Determination of the Absolute Concentration of Rayleigh Particles via Interferometric Scattering Microscopy

Nanoparticles play vital roles across healthcare, electronics, and energy, where their nanoscale dimensions enable unique functionalities, such as crossing biological barriers to enhance drug delivery. However, this same small size also raises safety concerns, making accurate characterization essential, especially in biomedical applications. Traditional techniques, including dynamic light scattering and mass spectrometry, often fall short in sensitivity, resolution, and versatility. To overcome these limitations, we introduce iCOUNT (interferometric concentration observer and ultrasmall nanoparticle tracker), a method that integrates interferometric scattering (iSCAT) microscopy with deep learning-based particle detection to enable the detection and quantification of nanoparticles smaller than 50 nm (Rayleigh particles, RPs), well below the diffraction limit and in the Rayleigh regime. Our machine learning-based analysis framework facilitates automated, accurate identification and robust tracking of individual RPs from high-frame-rate iSCAT image sequences. This label-free, real-time approach yields multiple key properties of RPs, including size, diffusion coefficients, scattering contrast, and absolute concentration in aqueous environments. iCOUNT reliably detects and tracks dielectric nanoparticles as small as 20 nm in diameter, achieving high spatial and temporal resolutions. We demonstrate its performance by characterizing diverse biological Rayleigh particles, such as immunoglobulin M, simian virus 40, and DNA origami. These results validate the method’s accuracy in size and concentration measurements and uncover single-particle heterogeneity in both optical and dynamic behavior. As a noninvasive optical technique, iCOUNT addresses critical shortcomings of conventional methods and provides a powerful, generalizable platform for nanoparticle analysis in native environments, with broad implications for diagnostics, therapeutics, and nanoscience.