Single-Pair FÖrster Resonance Energy Transfer With Multiparametric Excitation and Detection

Forster resonance energy transfer (FRET) between a donor and an acceptor dye molecule is a common method to study the distances between single molecules in living cells in the nanometer range. Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem is the incomplete fluorescent labeling of the molecules, which leads to the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescing acceptor dyes. Other problems occur due to spectral bleed through, direct acceptor excitation, and the difficulty to obtain the quantum efficiencies of the dyes and the detection efficiencies of the corresponding detectors. In order to correct these defects, the donor as well as the acceptor are excited alternatingly using pulsed interleaved excitation. Time-correlated single-photon counting enables the measurement of fluorescence lifetime; hence, the FRET efficiency can also be derived from the decrease of the donor fluorescence lifetime. The universal time-tagged time-resolved data format allows to retrieve all the necessary information from the fluorescence photons detected during the measurement.