Fluorescence intensity and lifetime fluctuations of single Cy5 molecules immobilized on the glass surface

Single molecule fluorescence spectroscopy is a powerful tool to reveal the fluorescence fluctuation dynamics of a fluorescence molecule immobilized on a surface. These fluctuations are correlated in time under different excitation intensities with different repetition rate, which may provide the information for us to describe the reasonable excited state processes as well as the surrounding environments. In this report, we showed the first results on the fluorescence fluctuations of a single Cy5 molecule at the air–glass interface excited with different conditions by using far-field confocal laser microscopy in conjunction with multichannel scale (MCS) and time-correlated single photon counting (TCSPC). We found that the on/off-time and count rates showed different behaviors under different excitation modes. It was found that the excitation with the high repetition rate and/or strong intensity may cause high count rate, and increase the possibility of reverse intersystem crossing from Tn to Sn state, which may lead to longer on-time and shorter off-time. Furthermore, the photoinduced dynamic processes excited with strong peak power described by a five-level model under continuous wave (CW) and pulsed excitations with high repetition rate, it can be roughly simplified by a three-level scheme under pulse laser at lower repetition rate.