ATP is the most universal and essential energy molecule in cells. This is due to its ability to store cellular energy in form of high energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, signaling, and for the turnover of biomolecules. Un-ACCEPTED MANUSCRIPT -CLEAN COPY 2 derstanding how ATP is produced and hydrolyzed with a spatiotemporal resolution is necessary to understand its functions both in physiological and pathological contexts. In this review, we will first describe the organization of the electron transport chain and ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths and weaknesses. Finally, we will explore the palette of geneticallyencoded biosensors designed for microscopy-based approaches, and show how their spatiotemporal resolution opened up the possibility to follow ATP levels in living cells.

ATP is the most universal and essential energy molecule in the eukaryotic cell. This is due to its ability to store energy in form of high energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, signalling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolysed with a spatiotemporal resolution is necessary to understand its functions both in physiological and pathological contexts. In this review, we will first describe the ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths and weaknesses. Then, we will explore the palette of genetically-encoded biosensors designed for microscope-based approaches and show how their spatiotemporal resolution opened up the possibility to follow ATP levels and production in living cells. Finally, we will comment on how ATP monitoring is used in preclinical practices, and to what extent genetically-encoded sensors could be used as a promising tool to elucidate pathologies in which ATP is implicated.