High-contrast optical imaging is achievable using phosphorescent labels to suppress the short-lived background due to the optical backscatter and autofluorescance.However,the long-lived phosphorescence is generally incompatible with high-speed laser-scanning imaging modalities.Here,we show that upconversion nanoparticles of structure NaYF4∶Yb co-doped with 8％ Tm (8T-UCNP) in combination with a commercial lassr-scanning multiphoton microscopy are uniquely suited for labeling biological systems to acquire high-resolution images with the enhanced contrast.In comparison with many phosphorescent labels,the 8T-UCNP emission lifetime of ～ 15 μs affords rapid image acquisition.The high-order optical nonlinearity of the 8T-UCNP (n ≈ 4,as confirmed experimentally and theoretically) afforded pushing the resolution limit attainable with UCNPs to the diffraction-limit.The contrast enhancement was achieved by suppressing the background using (i) bandpass spectral filtering of the narrow emission peak of 8T-UCNP at 455-nm,and (ii) time-gating implemented with a time-correlated single-photon counting system that demonstrated the contrast enhancement of ＞ 2.5-fold of polyethyleneimine-coated 8T-UCNPs taken up by human breast adenocarcinoma cells SK-BR-3.As a result,discrete 8T-UCNP nanoparticles became clearly observable in the freshly excised spleen tissue of laboratory mice 15-min post intravenous injection of an 8T-UCNP solution.The demonstrated approach paves the way for high-contrast,high-resolution,and high-speed multiphoton microscopy in challenging environments of intense autofluorescence,exogenous staining,and turbidity,as typically occur in intravital imaging.