Mapping Nanoscale Forces and Potentials in Live Cells with Microsecond 3D Single-Particle Tracking.

3D single-particle tracking has the potential to resolve the molecular-level forces that dictate particle motion in biological systems. However, the information gleaned from 3D single-particle tracking often cannot resolve underlying nanoscale potentials due to limited spatiotemporal resolution. To this end, we introduce an active-feedback 3D tracking microscope that utilizes silver nanoparticles (AgNPs) as probes to study intricate biophysical events in live cells at the nanometer and microsecond scales. Due to the extremely high, durable scattering photon flux of the plasmonic particles, a 1 MHz sampling frequency with nanometer precision in all three dimensions can be achieved over an unlimited observation duration. In this work, microsecond-sampling, active-feedback 3D single-particle tracking was applied to investigate the interaction between AgNPs and nanoscale filopodia on the live-cell surface. The nanometer precision and microsecond sampling revealed that TAT peptide-modified particles visit and dwell at local "hot spots" on the filopodium surface. The high sampling rate further enabled the calculation of local forces and potentials within these nanoscale hotspots on the cylindrical surfaces of live-cell filopodia. This study presents a promising tool for investigating intracellular biophysical events with unprecedented spatiotemporal resolution, along with a pipeline for studying nanoscale potentials within three-dimensional cellular structures.