The spatio-temporal localisation of molecular interactions within cells in situ and in particular in vivo is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localisation of protein complexes may be achieved by the detection of fluorescence (or Förster) resonance energy transfer (FRET) between protein-conjugated fluorophores. The use of fluorescence lifetime imaging microscopy (FLIM) to determine both intra- and intermolecular FRET has become an important and powerful technique for investigating biological systems. In this review we focus on the time-domain-based fluorescence lifetime sensing, using time-correlated single photon counting multiphoton microscopy. We give a description of the technique, including data acquisition and analysis, and discuss the different biological applications of this technology.