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Fluorescence lifetime imaging in cell biology

The measurement of fluorescence lifetimes offers the advantages of being independent of local intensity and concentration of the fluorophore, and can provide information regarding the molecular environment in a single living cell. Historically, measurements of fluorescence lifetimes have employed photomultipliers as detectors, providing high sensitivity but sacrificing spatial information. Fluorescence Lifetime Imaging Microscopy (FLIM) provides a 2- or 3D spatial map of the distribution of fluorescent lifetime(s) in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to fluorophore containing living cells on the stage of a fluorescent microscope, and images of the fluorescence emission at various times during the decay of the fluorescence lifetime are collected using a high speed nanosecond-gated multichannel plate image intensifier. FLIM promises to substantially enhance the information obtainable from living cells and tissues, and will allow observations of the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques.