Time-Resolved Confocal Fluorescence Microscopy: A Generalized Approach Enables New Directions for FLIM, FRET and FCS

Abstract

Fluorescence dynamics of single molecules can be followed on timescales from sub-nanoseconds to seconds and even beyond with a universal approach of time-resolved measurements. The underlying technique (Time-Tagged Time-Resolved (TTTR) Recording) allows one to simultaneously record timing and fluorescence intensity information, both spectrally and spatially, on a single photon basis. We apply photon sorting and weighting schemes determined from the nanosecond photon arrival times to extend and improve single-molecule fluorescence methodologies which up to now commonly utilize only intensity-based analysis, namely FCS and FRET.In Fluorescence Lifetime Correlation Spectroscopy (FLCS) photon weighting provides superior suppression of common parasitic contributions, e.g., Raman scattering and detector after-pulsing. Beyond this improvement of traditional FCS, FLCS also offers the possibility to accurately determine diffusion properties of different species only requiring that the species differ in their fluorescence lifetimes [1]. In 2-focus-FCS (2fFCS), the nanosecond timing information is used to identify the spatial origin of the photons by combining Pulsed Interleaved Excitation (PIE) with time-gated detection [2]. Thereby, 2fFCS dramatically improves the accuracy of measuring absolute diffusion coefficients. In addition to this, PIE can be used to identify artifacts and sub-populations in single-pair FRET measurements. Nanosecond time-resolved detection offers a complementary approach to donor/acceptor intensity based methods for calculating FRET efficiencies via quenching of the donor lifetime.[1] Benda A., Hof. M., Wahl M., Patting M., Erdmann R., Kapusta P., Rev. Sci. Instr., Vol.76, 033106 (2005)[2] Dertinger Th., Pacheco V., von der Hocht I., Hartmann R., Gregor I., Enderlein J, ChemPhysChem, Vol.8, p.433 (2007)