A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium Journal Article


Authors: North, A. J.; Sharma, V. P.; Pyrgaki, C.; S Y, J. L.; Atwal, S.; Saharat, K.; Wright, G. D.; Salje, J.
Article Title: A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium
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. © 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.
Keywords: confocal; confocal microscopy; cell membranes; fluorescence microscopy; animal cell culture; fluorescence imaging; image segmentation; deep learning; super-resolution imaging; structured illumination; intracellular bacteria; cell segmentation; 3d cell segmentation; 3d-sim; airyscan; isim; obligate intracellular bacteria; orientia tsutsugamushi; rickettsiales; sted; instant structured illumination microscopy; rickettsiale; stimulated emission depletion microscopy; super resolution imaging; three-dimensional structured illumination microscopy
Journal Title: Journal of Microscopy
ISSN: 0022-2720
Publisher: Wiley-Blackwell Publishing, Inc.  
Publication status: Online ahead of print
Date Published: 2024-12-09
Online Publication Date: 2024-12-09
Language: English
DOI: 10.1111/jmi.13376
PROVIDER: scopus
PUBMED: 39651611
DOI/URL:
Notes: Article -- Source: Scopus
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