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Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production

Hughes, Joshua G; Chisholm, David R; Whiting, Andrew; Girkin, John M; Ambler, Carrie A

Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production Thumbnail


Authors

Joshua Hughes joshua.g.hughes@durham.ac.uk
PGR Student Doctor of Philosophy



Abstract

Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers working on the challenge over the past 10–15 years without producing a definitive method. We report a new fluorescence microscopy-based technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching) technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and JC-1.

Citation

Hughes, J. G., Chisholm, D. R., Whiting, A., Girkin, J. M., & Ambler, C. A. (2023). Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production. Microscopy and Microanalysis, 29(2), 529-539. https://doi.org/10.1093/micmic/ozac040

Journal Article Type Article
Acceptance Date Nov 6, 2022
Online Publication Date Dec 22, 2022
Publication Date 2023-04
Deposit Date Jan 26, 2023
Publicly Available Date May 30, 2023
Journal Microscopy and Microanalysis
Print ISSN 1431-9276
Electronic ISSN 1435-8115
Publisher Cambridge University Press
Peer Reviewed Peer Reviewed
Volume 29
Issue 2
Pages 529-539
DOI https://doi.org/10.1093/micmic/ozac040
Keywords bullseye analysis, Apoptosis, Macrophages, reactive oxygen species (ROS), Reactive Oxygen Species, microscopy, photodynamic therapy (PDT), Coloring Agents, phototoxicity, Microscopy, Fluorescence, photosensitizer
Public URL https://durham-repository.worktribe.com/output/1181908

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Published Journal Article (4.5 Mb)
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Publisher Licence URL
http://creativecommons.org/licenses/by/4.0/

Copyright Statement
© The Author(s) 2022. Published by Oxford University Press on behalf of the Microscopy Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.







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