While fluorescence intensity informs us about the relative abundance of the light emitting objects, fluorescence lifetime reveals information about the immediate biochemical environment (e.g. oxidation / reduction, pH), molecular interactions (via FRET, binding or release of small molecules), and intra-molecular changes. By harnessing the power of quantitative analysis for complex fluorescence lifetime image and spectroscopic data, we can characterize functional fluorescent molecules or fluorescent protein based biosensors to understand the in vivo biochemistry of normal tissue homeostasis, disease mechanisms, and drug effects in the tissue.