摘要
High-contrast optical imagi ng is achievable using phosphoresce nt labels to suppress the short-lived background due to the optical backscatterand autofluoresce nee.However,the long-lived phosphorescence is generally incompatible with high-speed laser-scan ning imaging modalities.Here,we show that upc on versi on nan oparticles of structure NaYF4:Yb co-doped with 8%Tm(8T-UCNP)in combi nation with a commerciallaser-scanning multiphoton microscopy are uniquely suited for labeling biological systems to acquire high-resolution images with the enhancedcon trast.In comparison with many phosphoresce nt labels,the 8T-UCNP emission lifetime of-15μs affords rapid image acquisition.Thehigh-order optical nonlinearity of the 8T-UCNP(n=4,as confirmed experimentally and theoretically)afforded pushing the resolution limitattain able with UCNPs to the diffraction-limit.The contrast enha nceme nt was achieved by suppressing the backgro und using(i)ban dpassspectral 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 polyethyle neimine-coated 8T-UCNPs take n up by huma nbreast adeno carcinoma cells SK-BR-3.As a result,discrete 8T-UCNP nan oparticles became clearly observable in the freshly excised splee ntissue of laboratory mice 15-min post in trave nous injectio n of an 8T-UCNP solution.The dem on strated approach paves the way forhigh-contrast,high-resoluti on,and high-speed multiphot on microscopy in challe nging envir onments of i ntense autofluorescence,exogenous staining,and turbidity,as typically occur in intravital imaging.
