet S., Previte M.J., So P.T.C.
ISSN
1083-3668 (Print)
ISSN-L
1083-3668
Publication state
Published
Issued date
2006
Volume
11
Number
3
Pages
34017
Language
english
Abstract
The measurement of Forster 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.
Keywords
Algorithms, DNA/analysis, Fluorescence Resonance Energy Transfer/methods, Microscopy, Fluorescence, Multiphoton/methods, Reproducibility of Results, Sensitivity and Specificity
Pubmed
Web of science
Create date
25/09/2012 15:40
Last modification date
03/03/2018 18:56
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