eeFILM – Extended Excitation for Faster Measurement of Fluorescent Lifetimes

The estimation of excitation lifetimes based on fluorescence or phosphorescence emission is an essential technique in basic and applied science. It provides information on molecular interactions
as well as on physical properties. Measuring the emission lifetime with spatial resolution provides an imaging modality, which in case of fluorescence is called Fluorescence Lifetime Imaging (FLIM). FLIM is applied extensively in biological imaging in order to determine the conformation, activation, interactions and redistributions of key molecules involved in signal transduction. In biotechnology, fluorescence decay measurements are used for high-throughput screening (e. g. via binding assays) of prospective diagnostic or therapeutic molecules with designated targets.
The experimental difficulty in conventional time-domain measurements, in particular FLIM, is the need for short excitation light pulses (duration less to or comparable to the decay time), which deliver low energy, and the superposition of exponential emissive decay functions. Thus, data analysis of the detector response function for deriving the decay time requires deconvolution. Here, complex mathematical procedures based on iterative minimization are involved, requiring considerable computation time – even with current computers extending to minutes. Most FLIM applications are based on the mean decay lifetime computed from the individual decay times and their corresponding amplitudes. Deriving a mean value still requires a complete decay analysis in order to obtain the number of components and their parameters. This limitation applies to all current FLIM or single channel lifetime techniques.