An Enhanced Red Bioluminescent Indicator for Responsive Detection of Physiological Calcium Dynamics in Cells and Mice.

Calcium (Ca) is a crucial metal ion and signaling messenger. While bioluminescent indicators for Ca have emerged as powerful imaging tools, their performance has been suboptimal. In this study, we developed an nhanced ioluminescent red ndicator for a (eBRIC) by using a physiological Ca concentration range during library screening. Compared with its predecessors, this new sensor demonstrates substantially improved Ca responsiveness in protein-based assays, cultured cell lines, and primary neurons. We further demonstrated the utility of eBRIC for the in vivo recording of Ca dynamics in the brains of live mice, using both a microscope setup and a luminescent imaging dark box. Notably, by combining eBRIC with our recently developed water-soluble luciferin, we achieved minimally invasive, video-rate imaging of Ca activity in a defined brain region of awake mice. In a footshock-induced basolateral amygdala activation paradigm, eBRIC elicited approximately double the response compared to the previous BRIC indicator. The improved responsiveness offered by eBRIC underscores its potential as a powerful tool for investigating Ca dynamics in living systems.