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Deep tissue super-resolution microscopy mapping single nanoparticles inside multicellular spheroids

Organoid, an in vitro model to study cell behaviours in a living organism, holds great potential for human cellular biology study, especially in disease pathology, drug delivery and drug efficacy trials. However, it remains challenging to track subcellular features inside organoid, as organoid are clusters of high-density cells that highly scatters and absorbs both excitation and emission light. Here we report a strategy on nanoscopy that applying “non-diffractive” beam and near-infrared imaging probe to minimize the light scattering and absorption inside scattering bio-tissue. Using a single Bessel-doughnut beam excitation from a 980nm diode laser and detecting at 800nm, we achieved a near-infrared, “non-diffractive” nanoscopy with high resolution under-diffractive limit in water solution. We further demonstrate that this method can image single upconversion nanoparticles inside spheroids, as deep as half-100μm, with resolution of 113nm. This method provides simple solution to inspect inter-and intra-cellular trafficking and drug release of single nanoparticles in 3D biological systems.