Spontaneous photoblinking upconversion microscopy

Abstract

Super-resolution imaging has overcome the diffraction limit and enabled the nanoscale visualization of subcellular structures. However, the limited photostability of traditional fluorescent probes still poses challenges for stable, low-phototoxicity imaging. Here we introduce spontaneous photoblinking upconversion microscopy (SPUM) for super-resolution microscopy. SPUM exploits Yb3+/Ho3+-codoped upconversion nanoparticles (UCNPs) as photoblinking fluorescent emitters. We discover a spontaneous photoblinking phenomenon in such UCNPs under continuous near-infrared excitation. A Yb3+-mediated multiphoton process drives reversible transitions between emissive and dark states via defect-mediated energy trapping, producing persistent luminescent spikes that are ideal for high-precision localization. Unlike conventional photolabile fluorophores, these UCNPs resist photobleaching and enable unlimited blinking cycles for extended imaging durations. SPUM achieves a tenfold improvement in resolution, down to 35 nm for isolated emitters. We demonstrate the imaging of HeLa cell plasma membranes, as well as simultaneous live-cell tracking and the super-resolution imaging of endosomes with a spatial resolution of 30 nm for tens of minutes at a low excitation power density of 6.6 kW cm−2. Beyond improving the stability of super-resolution imaging, our findings provide a framework for engineering luminescent nanoprobes towards advanced optical imaging, nanoscale tracking and precision diagnostics in biomedical research and nanophotonics. Spontaneous photoblinking in Yb3+/Ho3+-codoped upconversion nanoparticles enables super-resolution imaging of biological cells with a spatial resolution down to 30 nm at excitation power densities of about 6 kW cm−2.