Introduction to Multi-dimensional TCSPC

Abstract

Classic time-correlated single photon counting (TCSPC) detects single photons of a periodic optical signal, determines the times of the photons relative to a reference pulse, and builds up the waveform of the signal from the detection times. The technique achieves extremely high time resolution and near-ideal detection efficiency. The modern implementation of TCSPC is multi-dimensional. For each photon not only the time in the signal period is determined but also other parameters, such as the wavelength of the photons, the time from the start of the experiment, the time after a stimulation of the sample, the time within the period of an additional modulation of the excitation light source, spatial coordinates within an image area, or other parameters which can either vary randomly or are actively be modulated in the external experiment setup. The recording process builds up a photon distribution over these parameters. The result can be interpreted as a (usually large) number of optical waveforms for different combination of the parameters. The advantage of multi-dimensional TCSPC is that the recording process does not suppress any photons, and that it works even when the parameters vary faster than the photon detection rate. Typical multi-dimensional TCSPC implementations are multi-wavelength recording, recording at different excitation wavelengths, time-series recording, combined fluorescence and phosphorescence decay recording, fluorescence lifetime imaging, and combinations of these techniques. Modern TCSPC also delivers parameter-tagged data of the individual photons. These data can be used to build up fluorescence correlation and cross-correlation spectra (FCS and FCCS), to record fluorescence data from single molecules, or to record time-traces of photon bursts originating from single molecules diffusing through a small detection volume. These data are used to derive multi-dimensional histograms of the changes in the fluorescence signature of a single molecules over time or over a large number of different molecules passing the detection volume. The chapter describes the technical principles of the various multi-dimensional TCSPC configurations and gives examples of typical applications.