The ONI Nanoimager is a bench top super resolution microscope optimised for single molecule localisation techniques and particle tracking. Using single molecule localisation, an X-Y resolution of 10-30 nm can be achieved. The ONI supports all techniques based on dye blinking, photoconversion or transient binding, such as dSTORM, PALM and PAINT. The single molecule sensitivity of the ONI permits particle tracking with time resolution in the millisecond range. In addition to particle tracks, diffusion co-efficient, size and number of particles is summarised for single or groups of tracks using the included analysis software. Offline analysis includes a dedicated extracellular vesicle profiling module optimised for quantifying size and biomarker distribution. The ONI is suitable for both fixed and live material, featuring high precision temperature control between 32° and 42° C. Hardware includes four laser lines, two simultaneous detection channels, a high precision X, Y, Z stage and real-time focus locking to prevent drift. Epifluorescence, Hi-Lo and TIRF imaging modalities are supported