Distinguishing Free Cytosolic and Vesicle-Confined Nanoparticle Motion by Fluorescence Correlation Spectroscopy

Understanding whether internalized nanoparticles remain confined within intracellular vesicles or are released into the cytosol is essential for evaluating their biological activity and potential use as delivery systems. However, this distinction is often difficult to establish by using fluorescence imaging alone, particularly at nanomolar probe concentrations. Here, we propose fluorescence correlation spectroscopy (FCS) combined with length-scale-dependent viscosity analysis and appropriate diffusion models as an approach for assessing nanoparticle uptake and intracellular localization. The method was evaluated in HeLa cells using fluorescent polystyrene nanoparticles, cell-penetrating peptide-coated nanoparticles, dextran of two molecular weights, and receptor-internalized transferrin. Cytosolic probes were identified by autocorrelation curves that fit an anomalous diffusion model and by diffusion coefficients consistent with those predicted by the cytoplasmic length scale-dependent viscosity profile. In contrast, vesicle-associated probes produced characteristic two-step autocorrelation curves that were modeled as the sum of probe diffusion within a confined space, vesicle translation, and directed intracellular transport. Bare nanoparticles, pinocytosed dextrans, and transferrin displayed predominantly vesicle-associated mobility, whereas microinjected dextrans and cell-penetrating-peptide-associated nanoparticles exhibited diffusion in the cytosol. Our results demonstrate that FCS can complement fluorescence imaging by detecting cytosolic probe populations and distinguishing between cytosolic release and vesicular retention.