jats:titleAbstract</jats:title>jats:pImage contrast is often limited by background autofluorescence in steady‐state bioimaging microscopy. Upconversion bioimaging can overcome this by shifting the emission lifetime and wavelength beyond the autofluorescence window. Here we demonstrate the first example of triplet‐triplet annihilation upconversion (TTA‐UC) based lifetime imaging microscopy. A new class of ultra‐small nanoparticle (NP) probes based on TTA‐UC chromophores encapsulated in an organic–inorganic host has been synthesised. The NPs exhibit bright UC emission (400–500 nm) in aerated aqueous media with a UC lifetime of ≈1 μs, excellent colloidal stability and little cytotoxicity. Proof‐of‐concept demonstration of TTA‐UC lifetime imaging using these NPs shows that the long‐lived anti‐Stokes emission is easily discriminable from typical autofluorescence. Moreover, fluctuations in the UC lifetime can be used to map local oxygen diffusion across the subcellular structure. Our TTA‐UC NPs are highly promising stains for lifetime imaging microscopy, affording excellent image contrast and potential for oxygen mapping that is ripe for further exploitation.</jats:p>