Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

High-Resolution Imaging of Bacterial Spatial Organization with Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI)

Version 1 : Received: 25 June 2021 / Approved: 28 June 2021 / Online: 28 June 2021 (10:38:11 CEST)

How to cite: Whitley, K.; Middlemiss, S.; Jukes, C.; Dekker, C.; Holden, S. High-Resolution Imaging of Bacterial Spatial Organization with Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI). Preprints 2021, 2021060639 (doi: 10.20944/preprints202106.0639.v1). Whitley, K.; Middlemiss, S.; Jukes, C.; Dekker, C.; Holden, S. High-Resolution Imaging of Bacterial Spatial Organization with Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI). Preprints 2021, 2021060639 (doi: 10.20944/preprints202106.0639.v1).

Abstract

Light microscopy is indispensable for analysis of bacterial spatial organization. However, imaging in bacteria is difficult due to their small sizes and the fact that most species are non-spherical, meaning they typically lie horizontally on a microscope coverslip. This is especially problematic when considering that many essential bacterial processes—such as cell division—occur along the short axes of these cells, and so are viewed side-on by standard microscopy. We recently developed a pair of methods to overcome this problem by forcing cells to stand vertically during imaging, named VerCINI (Vertical Cell Imaging by Nanostructured Immobilisation) and µVerCINI (Microfluidic VerCINI). The concept behind both methods is that cells are imaged while confined vertically inside cell traps made from a nanofabricated mould. By doing so, the short axes of the cells are rotated parallel to the microscope imaging plane and are imaged with high resolution. μVerCINI combines the vertical cell confinement with microfluidics so that vertical imaging can be done during fluid exchange, such as during antibiotic perturbations. Here, we provide a practical guide to implementing both VerCINI and µVerCINI, with detailed protocols and experience-based tips so that interested researchers can easily use one or both imaging methods to complement their current approaches.

Subject Areas

Microscopy; Bacterial cell biology; Nanofabrication; Microfluidics

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