Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules

The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Forster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique - FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.