Following FRET through five energy transfer steps: spectroscopic photobleaching, recovery of spectra, and a sequential mechanism of FRET

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Forde, T.S. and Hanley, Q.S. ORCID: 0000-0002-8189-9550, 2005. Following FRET through five energy transfer steps: spectroscopic photobleaching, recovery of spectra, and a sequential mechanism of FRET. Photochemical & Photobiological Sciences, 4 (8), pp. 609-616. ISSN 1474-905X

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Official URL: http://dx.doi.org/10.1039/b416478d

Abstract

We report the acquisition and analysis of spectrally resolved photobleaching data from a model system designed to exhibit FRET. Spectrally resolved photobleaching can be used to determine the presence of FRET in these systems and to investigate multi-step mechanisms of energy transfer. The model system was a previously described set of fluorescent beads consisting of a system of six fluorophores. In standard photobleaching experiments to determine FRET, bleaching of an acceptor molecule resulting in recovery of donor intensity or changes in photobleaching kinetics are used as indicators of FRET. Here, we use the Bateman equations to model growth and decay in a photobleaching experiment. Linked donor–acceptor growth and decay is used as an indicator of FRET. The apparatus required is relatively simple when compared to lifetime imaging systems. Several data analysis strategies, rigorous model building, global fitting procedures, and error analysis are presented. Using these procedures a five-step sequential mechanism of energy transfer was selected for these beads.

Item Type:

Journal article

Publication Title:

Photochemical & Photobiological Sciences

Creators:

Forde, T.S. and Hanley, Q.S.

Publisher:

Royal Society of Chemistry

Place of Publication:

Cambridge

Date:

2005