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The design and implementation of a time‐resolved fluorescence lifetime imaging microscope (TRFLIM) for the biomedical sciences are described. The measurement of fluorescence lifetimes offers many benefits, among which is that they are independent of local signal intensity and concentration of the fluorophore and they provide visualization of the molecular environment in a single living cell. Unlike single photon counting, which employs a photomultiplier as the detector, TRFLIM uses a nanosecond‐gated multichannel plate image intensifier providing a two‐dimensional map of the spatial distribution of fluorescent lifetime in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to the fluorophore inside living cells on the stage of a fluorescent microscope. Images of the fluorescence emission at various times during the decay of the fluorescence are collected using a high‐speed gated image intensifier and the lifetimes are calculated on a pixel‐by‐pixel basis. Lifetimes measured by TRFLIM are compared with those measured by conventional methods. © 1996 American Institute of Physics.