Abstract
Luminescence-based imaging is a fundamental tool for visualizing dynamic biological processes, primarily through bioluminescence, which generates light via enzyme-substrate reactions, and fluorescence, which requires external excitation. The recent emergence of self-sustaining systems like the fungal bioluminescence pathway (FBP) necessitates a renewed comparative analysis of these tracing technologies. Conventional tools for biological tracing, luciferases and fluorescent proteins, are limited by their dependence on external substrates or light excitation. This review systematically contrasts these methods with the self-sustaining FBP, an intrinsic metabolic circuit that enables substrate-free, autonomous bioluminescence. We conclude that integrating FBP with AI-driven protein design is the pivotal next step toward non-invasive, real-time monitoring in complex organisms, positioning FBP as a paradigm-shifting tool for future tracing technologies.