IPHT colloquium

Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC Technique and Applications

Dr. Wolfgang Becker

Becker & Hickl GmbH

A fluorescence lifetime imaging (FLIM) technique for biological imaging has to combine high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals, and supression of out-of-focus fluorescence and scattered light. We will show that the combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal laser scanning meets these requirements almost ideally. FLIM by multi-dimensional TCSPC is based on scanning the sample by a high-repetition rate pulsed laser beam and the detection of single photons of the fluorescence signal returning from the sample. In the simplest case, each photon is characterised by its time in the laser pulse period and the coordinates of the laser spot in the scanning area in the moment 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 has got a new push from the introduction of 64-bit data acquisition software. As a result, FLIM can be recorded at a spatial resolution in the megapixel range. This allows a large number of cells to be imaged simultaneously under identical experimental conditions and thus obtain exactly comparable FLIM parameters from them. The technique can further be extended by recording photon distribution over additional parameters of the photons. Such parameters can be 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 advanced techniques can be used to record FLIM Z stacks, multi-wavelength FLIM images, images of physiological effects occurring in the sample, and to simultaneously record fluorescence and phosphorescence lifetime images. Potential applications are the combination of metabolic imaging experiments with oxygen concentration measurement, and the measurement of fast changes in the Ca2+ concentration in neurons on electrical stimulation.