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Methods Enzymol. 2014;547:355-71. doi: 10.1016/B978-0-12-801415-8.00017-5.

Imaging changes in the cytosolic ATP-to-ADP ratio.

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Department of Chemistry, Purdue University, West Lafayette, Indiana, USA.
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. Electronic address:


Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.


ADP; ATP; Energy; Fluorescence; Genetically encoded; Imaging; Metabolism; Microscopy; Ratiometric; Sensor

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