<p>Optical imaging of tumor biomarkers provides key diagnostic information about tumor status. Light-induced afterglow (photoafterglow) imaging provides a higher signal to background ratio than typical fluorescence imaging; however, both modalities face challenges in detecting biomarkers in deep tissues owing to the limited penetration depth of light. Here we provide instructions for synthesizing ultrasound-induced afterglow (sonoafterglow) nanoprobes (SNAP) for the deep-tissue imaging of peroxynitrite (ONOO⁻), a biomarker specific for M1 macrophages and a proinflammatory tumor microenvironment. SNAPs are coassembled from initiators, afterglow substrates and amphiphilic polymers via the film rehydration method, a generic and facile approach that enables their rapid nanoconstruction (in 10 min), with high reproducibility, while also providing control over the nanoprobe concentration, which overcomes limitations of traditional nanoconstruction methods including solvent injection and emulsion–solvent evaporation. Following ultrasound stimulation, SNAPs emit sonoafterglow with bright near-infrared emission (peaking at 780 nm), with a long half-life (~2 min), and can be detected through biological tissues twice deeper than photoafterglow. We further develop SNAP into SNAP-M, which can be switched on only in the presence of ONOO⁻, allowing the real-time in vivo imaging of a proinflammatory tumor microenvironment at an unprecedented tissue depth for optical imaging. This Protocol can be implemented by users with expertise in material science in 1 week for nanoprobe construction and characterization, 1–2 week for cell assays and 3–4 weeks for animal experiments.</p>