Chapter 1 Introduction to Multi-dimensional TCSPC

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 multidimensional 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. W. Becker (&) Becker & Hickl GmbH, Berlin, Germany e-mail: becker@becker-hickl.com © Springer International Publishing Switzerland 2015 W. Becker (ed.), Advanced Time-Correlated Single Photon Counting Applications, Springer Series in Chemical Physics 111, DOI 10.1007/978-3-319-14929-5_1 1 1.1 Time-Resolved Optical Detection at Low Light Intensity When light propagates through matter it can interact with the molecules in various ways. In the simplest case, the photons of the incident light may be reflected, absorbed, or scatted. When photons are absorbed the molecules within a sample enter an excited state from which they may return by emitting a photon of longer wavelength. At high intensities several photons may interact with the molecules simultaneously, resulting in nonlinear effects like multiphoton excitation, or second second-harmonic generation. These interactions change the intensity, the spectral properties, and the temporal properties of the light. Light transmitted trough, scattered in, or otherwise emitted from a sample thus carries information on molecular parameters. In other words, optical techniques can be used to probe molecular parameters inside a sample. Often the effects involved in the conversion of the light are extremely weak, or the concentration of the molecules involved in the conversion processes is low. Moreover, the light intensity tolerated by the sample may be limited. As a result, the intensity of light signals to be detected may be very low. The situation is further complicated when signals are recorded at high time resolution. Optical signals have to be considered a stream of photons. At a given photon rate, the signal-to-noise ratio decreases with the square root of the detection bandwidth. A typical situation is shown in Fig. 1.1. An optical signal was detected by a photomultiplier tube at bandwidth of approximately 350 MHz, or a time resolution of 1 ns. The detected photon rate was approximately 10 photons/s. This is much higher than typically obtained in molecular imaging experiments in live sciences. As can be seen from Fig. 1.1, the signal is a random trace of extremely short pulses. The pulses represent the detection of single photons of the light arriving at the detector. Now consider the detection of a fast optical waveform, such as a fluorescence decay excited by short laser pulses. The fluorescence decay time is on the order of a Fig. 1.1 Optical signal detected at a time resolution of 1 ns. Detected photon rate 10 photons/s 2 W. Becker