In this work, we present a 3D single-particle tracking system that can apply tailored sampling patterns to selectively extract photons that yield the most information for particle localization. We demonstrate that off-center sampling at locations predicted by Fisher information utilizes photons most efficiently. When performing localization in a single dimension, optimized off-center sampling patterns gave doubled precision compared to uniform sampling. A ~20% increase in precision compared to uniform sampling can be achieved when a similar off-center pattern is used in 3D localization. Here, we systematically investigated the photon efficiency of different emission patterns in a diffraction-limited system and achieved higher precision than uniform sampling. The ability to maximize information from the limited number of photons demonstrated here is critical for particle tracking applications in biological samples, where photons may be limited.

3D 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.

The viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions as compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with one thousand localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long timescales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the whole life cycle of a single virus. Abstract Figure:

In this work, we present a 3D single-particle tracking system that can apply tailored sampling patterns to selectively extract photons that yield the most information for particle localization. We demonstrate that off-center sampling at locations predicted by Fisher information utilizes photons most efficiently. When performing localization in a single dimension, optimized off-center sampling patterns gave doubled precision compared to uniform sampling. A ~20% increase in precision compared to uniform sampling can be achieved when a similar off-center pattern is used in 3D localization. Here we systematically investigated the photon efficiency of different emission patterns in a diffraction-limited system and achieved higher precision than uniform sampling. The ability to maximize information from the limited number of photons demonstrated here is critical for particle tracking applications in biological samples, where photons may be limited.