An Adaptive Real‐Time 3D Single Particle Tracking Method for Monitoring Viral First Contacts

Abstract

Here, an adaptive real‐time 3D single particle tracking method is proposed, which is capable of capturing heterogeneous dynamics. Using a real‐time measurement of a rapidly diffusing particle's positional variance, the 3D precision adaptive real‐time tracking (3D‐PART) microscope adjusts active‐feedback parameters to trade tracking speed for precision on demand. This technique is demonstrated first on immobilized fluorescent nanoparticles, with a greater than twofold increase in the lateral localization precision (≈25 to ≈11 nm at 1 ms sampling) as well as a smaller increase in the axial localization precision (≈ 68 to ≈45 nm). 3D‐PART also shows a marked increase in the precision when tracking freely diffusing particles, with lateral precision increasing from ≈100 to ≈70 nm for particles diffusing at 4 µm2 s−1, although with a sacrifice in the axial precision (≈250 to ≈350 nm). This adaptive microscope is then applied to monitoring the viral first contacts of virus‐like particles to the surface of live cells, allowing direct and continuous measurement of the viral particle at initial contact with the cell surface.

Real-time 3D single molecule tracking

“…Feedback is applied using either piezoelectric nanopositioners or galvo mirrors 18 , meaning the target molecule or particle is followed at very high speed, rather than being physically confined. Pioneering work has shown that RT-3D-SPT can lock-on to a wide range of targets, from gold nanoparticles 19 , 20 and single quantum dots 21 – 23 all the way to viruses 24 – 26 , and is easily extended to live cell tracking 27 , 28 . This is because the “confinement” is not a physical impediment to the target, but rather a high-speed chase which follows the particle without perturbation.…”

Information-Efficient, Off-Center Sampling Results in Improved Precision in 3D Single-Particle Tracking Microscopy

“…Single-particle tracking (SPT) [ 1 ] has led to numerous advances in unveiling sophisticated intracellular biophysical events, including diffusion of membrane proteins [ 2 ], transportation of intracellular vesicles [ 3 ], and viral internalization events [ 4 , 5 ]. Despite the tremendous progress made in the field, there are limits to what can be gleaned from single-particle trajectories by the intrinsic localization precision.…”

Particle‐by‐Particle In Situ Characterization of the Protein Corona via Real‐Time 3D Single‐Particle‐Tracking Spectroscopy**

“…Real-time three-dimensional single-particle tracking (RT-3D-SPT) spectroscopy removes the surface-tethering requirement of imaging-based methods while preserving singleparticle sensitivity,d ue to its ability to capture and analyze individual freely diffusing nanoscale object in situ. [43][44][45] Herein, we introduce an RT-3D-SPT-based lock-on protein corona spectroscopy,b ased on ap reviously developed microscope called 3D Single-Molecule Active Real-time Tr acking (3D-SMART), [46][47][48] to lock-on to individual freely diffusing fluorescent polystyrene nanoparticle (PS NP) in three dimensions using optical feedback. Thea dsorbed protein coronas around PS NPs are continuously and simultaneously monitored particle by particle,primarily in bovine serum albumin (BSA) solutions employed here as amodel system (Figure 1, Figure S1-6, Movie S1).…”