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Lookup NU author(s): Dr Kevin WhitleyORCiD, Dr Stuart Middlemiss, Dr Calum Jukes, Dr Seamus Holden
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
Light microscopy is indispensable for analysis of bacterial spatial organization, yet the sizes and shapes of bacterial cells pose unique challenges to imaging. Bacterial cells are not much larger than the diffraction limit of visible light, and many species have cylindrical shapes and so lie flat on microscope coverslips, yielding low-resolution images when observing their short axes. In this protocol, we describe a pair of recently developed methods named VerCINI (vertical cell imaging by nanostructured immobilization) and µVerCINI (microfluidic VerCINI) that greatly increase spatial resolution and image quality for microscopy of the short axes of bacteria. The concept behind both methods is that cells are imaged while confined vertically inside cell traps made from a nanofabricated mold. The mold is a patterned silicon wafer produced in a cleanroom facility using electron-beam lithography and deep reactive ion etching, which takes ~3 h for fabrication and ~12 h for surface passivation. After obtaining a mold, the entire process of making cell traps, imaging cells and processing images can take ~2–12 h, depending on the experiment. VerCINI and µVerCINI are ideal for imaging any process along the short axes of bacterial cells, as they provide high-resolution images without any special requirements for fluorophores or imaging modalities, and can readily be combined with other imaging methods (e.g., STORM). VerCINI can easily be incorporated into existing projects by researchers with expertise in bacteriology and microscopy. Nanofabrication can be either done in-house, requiring specialist facilities, or outsourced based on this protocol.
Author(s): Whitley KD, Middlemiss S, Jukes C, Dekker C, Holden S
Publication type: Article
Publication status: Published
Journal: Nature Protocols
Year: 2022
Volume: 17
Pages: 847-869
Print publication date: 01/03/2022
Online publication date: 31/01/2022
Acceptance date: 19/11/2021
Date deposited: 23/02/2024
ISSN (print): 1754-2189
ISSN (electronic): 1750-2799
Publisher: Springer Nature
URL: https://doi.org/10.1038/s41596-021-00668-1
DOI: 10.1038/s41596-021-00668-1
Data Access Statement: Source data for all figures presented in the paper are available at figshare: https://doi.org/10.25405/data.ncl.c.5652010.v1
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