Fluorescence Lifetime Imaging (FLIM)

Fluorescence Lifetime Imaging (FLIM)

The fluorescence decay time of fluorescent molecules can be exploited for enhancing the contrast and the selectivity of fluorescence microscopy. The differences in the fluorescence decay behavior can often be associated with changes in the chemical environment of the fluorophore such as pH, ion concentration and binding. In addition the emission of specific fluorophores can be contrasted against an auto-fluorescence background arising from the same detected microscopic volume element. Moreover, fluorescence lifetime contrast imaging provides a discrimination of molecules with overlapping fluorescence emission bands, but with different fluorescence decays, in multi-labelling experiments. We have implemented fluorescence lifetime imaging using a pulsed laser in combination with time-gated techniques, see figure. The advantage of this simple approach is that reasonably fast image acquisition times can be achieved and changes in the fluorophore distribution on the timescale of seconds can be imaged. We measure the intensity decay of the fluorescence following excitation with a 70 femtosecond laser pulse, originating from a Titanium Sapphire laser. The fluorescence is collected in an 8 channel time gating module which can be set to a time width from 0.5 to 9000 nanoseconds.