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Sci Rep. 2018 Sep 19;8(1):14038. doi: 10.1038/s41598-018-32335-z.

Bacterial cell wall nanoimaging by autoblinking microscopy.

Author information

1
Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France.
2
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France.
3
Institut Interdisciplinaire de Neurosciences, University of Bordeaux, Bordeaux, France.
4
Centre National de la Recherche Scientifique, UMR5297, Bordeaux, France.
5
Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France. dominique.bourgeois@ibs.fr.
6
Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France. Joanna.timmins@ibs.fr.

Abstract

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this 'autoblinking' phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.

PMID:
30232348
PMCID:
PMC6145920
DOI:
10.1038/s41598-018-32335-z
[Indexed for MEDLINE]
Free PMC Article

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