Accurate Nanoparticle Localization in Interferometric Scattering Microscopy

Interferometric scattering (iSCAT) microscopy is a powerful label-free technique for the sensitive detection of nanomaterials. However, conventional image processing methods based on image subtraction or mitigation can introduce localization errors when the image background (reference field) is nonuniform. Here, we present an optimized processing approach that eliminates such errors by normalizing the differential image with the amplitude (rather than the intensity) of the reference field, thereby isolating the pure scattering field for precise nanoparticle positioning. Through rigorous numerical simulations, we demonstrate that this method accurately localizes nanoparticles under both focused and defocused conditions. Experimental validation demonstrates that our approach achieves a maximum localization deviation correction of 22.3 nm, representing a significant improvement over conventional methods.