New method for fluorescence lifetime imaging in bilateral-confocal microscopy by double-pulse excitation

A new technique for the measurement of fluorescence lifetimes relies on the (near steady state) excitation with short optical pulses. The novel technique has the potentiality to provide high time resolution - since it relies on the laser pulse duration, rather than on electronic gating techniques - and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. Combined with confocal microscopy it enables the spatial determination of the fluorescence lifetimes, the value of which is influenced by the local environment of fluorescent probe molecules in biological samples. The principle of the technique is discussed within a theoretical framework taking into account various secondary effects.