Fig. 2
The schematic diagram of the principle of time-gated detection for FLIM. After exciting an ultrashort laser pulse, a set of fluorescence intensity images are obtained by strobing the image intensifier or CCD camera at different times (time window). When conditions permit, multi-gated detection is usually adopted, that is, multiple time windows (t1, t2, t3,…,tn) are selected to obtain multiple intensity images (usually 5 to 10). Using the formula (1), the fluorescence lifetime of each point on the sample is calculated point by point to capture the fluorescence image. The excitation light pulse is in dark blue, with the fluorescence emission in light blue. Currently, most of the devices available are capable of a minimum time gate of 3–5 ns, much faster devices, exhibiting a minimum time gate down to 80–100 ps, have been developed recently. Typical fluorescence decay times of organic compounds fall between a few hundreds of picoseconds and several nanoseconds [34]. Δt: gate width