Assessing the real-time metabolism and bioenergetics of single cells using fluorescence biosensors

Abstract Cellular metabolism plays a crucial role in all living organisms as almost every cellular process requires the presence of adenosine triphosphate (ATP) not only as primary energy currency but also as signaling molecule. Given the importance, monitoring cellular bioenergetics will bring about a more complete understanding of a large variety of biologically important reactions in both physiological and pathophysiological situations. Although many advances have been made in cell-based assays, fluorescence probes show an important advantage by enabling high-resolution visualization of spatiotemporal dynamics of intracellular nucleotides in real time of individual intact live cells, in particular using genetically encoded fluorophores and their variants. By targeting biosensors, any organelle or subcellular compartment of interest can be examined; thus, further elucidating compartmentalized cellular metabolism and explaining more fully how such compartmentation of ATP may influence some pivotal biological events. Here, we introduce and discuss the development and use of fluorescence biosensors for measuring cellular energetics.