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.