We have previously shown that formation of triplet states and other photo-induced states can be controlled by
modulating the excitation with pulse widths and periods in the range of the transition times of the involved states.
However, modulating the excitation in fluorescence correlation spectroscopy (FCS) measurements normally destroys
correlation information and induces ringing in the correlation curve. We have introduced and experimentally verified a
method to retrieve the full correlation curves from FCS measurements with modulated excitation and arbitrarily low
fraction of active excitation. Modulated excitation applied to FCS experiments was shown to suppress the triplet build-up
more efficiently than reducing excitation power with continuous wave excitation. The usefulness of the method was
demonstrated by measurements done on fluorescein at different pH, where suppression of the triplet significantly
facilitates the analysis of the protonation kinetics. Using a fluorophore where the protonation-coupled fluorescence
intensity fluctuations are due to spectral shifts, introduction of two-color alternating excitation and spectral crosscorrelation
can turn the protonation component of the correlation curve into an anti-correlation and further facilitate the
distinction of this component from those of other processes.
Photoinduced transient dark states are exhibited by practically all common fluorophores. However, their information
content has to date only been sparsely exploited due to methodological constraints. Here, a new concept is presented and
verified that can monitor and image these states via their photodynamic fingerprints. It unites the environmental
sensitivity of these states with the sensitivity of
fluorescence-based detection. For demonstration, triplet state images of
liposomes in different environments were generated, showing how local environmental differences can be resolved, not
clearly distinguishable via other fluorescence parameters. The concept can provide several new, useful and independent
fluorescence-based parameters in biomolecular imaging.
We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.