Liquid phase electron microscopy of bacterial ultrastructure

Abstract

Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometre resolutions, paving the way for live-cell imaging using electron microscopy. However, this technique is often hampered by the inherent thickness of whole cell samples and damage from electron beam irradiation. These restrictions degrade image quality and resolution, impeding biological interpretation. Here we detail the use of graphene encapsulation, STEM, and energy-dispersive X-ray (EDX) spectroscopy methods to mitigate these issues, providing unprecedented levels of intracellular detail in aqueous specimens. We demonstrate the feasibility of our approach using a radiation resistant, gram-positive bacterium, Deinococcus radiodurans, chosen specifically for its tolerance to the highly oxidative environments created by electron irradiation. This work shows the potential of LP-STEM to examine internal cellular structures in thick biological samples and identify key ultrastructure in these samples using a variety of imaging techniques.