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High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI)

Lookup NU author(s): Dr Kevin WhitleyORCiD, Dr Stuart Middlemiss, Dr Calum Jukes, Dr Seamus Holden

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

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.


Publication metadata

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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Funding

Funder referenceFunder name
a Wellcome Trust & Royal Society Sir Henry Dale Fellowship [206670/Z/17/Z]
ERC Advanced Grant [669598],
ERC Advanced Grant [883684]

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