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Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM) can provide high resolution and high imaging frame during mature FLIM methods. An abstract time-gated FLIM model was given, and important temporal parameters are shown as well. Aiming at different applications of steady and transient fluorescence processes, two different operation modes, timing and lifetime computing algorithm are designed. High resolution and high frame can be achieved by one-excitation one-sampling mode and least square algorithm for steady imaging applications. Correspondingly, one-excitation two-sampling mode and rapid lifetime determination algorithm contribute to transient fluorescence situations.

Timing and Operating Mode Design for Time-Gated Fluorescence Lifetime Imaging Microscopy