Improved Localization Precision in 3D Single-Particle Localization Microscopy via Off-Center Sampling and Its Applications in Living Systems

Abstract

Optimization of sampling patterns in 3D Real-time Single Particle Tracking (3D-RT-SPT) systems is crucial due to the limited photon yield and irreversible photobleaching of fluorescent materials commonly used in biological imaging. This study focuses on the evaluation of different sampling patterns in a highly flexible and robust 3D Single Molecule Active Real-time Tracking (3D-SMART) microscope, a representative 3D-RT-SPT system, and further application of an information-efficient version of 3D-SMART. The investigation reveals that optimal sampling patterns in the XY-plane and Z-axis, are off-centered, leading to doubled precision. Theoretical analysis further demonstrates that information-efficient sampling patterns coincide with areas of high Fisher information. These findings not only enhanced the precision of the 3D-SMART system but also established a roadmap for achieving information-efficient sampling in similar 3D-RT-SPT methodologies. Utilizing the information-efficient 4-Corners sampling pattern implemented in the 3D-SMART system, further investigations focused on intracellular events, specifically filopodium dynamics, by employing silver nanoparticles (AgNPs) as probes. Filopodia are thin extracellular protrusions that have important functions in various biological events. Due to their small scale and highly dynamic nature, it is difficult to investigate their activities in a physiologically relevant condition. AgNPs offer advantages over conventional fluorescent materials, providing ample photon flux due to their unique photoluminescent mechanisms known as surface plasmon resonance. Upon incubation with live cells, AgNPs exhibited constrained cylindrical diffusion, revealing significant heterogeneity in residence time across the cylindrical surface. Notably, localized "hot spots" repeatedly visited by AgNPs were observed. Force analysis indicated that these hot spots corresponded to local potential wells, where diffusion speed remained relatively stable. These findings provided evidence of nanoscale structures (~50 nm scale) on filopodia, demonstrating charge-dependent interactions with AgNPs. Furthermore, this study presented a framework for extracting hidden information from 3D trajectories of particles interacting with cylindrical structures, surpassing the capabilities of conventional techniques.