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

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...

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Autores principales: Amselem, Elias, Broadwater, Bo, Hävermark, Tora, Johansson, Magnus, Elf, Johan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10008558/
https://www.ncbi.nlm.nih.gov/pubmed/36906676
http://dx.doi.org/10.1038/s41467-023-36879-1
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author Amselem, Elias
Broadwater, Bo
Hävermark, Tora
Johansson, Magnus
Elf, Johan
author_facet Amselem, Elias
Broadwater, Bo
Hävermark, Tora
Johansson, Magnus
Elf, Johan
author_sort Amselem, Elias
collection PubMed
description 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.
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spelling pubmed-100085582023-03-13 Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization Amselem, Elias Broadwater, Bo Hävermark, Tora Johansson, Magnus Elf, Johan Nat Commun Article 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. Nature Publishing Group UK 2023-03-11 /pmc/articles/PMC10008558/ /pubmed/36906676 http://dx.doi.org/10.1038/s41467-023-36879-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Amselem, Elias
Broadwater, Bo
Hävermark, Tora
Johansson, Magnus
Elf, Johan
Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
title Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
title_full Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
title_fullStr Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
title_full_unstemmed Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
title_short Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
title_sort real-time single-molecule 3d tracking in e. coli based on cross-entropy minimization
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10008558/
https://www.ncbi.nlm.nih.gov/pubmed/36906676
http://dx.doi.org/10.1038/s41467-023-36879-1
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