Bacterial cell wall nanoimaging by autoblinking microscopy
Sci Rep. 2018-09-19; 8(1):
Lire sur PubMed
1. Sci Rep. 2018 Sep 19;8(1):14038. doi: 10.1038/s41598-018-32335-z.
Bacterial cell wall nanoimaging by autoblinking microscopy.
Floc’h K(1), Lacroix F(1), Barbieri L(1), Servant P(2), Galland R(3)(4), Butler
C(3)(4), Sibarita JB(3)(4), Bourgeois D(5), Timmins J(6).
(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,
(4)Centre National de la Recherche Scientifique, UMR5297, Bordeaux, France.
(5)Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France.
(6)Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France.
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.