Fluorescent reporters have emerged as valuable tools for visualizing neurotransmitterswith exceptional spatial and temporal precision. In this study, we introduce a strategy that
harnesses thermophilic protein sequences to identify stabilizing mutations, complemented by an
efficient unfolding screening assay to evaluate the stability of genetically encoded fluorescent
reporters. Our findings include three novel variants of the intensity-based GABA-sensing
fluorescent reporter (iGABASnFR) with improved brightness and physiological sensitivity in
vitro, while also demonstrating potential for in vivo applications.