A comparison of super‐resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium.
Saved in:
| Title: | A comparison of super‐resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium. |
|---|---|
| Authors: | North, Alison J.1 (AUTHOR), Sharma, Ved P.1 (AUTHOR), Pyrgaki, Christina1,2 (AUTHOR), S.Y., John Lim3 (AUTHOR), Atwal, Sharanjeet4 (AUTHOR), Saharat, Kittirat5 (AUTHOR), Wright, Graham D.6 (AUTHOR), Salje, Jeanne5,7,8,9 (AUTHOR) jss53@cam.ac.uk |
| Source: | Journal of Microscopy. Feb2026, Vol. 301 Issue 2, p187-205. 19p. |
| Subjects: | Fluorescence microscopy, Three-dimensional imaging, Microscopy, Cell segmentation, Bacteria, High resolution imaging, Intracellular pathogens |
| Abstract: | Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three‐dimensional Structured Illumination Microscopy (3D‐SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half‐maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D‐SIM microscopy, in combination with the shortest‐wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D‐STED to be the most successful method for this. We then combined this 3D‐STED approach with a custom 3D cell segmentation and analysis pipeline using the open‐source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super‐resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed. [ABSTRACT FROM AUTHOR] |
| Copyright of Journal of Microscopy is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
|
Full text is not displayed to guests.
Login for full access.
|
|
| Abstract: | Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three‐dimensional Structured Illumination Microscopy (3D‐SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half‐maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D‐SIM microscopy, in combination with the shortest‐wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D‐STED to be the most successful method for this. We then combined this 3D‐STED approach with a custom 3D cell segmentation and analysis pipeline using the open‐source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super‐resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed. [ABSTRACT FROM AUTHOR] |
|---|---|
| ISSN: | 00222720 |
| DOI: | 10.1111/jmi.13376 |
