time-correlated single photon counting

When studying the kinetics of fluorescence emission, we are interested in a number of things. One is how quickly the fluorescence decays after excitation (the rate constant, or its inverse, the lifetime). This is dependant on the probability of the transition from the excited state to the ground state. If the probability is low, the molecule in question will, on the average, stay in the excited state a long time, resulting in a long lifetime. A second parameter is the number of lifetime parameters needed to accurately describe the decay. If you are using a discrete exponential model, a homogenious system should have a monoexponential decay. If there are multiple states in the system, then you may be able to resolve separate decay kinetics for each using a non-linear least squares algorithm to fit the decay curve. If multiple processes are occuring, then the third parameter one wants to determine is the relative contribution of each process (the pre-exponential factor).

Time-correlated single photon counting is an experimental technique used to collect the data required to obtain the above parameters. The light source is usually a laser, tuned to the absorption band of the molecule being studied. A short (picosecond duration, sometimes shorter) pulse of light hits the sample, driving molecules into the excited state. At the time of excitation, a reference signal is sent to a computer, so that it knows when the pulse started. Then, a photomultiplier (photon detector) senses how long it takes for a photon to be emitted after excitation. This time is also reported to the computer. The computer then subtracts the time of the reference signal from the time of the emitted photon, thereby determining the lifetime of the excited state for that single event. This process is repeated thousands to millions of times, in order to build a good statistical data set of decay kinetics. The final decay curve is just a histogram of single decay times.

The advantages of this technique are the extreme sensitivity and accuracy of timing one can obtain. It is important that only one photon reaches the detector after each excitation event, otherwise one may see artifacts from photon pileup, where the photomultiplier recieves two or more photons, but only counts them as one event. This ends up making the lifetime seem shorter than it really is.

Once one has acceptable estimates for the various parameters, one can then interpret the decay kinetics in the context of the system being studied. Proteins which show multiple decay components, may exist in several distinct conformations, each one with its own decay rate. This allows you to see heterogeneity in the sample which could not otherwise be detected by steady state methods. Furthermore, because the lifetime of fluorescent probes are often in the nanosecond range, their emission kinetics can be used to monitor very fast motions in proteins.