Functional principles of genetically encoded fluorescent biosensors for metabolism and their quantitative use

Genetically encoded fluorescent biosensors provide an attractive means of measuring chemical changes in single cells on fast timescales (milliseconds to seconds). While their most prominent application has been in tracking neural activity and neurotransmitter release, there has been growing interest in developing and deploying new versions of these tools to study brain metabolism. However, the careful use of these tools and the interpretation of the data they provide remain challenging. Many biosensors are subject to interferences that can alter sensor responses within a single cell or between cells, producing ambiguous results. This presents a challenge for quantitation and for our ability to accurately interpret sensor responses. This review describes current methods of sensor quantitation, with a focus on cellular interferences that commonly affect sensor performance, ways to avoid false inferences, and recent advances in sensor optimization to make them more robust.