Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes
Quantitative fluorescence lifetime imaging microscopy (FLIM) is limited by a lack of probes that combine single-residue specificity, minimal steric perturbation, visible-light excitation, and strong environment-dependent lifetime contrast. We introduce benzodiazole-based non-canonical amino acids (BD-ncAAs) as compact, genetically encoded sensors for site-specific FLIM. Using an engineered pyrrolysyl-tRNA synthetase pair, we incorporate three different BD-ncAAs into proteins in living cells. Site-specific incorporation of BD-ncAAs into a nanobody at different positions preserved native structure and antigen binding. The lead probe, cyano-substituted CBDS, exhibits visible-light excitation, large Stokes shifts, and pronounced environment-dependent fluorescence lifetimes spanning 1 to 24 ns. Under wash-free, live-cell FLIM conditions, CBDS quantitatively resolves localized structural microenvironments and transmembrane hydration gradients, while also distinguishing distinct states during protein secretion in eukaryotes. BD-ncAAs present genetically encoded, minimally perturbing reporters for tracking target proteins, detecting interactions with binding partners, and quantifying site-specific solvent accessibility in living cells by quantitative FLIM.