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. 2023 Nov 24;72(6):515-519.
doi: 10.1093/jmicro/dfad027.

A preparation of bacterial outer membrane with osmium tetroxide and uranyl acetate co-stain enables improved structural determination by transmission electron microscopy

Aadil Sheikh  1 , Bernd Zechmann  2 , Christie M Sayes  3 , Joseph H Taube  1 , K Leigh Greathouse  1   4
Affiliations

A preparation of bacterial outer membrane with osmium tetroxide and uranyl acetate co-stain enables improved structural determination by transmission electron microscopy

Aadil Sheikh et al. Microscopy (Oxf). .

Abstract

Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we report a novel method to prepare OMVs for TEM imaging. To preserve vesicular shape and structure, we developed a dual fixation protocol involving osmium tetroxide incubation prior to negative staining with uranyl acetate. Combining osmium tetroxide with uranyl acetate resulted in preservation of sub-50 nm vesicles and improved morphological stability, enhancing characterization of lipid-based nanoparticles by TEM.

Keywords: bacteria; nanoparticles; osmium tetroxide; outer membrane vesicles; transmission electron microscopy; uranyl acetate.

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Figures

Fig. 1.
Fig. 1.
OMV staining with UA alone. TEM micrographs of ETBF (a and b) and NTBF OMVs (c and d) stained with UA. Arrows denote vesicles that appear collapsed or that experienced a loss of structure. Scale bars, 100 nm. ×50 000 magnification.
Fig. 2.
Fig. 2.
OMV staining with OsO4 and UA. TEM micrographs of ETBF (a and b) and NTBF (c and d) OMVs stained with osmium tetroxide and UA. Vesicles are expected to be spherical in shape. Scale bars, 100 nm. ×50 000 magnification.
Fig. 3.
Fig. 3.
Quantitative analyses of OMV size (a and b) and roundness (c and d). Comparisons between different staining methods are shown as a box blot (a and c) and density plot (b and d). Osmium tetroxide and UA co-stain was able to visualize smaller vesicles and rounder vesicles in greater quantity. Statistics were calculated using Welch’s t-test.
See this image and copyright information in PMC

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