Bioorthogonally Activatable Chemiluminescence for the <i>N</i> ‐Methyl‐ <scp>d</scp> ‐aspartate Receptors Intravital Imaging

The signal attenuation caused by skull/vertebrae remains a challenge in central nervous system (CNS) receptor imaging. Chemiluminescence (CL), free from external excitation, offers unparalleled tissue penetration in optical imaging. However, existing 1,2-dioxetane CL systems are shackled by two limitations: (i) short half-lives (<2 h) from rapid dioxetane decomposition and (ii) dependence on reactive biomolecules such as reactive oxygen species and enzymes to trigger dioxetane decomposition, rendering them incompatible with imaging nonreactive biomolecules like receptor proteins. Here we report a bioorthogonally activatable chemiluminescence (BACL) strategy that integrates click-to-release reactions with 1,2-dioxetane luminophores to enable tetrazine-triggered OFF-ON CL signals and bioorthogonally tunable half-lives (5.2-18 h). The tissue penetration depth was up to 6 cm. Through a tetrazine-conjugated specific ligand, BACL imaged N-methyl-d-aspartate receptors (NMDARs) in vivo with a signal background ratio of ∼182, allowing clear differentiation of NMDAR expression levels between Alzheimer's disease model mice and normal controls. Beyond imaging, the bioorthogonally spatiotemporally controlled CL emission positions BACL as a potential internal light source for deep-tissue precision phototherapeutics, bypassing external irradiation.