Unlocking chloride sensing in the red at physiological pH with a fluorescent rhodopsin-based host.

Chloride is a vital ion for all forms of life. Protein-based fluorescent biosensors can enable researchers to visualize chloride in cells but remain underdeveloped. Here, we demonstrate how a single point mutation in an engineered microbial rhodopsin results in ChloRED-1-CFP. This membrane-bound host is a far-red emitting, ratiometric sensor that provides a reversible readout of chloride in live bacteria at physiological pH, setting the stage to investigate the roles of chloride in diverse biological contexts.