Genetically‐Encoded ATP Sensors for Fluorescence Lifetime Measurements

Fluorescent protein‐based genetically‐encoded sensors of adenosine triphosphate (ATP) have proven valuable for the detection and imaging of both intracellular and extracellular ATP. Current ATP sensors employ either a Förster‐type resonance energy transfer (FRET) fluorescent protein pair or circularly‐permuted fluorescent proteins to report changes in ATP levels. Steady‐state fluorescence spectroscopy and imaging has been the predominant mode of measurement, but both single‐channel intensity and two‐channel ratiometric signals are subject measurements artifacts from a variety of confounding factors such as variance in sensor expression levels and wavelength‐dependent scattering. Recently, we demonstrated that time‐resolved measurements of fluorescence lifetime offer a robust and quantitative alternative mode of detection that relies only on an intrinsic photophysical parameter. Here, we characterize the fluorescence lifetime responses of a panel of the currently available ATP sensors that span different protein architectures. We find that ATP sensors can provide large fluorescence lifetime dynamic ranges, with some sensors exhibiting nearly 1 ns changes in response to ATP. Thus, our study demonstrates that time‐resolved measurements expand the application space of genetically‐encoded fluorescent protein ATP sensors and will facilitate multicolor, multiplexed quantitation of live‐cell energy metabolism and purinergic signaling dynamics.