Organic afterglow probes: mechanisms, chemical design, performance optimization, and biomedical applications

Afterglow imaging shows distinct advantages for bioimaging, such as significantly reduced background signals, an enhanced signal-to-background ratio, and excitation-free in situ detection. To achieve real-time molecular visualization and improve diagnostic specificity, various activatable afterglow probes have been developed that selectively respond to biological targets or microenvironmental stimuli. This Review systematically summarizes organic activatable afterglow probes. We introduce their main material categories and luminescence mechanisms, and analyze chemical design strategies for constructing responsive afterglow probes, including target-cleavable linkers, conformational switching, and energy transfer regulation. We also discuss strategies to optimize imaging performance, such as extending emission lifetime, red-shifting emission to tissue-transparent windows, improving luminescence brightness, and using alternative excitation modes (e.g., X-ray or ultrasound) to overcome optical penetration limitations. Representative applications of activatable afterglow nanoprobes are highlighted, covering analyte sensing, lymph node mapping, immune cell imaging, tumor detection, inflammation imaging, and image-guided therapy. Finally, we address current challenges including oxygen dependence, biocompatibility, unified evaluation standards, and scalability, and propose future directions to promote the translation of afterglow probes in precision biomedicine. This review is expected to provide practical references for the rational design and comparative evaluation of such probes.