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