Single-molecule detection of fluorescence resonance energy transfer using confocal microscopy

We demonstrated single-molecule fluorescence resonance energy transfer (FRET) from single donor-acceptor dye pair attached to a DNA with a setup based on a confocal microscope. Single-strand DNAs were immobilized on a glass surface with suitable inter-dye distance. Energy transfer efficiency between the donor and the acceptor dyes attached to the DNA was measured with different lengths of DNA. Photobleaching of single dye molecule was observed and used as a sign of single-molecule detection. We could achieve high enough signal-to-noise ratio to detect the fluorescence from a single-molecule, which allows real-time observation of the distance change between single dye pairs in nanometer scale.