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In this study, we imaged intracellular bacteria from the species\n                    <jats:italic>Orientia tsutsugamushi<\/jats:italic>\n                    (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three\u2010dimensional Structured Illumination Microscopy (3D\u2010SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (\n                    <jats:italic>xy<\/jats:italic>\n                    ) axis, using full width half\u2010maximum (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\u2010SIM microscopy, in combination with the shortest\u2010wavelength 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 (\n                    <jats:italic>z<\/jats:italic>\n                    ) direction and found 3D\u2010STED to be the most successful method for this. We then combined this 3D\u2010STED approach with a custom 3D cell segmentation and analysis pipeline using the open\u2010source, 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\u2010resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.\n                  <\/jats:p>","DOI":"10.1111\/jmi.13376","type":"journal-article","created":{"date-parts":[[2024,12,9]],"date-time":"2024-12-09T06:10:20Z","timestamp":1733724620000},"page":"187-205","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A comparison of super\u2010resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium"],"prefix":"10.1111","volume":"301","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0577-7949","authenticated-orcid":false,"given":"Alison J.","family":"North","sequence":"first","affiliation":[{"name":"Bio\u2010Imaging Resource Center The Rockefeller University New York New York 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