<p>Nanoparticles (NPs)
adsorb proteins when exposed to biological fluids, forming a dynamic protein
corona that affects their fate in biological environments. A comprehensive
understanding of the protein corona is lacking due to the inability of current techniques to precisely measure the full
corona <i>in situ</i> at the single particle level. Herein, we introduce a 3D
real-time single-particle tracking spectroscopy to "lock-on" to
single freely-diffusing polystyrene NPs and probe their individual protein
coronas. The diffusive motions of the tracked NPs enable quantification of the "hard
corona" using mean-squared displacement analysis. Critically, this
method's particle-by-particle nature enabled a lock-in-type frequency filtering
approach to extract the full protein corona, despite the typically confounding
effect of high background signal from unbound proteins. From these results, the
dynamic <i>in situ </i>full protein corona is observed to contain double the
number of proteins than are observed in the <i>ex situ</i> measured "hard"
protein corona.</p><br>
Particle-by-Particle in Situ Characterization of the Protein Corona via Real-Time 3D Single Particle Tracking Microscopy