Locating and identifying viruses in in vitro optical measurements is desirable for disease control; however, the sub-diffraction-limit dimensions and low refractive index contrast of viruses make this challenging. Here, we introduce a 3D positional nanometrology for a virus-like 100 nm polystyrene sphere that can reveal the position of the nanoparticle with deeply sub-wavelength accuracy using topologically structured light illumination and machine learning. Scattering of topologically structured light is highly sensitive to the position of a nano-object. Exploiting this, our experiments demonstrate deeply sub-wavelength (λ) precision reaching 5 nm (λ/91). Our findings indicate a route toward label-free in vitro nanometrology of viruses and similar nano-objects with precision far beyond the Abbe–Rayleigh diffraction limit.
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