Principle of TCSPC FLIM
When TCSPC is combined with a scanning technique fluorescence lifetime imaging (FLIM) can be performed. FLIM by multi-dimensional TCSPC was introduced by Becker & Hickl (bh) in 1996. The speed of FLIM with scanning often exceeds the users requirements and expectations. For applications with the need of extreme short acquisition times we developed a fast FLIM system. We call it FASTAC FLIM.
In more detail, the TCSPC FLIM technique is based on scanning the sample by a high-repetition rate pulsed laser beam and detecting single photons of the fluorescence signal returned from the sample. Each photon is characterized by its time in the laser pulse period and the coordinates of the laser spot in the scanning area in the moment of its detection. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve in a large number of time channels.
TCSPC FLIM delivers a near-ideal photon efficiency, excellent time resolution, and is independent of the speed of the scanner. The signal-to-noise ratio depends only on the total acquisition time and the photon rate available from the sample.
The technique can be extended by including additional parameters in the photon distribution. These can be the depth of the focus in the sample, the wavelength of the photons, the time after a stimulation of the sample, or the time within the period of an additional modulation of the laser. These techniques are used to record Z stacks or mosaics of FLIM images, multi-wavelength FLIM images, images of physiological effects occurring in the sample, or to record simultaneously fluorescence and phosphorescence lifetime images.
As an option, e.g. for thin specimen, also a widefield TCSPC camera can be employed.