RESUMEN
Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometer 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. Using graphene encapsulation, scanning transmission electron microscopy (STEM), and energy-dispersive X-ray (EDX) spectroscopy to mitigate these issues provides unprecedented levels of intracellular detail in aqueous specimens. This study demonstrates the potential of LP-STEM to examine and identify internal cellular structures in thick biological samples. Specifically, it highlights the use of LP-STEM to investigate the radiation resistant, gram-positive bacterium, Deinococcus radiodurans using various imaging techniques.
RESUMEN
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1-2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and manual segmentation are two critical problems that limit the more extensive use of FIB-SEM technology. Here, we present an easily accessible robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for semi-automated data segmentation using the open-source machine learning Weka segmentation software, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others.