Fluorescence lifetime distribution of single molecules undergoing Fِrster energy transfer

The Förster energy transfer process of a donor–acceptor system was investigated at the single-molecule level as well as in bulk by confocal fluorescence lifetime microscopy. While the fluorescence decay of bulk tetramethylrhodamine ethyl ester (TMR) was nonexponential, the single TMR molecules exhibit single exponential decay in PMMA in the absence or presence of azulene. The lifetime distribution of single TMR molecules undergoing fluorescence energy transfer shows some deviation from that deduced from the Förster equation. Although the bulk data are well fitted by two exponentials, the measured distribution shows no physical basis for that assumption.