Photon coincidence analysis is nowadays a widely used technique to study fluorescence intensity fluctuations, taking place on a timescale from seconds down to picoseconds. Photon bursts in the microsecond regime are e.g. used to study diffusion properties via Fluorescence Correlation Spectroscopy (FCS). Photon bunching in the microsecond regime allows to study fast conformational changes as well as internal photophysics like singulett-triplet transitions. Interphoton delay times in the ns regime carry information about the fluorescence lifetime and can also be used to characterise molecular rotation. Down in the picosecond regime, photon antibunching is used to quantify a small number of emitters and especially to proof the existence of a single emitting dye molecule. All of these methods can be carried out with the single molecule sensitive confocal fluorescence microscope MicroTime 200 and are based on time-correlated single photon counting (TCSPC). We developed a generalized approach to store the individual photon arrival time information with ps accuracy on a timescale up to hours which allows to study all mentioned phenomena in a single measurement (Full Correlation Analysis). Using the new HydraHarp 400 TCSPC unit we can now acquire photon information in 4 completely independent detection channels. This paper present the straightforward experimental concept as well as typical results and recent application examples.

Date Published: 24 February 2009
PDF: 8 pages
Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 71850Q (24 February 2009); doi: 10.1117/12.807803

Published in SPIE Proceedings Vol. 7185:
Single Molecule Spectroscopy and Imaging II
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)