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High-contrast optical imaging is achievable using phosphorescent labels to suppress the short-lived background due to the optical backscatter and autofluorescence. 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 laser-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.
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