Direct Measurement of Protein Corona Thickness on Nanomaterials Using Dual‐Color Stochastic Optical Reconstruction Microscopy (STORM)

Nanoparticles introduced to biological media rapidly adsorb proteins to form a protein corona that alters their surface identity and governs biological interactions. While protein corona composition has been extensively studied, its spatial structure, particularly thickness at the single-particle level, remains poorly resolved. Here, we establish and validate a strategy using dual-color stochastic optical reconstruction microscopy (STORM) for the direct measurement of hard corona thickness on individual nanoparticles. The nanoparticle surface and retained protein corona were labeled with spectrally distinct fluorophores, allowing the radial separation between the nanoparticle and adsorbed protein layer to be determined from single-particle localization distributions. Applied to dense silica, mesoporous silica, and polystyrene nanoparticles, the method revealed a remarkably consistent hard corona thickness of approximately 50 nm, with particle-level standard deviations ranging from 12 to 27 nm. Sensitivity analysis, localization precision assessment, control experiments, and day-to-day reproducibility measurements supported the robustness of the measurement strategy. Proteomic analysis showed similar molecular weight distributions across all nanoparticle classes, consistent with the comparable external surface chemistries used here.