Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.

ABSTRACT: Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for cross-talk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from

SUBMITTER: Lee NK 

PROVIDER: S-EPMC1282518 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature