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

Serge Blaise Pelet; Michael John Robert Previte; Peter T. C. So

doi:10.1117/1.2203664

1 May 2006 Comparing the quantification of Förster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging

Serge Blaise Pelet, Michael John Robert Previte, Peter T. C. So

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Journal of Biomedical Optics, Vol. 11, Issue 3, 034017 (May 2006). https://doi.org/10.1117/1.2203664

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.

©(2006) Society of Photo-Optical Instrumentation Engineers (SPIE)

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Serge Blaise Pelet, Michael John Robert Previte, and Peter T. C. So "Comparing the quantification of Förster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging," Journal of Biomedical Optics 11(3), 034017 (1 May 2006). https://doi.org/10.1117/1.2203664

Published: 1 May 2006

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KEYWORDS

Fluorescence resonance energy transfer

Capillaries

Luminescence

Fluorescence lifetime imaging

Imaging spectroscopy

Photons

Microscopes

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