The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy

Abstract

Expansion Microscopy (ExM) is a novel tool improving the resolution of fluorescence microscopy by linking the sample into a hydrogel that gets physically expanded in water. Previously, we have used ExM to visualize the intracellular Gram-negative pathogens <i>Chlamydia trachomatis</i>, <i>Simkania negevensis</i>, and <i>Neisseria gonorrhoeae</i>. Gram-positive bacteria have a rigid and thick cell wall that impedes classic expansion strategies. Here we developed an approach, which included a series of enzymatic treatments resulting in isotropic 4× expansion of the Gram-positive pathogen <i>Staphylococcus aureus.</i> We further demonstrate the suitability of the technique for imaging of planktonic bacteria as well as endocytosed, intracellular bacteria at a spatial resolution of approximately 60 nm with conventional confocal laser scanning microscopy.