Chemiluminescence microscopy single cell imaging using esterase triggered 1,2-dioxetanes

Chemiluminescence microscopy using emerging 1,2-dioxetane chemiluminescent probes is an attractive prospect for low background biological molecular imaging. Elimination of an external light source reduces autofluorescence, light scattering, and complications from fluorophore photobleaching, providing enticing advantages compared to fluorescence imaging. While bioluminescence microscopy with genetically modified luciferase expressing organisms has been successful, examples of chemiluminescence microscopy using 1,2-dioxetanes are limited due to challenges with cell uptake, signal brightness, and microscopy apparatus that are not optimized for these molecules. To address this gap, we herein describe a home-built chemiluminescence microscope capable of imaging nanomolar concentrations of luminescent 1,2-dioxetanes. Using acetoxymethyl ester functionalized 1,2-dioxetanes, 2 and 3, which display uptake and retention into unmodified A549 cells for imaging intracellular esterase activity, we characterize the luminescence imaging of A549 human lung epithelial cells via chemiluminescence microscopy. This study provides a complementary chemiluminescent 1,2-dioxetane and microscopy system, thereby introducing a generalizable framework for imaging a wide variety of clinically relevant samples.