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

Détails

ID Serval
serval:BIB_85110E93ED03
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging.
Périodique
Journal of Biomedical Optics
Auteur(s)
Pelet S., Previte M.J., So P.T.C.
ISSN
1083-3668 (Print)
ISSN-L
1083-3668
Statut éditorial
Publié
Date de publication
2006
Volume
11
Numéro
3
Pages
34017
Langue
anglais
Résumé
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.
Mots-clé
Algorithms, DNA/analysis, Fluorescence Resonance Energy Transfer/methods, Microscopy, Fluorescence, Multiphoton/methods, Reproducibility of Results, Sensitivity and Specificity
Pubmed
Web of science
Création de la notice
25/09/2012 15:40
Dernière modification de la notice
03/03/2018 18:56
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