The study of cellular metabolism is undergoing a renaissance. Although long considered by many biologists to be a set of rather boring and well-understood "housekeeping" processes that simply set the stage for the more interesting things that cells do, it is now appreciated that cellular metabolism is dynamically regulated and fundamentally integrated into most cell signaling pathways.

Another important realization is that location matters. The cell is not just a bag full of enzymes: even the apparently amorphous cytoplasm has many multi-protein complexes that organize and localize the function of both metabolic enzymes and signal transducers.

To learn more about the spatial and temporal organization of cell metabolism, we are developing a series of fluorescent biosensors to report on key cellular metabolites. We have developed Perceval, a reporter of the ATP:ADP ratio; Peredox for NADH:NAD⁺ ratio (redox cofactors that play a central role in cellular metabolism); a red pH sensor, pHRed; a lactate sensor, LiLac; and a lifetime-readout sensor iGlucoSnFR-TS.

These new fluorescent biosensors will allow us and others to investigate the dynamic changes in metabolism that occur in living cells in response to stimulation (biochemical or electrical) and to changes in fuel source. They will also permit the exploration of metabolic compartmentation:

• between cells (e.g. between adjacent neurons and astrocytes in the brain)
• between parts of a cell (e.g. between dendrite and soma, or locally within a dendrite)
• between microscopic subcellular compartments (e.g. between submembrane space and cytoplasm)

We image the sensors in brain slices, using two-photon microscopy and fast FLIM (fluorescence lifetime microscopy), to get a dynamic, quantitative picture of metabolism in resting and stimulated brain cells.