Lophine, the first chemiluminescent compound discovered in history, has rarely been applied for in vivo imaging since its discovery in 1877. In this report, we demonstrate that lophine's chemiluminescence emission could be significantly enhanced by caging the imidazole moiety via molecular afterglow mechanism. Notably, our study revealed a rare superoxide anion-mediated luminescence. Our novel probes JIMI-11 and JIMI-12 could be used for in vivo mouse imaging. Compared to its uncaged form JIMI-6, JIMI-11 exhibited a significant enhancement (126-fold) in vitro and a 190-fold higher emission signal in vivo. JIMI-11 selectively accumulates in white adipose tissues (WAT) and can be used to monitor changes in WAT mass in a mouse model of type-1 diabetes. Additionally, it can assess the therapeutic effects of Semaglutide in a mouse model of diet-induced obesity. Lastly, we designed JIMI-12 with a reactive oxygen/nitrogen species (ROS/RNS) responsive moiety as the caging group and demonstrated its utility for in vivo imaging of ROS in a lipopolysaccharide (LPS)-induced inflammatory mouse model. Our studies suggest that re-designing lophine-based probes could unlock their potential for both in vitro and in vivo applications. The ability to switch from chemiluminescence to molecular afterglow introduces a novel approach to designing imaging probes.