The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio-probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (Re^I) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, Re^I-diimine complex already exhibits excellent emission yield (34%, λ_em = 583 nm) and large two-photon absorption cross-sections (σ₂ = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen-bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post-extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial-empowered phosphorescence sensing and imaging.