Highly sensitive and specific non-invasive molecular imaging methods are particularly desirable for the early detection of cancers. Here we report a near-infrared optical imaging probe highly specific to the hypoxic tumour microenvironment to detect tumour and cancer cells with the sensitivity to a few thousands cancer cells. This oxygen-sensitive, near-infrared emitting and water-soluble phosphorescent macromolecular probe can not only report the hypoxic tumour environment of various cancer models, including metastatic tumours in vivo, but can also detect a small amount of cancer cells before the formation of the tumour based on the increased oxygen consumption during cancer cell proliferation. Thus, the reported hypoxia-sensitive probe may offer an imaging tool for characterizing the tumour microenvironment in vivo, detecting cancer cells at a very early stage of tumour development and lymph node metastasis.

Afterglow luminescent probes with high signal-to-background ratio show promise for in vivo imaging; however, such probes that can be selectively delivered into target sites and switch on afterglow luminescence remain limited. We optimize an organic electrochromic material and integrate it into near-infrared (NIR) photosensitizer (silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) and (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) containing nanoparticles, developing an H₂S-activatable NIR afterglow probe (F1²⁺-ANP). F1²⁺-ANP displays a fast reaction rate (1563 ± 141 M⁻¹ s⁻¹) and large afterglow turn-on ratio (~122-fold) toward H₂S, enabling high-sensitivity and -specificity measurement of H₂S concentration in bloods from healthy persons, hepatic or colorectal cancer patients. We further construct a hepatic-tumor-targeting and H₂S-activatable afterglow probe (F1²⁺-ANP-Gal) for noninvasive, real-time imaging of tiny subcutaneous HepG2 tumors (<3 mm in diameter) and orthotopic liver tumors in mice. Strikingly, F1²⁺-ANP-Gal accurately delineates tumor margins in excised hepatic cancer specimens, which may facilitate intraoperative guidance of hepatic cancer surgery.