Full correlation from picoseconds to seconds by time-resolved and time-correlated single photon detection

Abstract

We present an advanced time-correlated single photon counting (TCSPC) technique that delivers traditional fluorescence correlation (FCS) or cross correlation (FCCS) and fluorescence lifetime data simultaneously. Newly developed electronics allow for detection and registration of single photon events over time periods of hours with picoseconds accuracy. Subsequent software-correlation yields correlation curves covering more than 12 orders of magnitude in time. At the same time, the original data, containing all information accessible by single photon counting techniques, can be analyzed conventionally according to common single molecule fluorescence techniques. Potential applications of the new technique using pulsed or cw laser excitation are discussed.

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

“…[22,23] By combining macroscopic times, given by the repetition rate of the laser pulses (with 10-100 ns resolution), with the microscopic arrival time of the fluorescence photon with respect to the laser pulse as measured by TCSPC, an overall time resolution of picoseconds can be achieved even over very long measurement times of hours. [24] Moreover, the information on its color, polarization and the delay between excitation and emission of the fluorescence photon is recorded for each photon. All photons detected from a single molecule burst are analyzed making it possible to determine burst-integrated fluorescence parameters: FRET efficiency from the spectral information, an average fluorescence lifetime from the TCSPC data and anisotropy from the polarization information.…”

Wide-field TCSPC: methods and applications

“…Poisson noise which is given by the square root of the number of counts [34]. It is important to note that due to Poisson statistics, there is always a chance to detect two photons after one excitation pulse-this probability can never be exactly zero, and it is independent of the time scale used, picoseconds or microseconds, and it is also independent of the technological implementation of TCSPC, using a single TAC, multiple TACs, [35], TDCs [36,37] or imaging. It is just a question of how much pile-up can be tolerated, for low peak counts more than for very high peak counts [38].…”

Phosphorescence decay time measurements using intensity correlation spectroscopy

“…(Lakowicz, 1999) The combination with the decay time measurement system is recently developed. (Felekyan et al, 2005) Usually, the decay time measurements require different detection system, such as the time-correlated single photon counting (TCSPC) system and the pulsed or modulated light source. Therefore, the extension of the commercially available FCS system tends to become very complex and expensive.…”