All measurements were perfomed with the single molecule sensitive, inverse Time-resolved Fluorescence Microscope MicroTime200

On the right: FRET analysis of freely diffusing oligopeptids in water labeled with single Alexa Fluor 488 and Alexa Fluor 594 chromophores as donor and acceptor, respectively. The upper part shows in the MCS trace the corresponding photon bursts, recorded simultaneously in two spectrally separated detection channels. The blue trajectory shows the remaining fluorescence of the laser excited FRET donor and the red trace the emission intensity of the FRET acceptor. The bursts can be identified with a threshold criterion to exctract the photon numbers of each peptide which are then used to calculate its individual FRET efficiency. The spread of individual FRET efficiencies over all investigated species from a 900 s measurement is displayed in the lower histogram.

On the left: Intensity and lifetime fluctuations of a single Cy5 molecule adsorbed on a glass coverslip surface. The MCS trace in the upper half depicts spontaneous decreases and total interuptions of the fluorescence emission (photons binned in 100 ms). The corresponding fluctuations of the fluorescence lifetime in the lower half was extracted from succesive fractions (1.25 s each) of the emitted photons, which are collected in a TCSPC histogram and fitted with an exponential decay. Two representative histograms are shown on the right side and indicate that the same molecule can exhibit a fast (0.6 ns) and a slow (1.65 ns) fluorescence decay at different times of the acquisition.

On the right: Time-resolved fluorescence measurements of freely diffusing ATTO655 molecules in different solvents (about 100 pmol/l): The partial view of the MCS trace in the upper half depicts the typical fluorescence bursts of single molecules which diffuse through the confocally defined investigation volume. The lower half shows the related FCS curve of ATTO655 molecules in two solvents with a different viscosity, which affects the mobility of the fluorophores. The fluorescence intensity auto-correlation was directly calculated from the TTTR raw data.