Advances in Real-time 3D Single Particle Tracking Microscopy for Particle-by-Particle In-Situ Characterization of the Nanoparticle Protein Corona

Xiaochen Tan, Ph.D. Candidate

Kevin Welsher, Ph.D., Advisor

Abstract: Single-molecule spectroscopic (SMS) measurements have revolutionized biological science by removing the ensemble average and revealing molecular heterogeneities. Unfortunately, traditional SMS techniques require removing a molecule from its native environment and tethering it to a surface for extended observation. This isolation removes critical context from SMS measurements. To address this gap, real-time feedback single particle tracking (RT-3D-SPT) has been developed to directly monitor individual freely diffusing particles in the solution phase by applying active feedback to keep the particle in the detection volume. This technique allows synchronized diffusion and spectroscopic analysis to study particle dynamics at a high spatiotemporal resolution. However, current RT-3D-SPT methods have limited ability to track dim and fast diffusing objects and lack rapid concurrent 3D contextualization to observe fast processes happening around the tracked objects. These rapid processes include the protein corona adsorption onto nanoparticles, a critical roadblock the biological application of engineered nanoparticles. Herein, we present a new RT-3D-SPT technique, called 3D real-time ultrafast local microscopy (3D-RULM), to track fast diffusing objects while simultaneously imaging their surroundings in three dimensions. We show that 3D-RULM can be applied to quantitatively characterize the protein corona on individual nanoparticles in situ, with single protein sensitivity at signal-to-background ratios down to 1%. To expand this method to smaller NP-protein systems, we have further implemented a Galvo mirror as a control actuator with a response time five times faster than the currently used piezoelectric stage, opening the possibility to study transient NP-protein interactions and other fast biological phenomena in situ and in vivo.