This second article in the series shows how fluorescence lifetime imaging allows natural biochemical and physiological properties of tissues to act as contrast agents and so provide a basis for distinguishing normal and diseased tissue components. When combined with methods for imaging through non-transparent tissues and tomographic reconstruction it shows promise as a new optical biopsy technique. In addition to this, specially designed vital fluorescent probes of specific biochemical, secondary messenger and receptor activity in living cells may be imaged using FLIM. This is the youngest of the techniques covered in these review articles on imaging, the first FLIM images of cells having been produced in 1994.