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

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.

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Fig. 1: Concept of VerCINI and comparison with conventional imaging of division protein dynamics.
Fig. 2: Concept of VerCINI and demonstration to conventional imaging of polar protein dynamics.
Fig. 3: Concept of μVerCINI and demonstration of imaging division protein dynamics during rapid antibiotic perturbation.
Fig. 4: Design of micropillar wafer.
Fig. 5: Development and etching of micropillar wafer.
Fig. 6: Sample preparation for VerCINI.
Fig. 7: Device assembly and cell loading for μVerCINI.
Fig. 8: Image processing and analysis for VerCINI and μVerCINI.

Data availability

Source data for all figures presented in the paper are available at figshare (https://doi.org/10.25405/data.ncl.c.5652010.v1).

Code availability

Custom software is available on the Holden lab GitHub page or Zenodo: https://github.com/HoldenLab/VerCINI_nanofab25, https://github.com/HoldenLab/DeepAutoFocus37, https://github.com/HoldenLab/VerciniAnalysisJ38 and https://github.com/HoldenLab/ring-fitting230.

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Acknowledgements

We acknowledge S. Pud (TU Delft; now at Twente) for help with wafer design and nanofabrication, S. Deshpande (TU Delft; now at Wageningen) for help with nanofabrication and soft lithography, M. Zuiddam (TU Delft) for help with wafer etching, H. Strahl (Newcastle) for strains and helpful discussions, and J. Fritzsche (ConScience AB, Sweden) for helpful discussions. S.H., K.D.W., C.J. and S.M. acknowledge funding support by a Wellcome Trust & Royal Society Sir Henry Dale Fellowship [206670/Z/17/Z]. C.D. acknowledges funding support by from the ERC Advanced Grants [883684] and [669598], and the NanoFront and BaSyC programs. S.M. is supported by a UK Biotechnology and Biological Sciences Research Council doctoral studentship.

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Authors

Contributions

K.D.W., S.M. and C.J. performed the experiments. K.D.W., S.M., S.H. and C.D. wrote the paper.

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Correspondence to Kevin D. Whitley or Séamus Holden.

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The authors declare no competing interests.

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Nature Protocols thanks Cecile Morlot and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Bisson-Filho, A. W. et al. Science 355, 739–743 (2017): https://doi.org/10.1126/science.aak9973

Perez, A. J. et al. Proc. Natl. Acad. Sci. USA 116, 3211–3220 (2019): https://doi.org/10.1073/pnas.1816018116

Whitley, K. D. et al. Nat. Commun. 12, 2448 (2021): https://doi.org/10.1038/s41467-021-22526-0

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Methods.

Reporting Summary

Supplementary Video 1

Suboptimal cell trapping visualized by bright-field microscopy. B. subtilis PY79 cells improperly trapped in microhole arrays where microhole width (>1.1 µm) is too large to immobilize most cells. Lateral diffusive motion in the holes (wobbling) can be observed in ~50% of trapped cells. The video shows acceptable but not excellent cell loading efficiency. A few cells can be seen sitting on top of rather than within microholes, likely those that did not wash off during sample preparation. Video was recorded at 100 Hz and plays in real time. Scale bar, 10 µm.

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Whitley, K.D., Middlemiss, S., Jukes, C. et al. High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI). Nat Protoc 17, 847–869 (2022). https://doi.org/10.1038/s41596-021-00668-1

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