Automated Five-Dimensional Single Particle Tracking by Bifocal Parallax Dark-Field Microscopy with Electronic Tunable Lens.

We present a novel method for the precise tracking of plasmonic gold nanorods (AuNRs) in live cells, enabling a comprehensive understanding of the nanocargo's cellular dynamics. Traditional single particle tracking (SPT) struggles with accurately determining all five spatial parameters ( x , y , z , ϕ, and θ) in live cells due to various challenges. Our innovation combines electronic tunable lens (ETL) technology with bifocal parallax dark-field (DF) microscopy, allowing continuous adjustment of the imaging focal plane for automatic tracking of both translational and rotational movements of AuNRs. This 5D single-particle orientation and rotational tracking (5D SPORT) method achieves remarkable precision, with 3D localization precisions of 9 ( x ), 10 ( y ), and 15 nm ( z ) and angular resolutions below 2°. To showcase its applicability, we investigated intracellular transport of nanocargos using transferrin-modified AuNRs as the imaging probe. Differentiated transport stages, such as active transport and pause period, were clearly unveiled from the observed dynamics in 5D. This advancement in single particle tracking holds promise for a wide range of applications in biomedical research, particularly when combined with other imaging modalities, such as light sheet fluorescence microscopy.