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High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI)
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Whitley, Kevin D., Middlemiss, Stuart, Jukes, Calum, Dekker, Cees and Holden, Séamus (2022) High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI). Nature Protocols, 17 (3). pp. 847-869. doi:10.1038/s41596-021-00668-1 ISSN 1750-2799.
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Official URL: http://dx.doi.org/10.1038/s41596-021-00668-1
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.
Item Type: | Journal Article | ||||||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||
Journal or Publication Title: | Nature Protocols | ||||||||
Publisher: | Nature Publishing Group | ||||||||
ISSN: | 1750-2799 | ||||||||
Official Date: | March 2022 | ||||||||
Dates: |
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Volume: | 17 | ||||||||
Number: | 3 | ||||||||
Page Range: | pp. 847-869 | ||||||||
DOI: | 10.1038/s41596-021-00668-1 | ||||||||
Status: | Peer Reviewed | ||||||||
Publication Status: | Published | ||||||||
Access rights to Published version: | Restricted or Subscription Access |
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