Mapping nanoscale forces and potentials in live cells with microsecond 3D single-particle tracking

Abstract

Abstract3D single-particle tracking has the potential to resolve the molecular level forces which 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 this extremely high and durable scattering photon flux of the plasmonic particles, 1 MHz sampling frequency at nanometer precision in all three dimensions can be achieved over an unlimited observation times. In this work, we applied microsecond-sampling, active-feedback 3D single-particle tracking to investigate the interaction between AgNPs and nanoscale filopodium 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 the local forces and potentials within these nanoscale hotspots on the cylindrical surface of live cell filopodia. This study presents a promising tool to investigate intracellular biophysical events with unprecedented spatiotemporal resolution and a pipeline to study nanoscale potentials on three-dimensional cellular structures.

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

“…The long time scale trajectories traced out the shape of physical cellular structures such as the cytoskeleton and membrane protrusion, demonstrating that SVT is also a tool for studying intracellular structures that play a role in viral infection . Previous research using 3D-SPT revealed cylindrical patterned trajectories along cell surface microspikes, hemispherical diffusion on membrane blebs, and linear motions along a membrane protrusion. ,, In the highly sampled trajectories reported here, directed linear motion could be observed, consistent with viral trafficking along the microtubule network. Uniquely, the extremely long time scale of observation here, combined with high-speed 3D sampling, allowed the large scale geometry of the underlying network to be resolved.…”

“…For the particles described below, which have emission intensities in the 50 to 100 kHz range, the position could be updated every 10 to 20 μs, though with an intrinsic trade-off between temporal resolution and localization precision. If there were infinite amount of photons, the bin time could be further narrowed down and the temporal resolution would only be ultimately limited to the photon collection rate . The additional photons provided by the StayGold fluorescent proteins are critical to increasing both the observation window and the sampling rate in SVT.…”

“…If there were infinite amount of photons, the bin time could be further narrowed down and the temporal resolution would only be ultimately limited to the photon collection rate. 36 The additional photons provided by the StayGold fluorescent proteins are critical to increasing both the observation window and the sampling rate in SVT. To provide label-free imaging of the surrounding cellular environment, a brightfield imaging setup was integrated with the 3D-SMART microscope for hour-long contextualization of 3D viral trajectories with kHz sampling rate (Figure 2).…”

Hour-long, Kilohertz Sampling Rate 3D Single-virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions

“…32 Previous research using 3D-SPT revealed cylindrical patterned trajectories along cell surface microspikes, hemispherical diffusion on membrane blebs, and linear motions along a membrane protrusion. 7, 29, 33 In the highly-sampled trajectories reported here, directed linear motion could be observed, consistent with viral trafficking along the microtubule network. Uniquely, the extremely long timescale of observation here, combined with high-speed 3D sampling, allowed the large scale geometry of the underlying network to be resolved.…”

“…If there were infinite amount of photons, the bin time could be further narrowed down and the temporal resolution would only be ultimately limited to the photon collection rate. 29 The additional photons provided by the StayGold fluorescent proteins are critical to increasing both the observation window and sampling rate in SVT.…”