Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution.

Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.