Fluorescence Correlaton Spectroscopy (FCS) down to picosecond lagtimes

While conventional hardware correlators are designed for a few nanoseconds resolution, the PicoHarp 300 in T2 mode makes it possible to perform FCS with a resolution of 4 ps ("total correlation").

The following example measurement was perfomed using a Zeiss LSM 510 Meta with custom FCS Detection unit (see Ries, J. & Schwille, P. Biophys J, 2006, 91, 1915-1924). Light was collected through a 40x-C-Apochromat UV-VIS-IR objective by two Perkin Elmer SPAD modules SPCM-CD 3017. Using two detectors with cross correlation in T2 mode eliminates dead time as well as afterpulsing. The excitation laser power was approximately 80µW at 488 nm and the measurement time was 5 min.

The sample was a solution of Alexa 488 in water with a concentration on the order of 30 nM. The solution was kept in a closed chamber preventing evaporation. The resulting correlation curve shows nicely the antibunching dip as well as the classical diffusion part. Note that the noisiness at the high resolution side is purely statistical, due to the low probability of photon coincidences in picosecond time bins. Better statistics can be obtained by longer measurement times.

Data collection: courtesy of J. Ries, Institut für Biophysik, TU Dresden, Tatzberg 47-51, 01307 Dresden.

Data processing: SymPhoTime Software v4.2, PicoQuant 2006.