Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems. Biosensors often report relative rather than absolute values. Here the authors report a method that utilises the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity: photochromism-enabled absolute quantification (PEAQ) biosensing.

Automated summary

The authors present a method, called photochromism-enabled absolute quantification (PEAQ) biosensing, that utilizes the photochromic properties of biosensors to provide an absolute measure of analyte concentration or activity. By developing photochromic variants of the popular GCaMP family of Ca2+ biosensors, they are able to resolve dynamic changes in the absolute Ca2+ concentration in live cells. They also demonstrate how this method can be applied to other biosensors with photochromic properties, expanding the possibilities for fully quantitative measurements in complex and dynamic systems.