Functional nanoparticles,including natural viruses and synthetic nanomedicine have received much attention due to their great importance for biomedical diagnosis,nanomedicine,material science,and global environment.For most of the applications,rapid,high-resolution,and multiparameter characterization of functional nanoparticles is critical.Single particle techniques can reveal the heterogeneity or rare events that are otherwise masked by ensemble-averaged measurements.However,the small size and low refractive index of viruses and nanomedicine render single-particle analysis extremely challenging.Our laboratory has developed high sensitivity flow cytometry(HSFCM)that allows real-time light scattering detection of single low-refractive-index nanoparticles as small as 24-nm in diameter.With the superior sensitivity,we achieved the detection of single levivirus MS2,a very small RNA bacteriophage(27 nm in diameter).In this presentation,high resolution differentiation of virus mixtures is demonstrated,accurate sizing and concentration measurement of viruses is attained.Upon nucleic acid staining,we demonstrate clear discrimination of individual virions from empty viral capsids and free viral DNA with bacteriophage T7 as the model.In terms of nanomedicine analysis,by taking siRNA loaded lipid nanoparticles as model system,the precise particle count,the fraction of siRNA loaded particles as well as the siRNA content per particle are determined.By providing quantitative multipatameter analysis at a throughput of up to 10,000 particles per minute,HSFCM represents a valuable tool for functional nanoparticle characterization.