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 successfully apply these methods to track the Trigger Factor protein in living bacterial cells. Overall, our results show that while it is possible to reach sub-millisecond live-cell single-molecule tracking, it is still hard to resolve state transitions based on diffusivity at this time scale.

Dynamic binding of the bacterial chaperone Trigger factor to translating ribosomes

“…HaloTag covalently binds chloroalkane ligands 26 such as the chloroalkane-activated organic fluorescent Janelia-Fluor (JF) dyes 27 . We have previously confirmed that the TF-Halo fusion is functional in vivo (see Supplementary Note 10B in reference 28 ). We applied our SPT method previously used for HaloTag-labeled ribosomes in live E. coli 29 .…”

“…This result suggests that the cellular level of TF-Halo is lower than that of wt TF when they are expressed from the same position on the chromosome, or, alternatively, that TF-Halo has slightly reduced binding capacity to RNC’s compared to the wt TF. However, since TF-Halo is functional 28 , and since the overall RNC binding dynamics of TF-Halo is highly similar in all three scenarios (Fig. 4b, average bound-state dwell times 130-180 ms, and free-state dwell time 20-40 ms), we believe that our measurements represent the binding kinetics of TF to RNCs well.…”

“…Similarly, the TF FRK/AAA -HaloTag mutant was expressed from the same vector (pQE30lacIq_tig_halotag_frk_aaa) in the TF knockout background. Construction of MG1655Δtig::kan and the two plasmids are described in Amselem et al 2023 28 . For low constitutive expression of TF-Halo from a plasmid (p w TF-Halo), inverse PCR was performed on pQE30lacIq_tig_halotag to exchange the T5 promoter to the weak constitutive apFAB124 promoter and to switch to a weak Shine-Dalgarno sequence (TTCTCA instead of AGGAGG 29 ) using primers pQE_SDw_F and p124_R (pQE30lacIq_p124_SDw_tig_halotag).…”

Glycogen phase separation drives macromolecular rearrangement and asymmetric division inE. coli

“…This anticorrelated pattern is thought to be mediated by steric exclusion of these two macromolecules. In contrast, smaller components such as free ribosomal subunits and cytoplasmic proteins can diffuse throughout the nucleoid unimpeded (Amselem et al , 2023; Bakshi et al , 2012; Sanamrad et al , 2014), resulting in homogeneous distribution throughout the cytoplasm. This archetypical macromolecular organization is reproduced every generation.…”

Three-dimensional localization of fluorescent proteins in livingEscherichia coli

“…SMLM has also been used to study molecular diffusion properties and their statetransitions in living cells [6][7][8]. Molecules of interest are labeled with bright dyes and imaged in time-lapse to construct molecular motion trajectories for further analysis.…”