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In recent years time-resolved fluorescence measurement and analysis techniques became a standard in single molecule
microscopy. However, considering the equipment and experimental implementation they are typically still an add-on and
offer only limited possibilities to study the mutual dependencies with common intensity and spectral information. In
contrast, we are using a specially designed instrument with an unrestricted photon data acquisition approach which
allows to store spatial, temporal, spectral and intensity information in a generalized format preserving the full
experimental information. This format allows us not only to easily study dependencies between various fluorescence
parameters but also to use, for example, the photon arrival time for sorting and weighting the detected photons to
improve the significance in common FCS and FRET analysis schemes. The power of this approach will be demonstrated
for different techniques: In FCS experiments the concentration determination accuracy can be easily improved by a
simple time-gated photon analysis to suppress the fast decaying background signal. A more detailed analysis of the
arrival times allows even to separate FCS curves for species which differ in their fluorescence lifetime but, for example,
cannot be distinguished spectrally. In multichromophoric systems like a photonic wire which undergoes unidirectional
multistep FRET the lifetime information complements significantly the intensity based analysis and helps to assign the
respective FRET partners. Moreover, together with pulsed excitation the time-correlated analysis enables directly to take
advantage of alternating multi-colour laser excitation. This pulsed interleaved excitation (PIE) can be used to identify
and rule out inactive FRET molecules which cause interfering artefacts in standard FRET efficiency analysis. We used a
piezo scanner based confocal microscope with compact picosecond diode lasers as excitation sources. The timing
performance can be significantly increased by using new SPAD detectors which enable, in conjunction with new TCSPC
electronics, an overall IRF width of less than 120 ps maintaining single molecule sensitivity.
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