Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM systems have been implemented both in the frequency domain, using sinusoidally intensity-modulated excitation light and modulated detectors, and in the time domain, using pulsed excitation sources and time-correlated or time-gated detection. In this review we describe the different modes in which both frequency-domain and time-domain FLIM instruments have been constructed in wide-field and in point-scanning (confocal) microscopes. Also, novel additional strategies for constructing FLIM-instruments are discussed. In addition to technical implementation, this chapter gives an overview of the application of FLIM in cell biological en biomedical studies. Especially for in situ protein-protein interaction studies using fluorescence resonance energy transfer (FRET), FLIM has proven to be a robust and established technique in modern cell biology. Other application areas, including usage of lifetime contrast for ion-imaging, quantitative imaging, tissue characterization and medical applications, are discussed.