Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization.

ABSTRACT: Reaching sub-millisecond 3D tracking of individual molecules in living cells would enable direct measurements of diffusion-limited macromolecular interactions under physiological conditions. Here, we present a 3D tracking principle that approaches the relevant regime. The method is based on the true excitation point spread function and cross-entropy minimization for position localization of moving fluorescent reporters. Tests on beads moving on a stage reaches 67 nm lateral and 109 nm axial precision with a time resolution of 0.84 ms at a photon count rate of 60 kHz; the measurements agree with the theoretical and simulated predictions. Our implementation also features a method for microsecond 3D PSF positioning and an estimator for diffusion analysis of tracking data. Finally, we successf

SUBMITTER: Amselem E 

PROVIDER: S-EPMC10008558 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature