We describe the characterisation of a hyperspectral fluorescence lifetime imaging microscope that exploits high-speed time-gated imaging technology and a tunable continuum source for 6-D fluorescence imaging. This line-scanning confocal microscope can record the full spectral-temporal (i.e. excitation-emission-lifetime) fluorescence matrix at each pixel in a three dimensional (x-y-z) sample. This instrument has been applied to biological samples including model membranes and live cells labelled with the phase-sensitive membrane dye di-4-ANEPPDHQ, for which significant variation of lifetime with emission wavelength is observed.© (2007) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.