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Time correlated single photon counting

Cuvet based TCSPC is used for time-resolved fluorescence measurements. It can quantify the amount of FRET, or resolve the ultrafast events in photosynthesis. It covers the range from tryptophan (UV) to chlorophyll (NIR).

The fluorescence of a sample can be monitored as a function of time after excitation by a flash of light. We use the Time Correlated Single Photon Counting (TCSPC) method to record the fluorescence time trace. Processes in the excited state(s) of fluorophores can be followed: e.g. energy transfer, solvent relaxation, etc. The rates of these processes can be determined from the lifetimes of the fluorescent intermediate and final states. Using our TCSPC set-up, lifetimes between 20 ps up to 20 ns can be measured, and it covers the wavelength range from tryptophan (UV) to chlorophyll (NIR).

Furthermore, the set-up is equipped with polarizers for time-resolved fluorescence anisotropy measurements. During the fluorescence lifetime, the changes in the orientation of the molecules can be probed. Time-resolved fluorescence anisotropy is used to obtain information about molecular rotation and homo-FRET between proteins. The rotational dynamics are determined by e.g. the shape of the protein and its aggregation state, or the viscosity of the medium surrounding the fluorescent molecule.

Typical applications: photosynthesis research, solvent environment probing, protein conformation and inter/intra-molecular distance measurements (FRET).


After absorption of light, a molecule is in one of its excited states. It can now undergo vibrational relaxation, internal conversion, solvent relaxation, energy transfer and/or fluorescence. All processes have their own rate (in s-1), and consequently all intermediate states have the own lifetime. When such an intermediate state can fluoresce, the lifetime of that state can measured. By recording the time-resolved fluorescence decay at the emission wavelength of these states (for example the blue, green and red in the figure), the lifetime of that state can be determined. The rate of the population of the state (ingrowth) and depopulation (decay) can be calculated from the traces.


The fluorescence decay is measured by time-correlated single photon counting (TCSPC). In TCSPC, the time differences between the detection of the individual photons (fluorescence) and the subsequent next laser pulses are recorded. All these time differences are plotted in a histogram. The number of detected counts per second is kept much lower than the number of excitation pulses (1:100). A second photon in the same cycle cannot be processed and neglecting that photon will lead to (pile-up) distortion. 


For excitation a Ti:sapphire laser is used in combination with a pulse picker and frequency converters (OPO or harmonic generator) to create 4 MHz repetition rate pulses with variable wavelength. For detection, a multichannel plate photomultiplier tube (MCP-PMT) is used.

Excitation source.png

Key specifications:

Available excitation wavelengths: 250-665 nm

Available interference / cut-of filters: ranging from 300 to 850 nm

Repetition rate: 4 MHz

Instrument response: 45 ps FWHM

Can measure lifetime from 20 ps up to 20 ns


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