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A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 μs for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of ∼2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5−15% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.
Tinnefeld et al. (Wed,) studied this question.