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Three-dimensional transmission and scanning electron microscopy of molecular and cellular structures Caffrey, Brian James

Abstract

Unravelling the complex spatial arrangement of networks and interfaces between proteins, cells and tissues is fundamental to our understanding of healthy and pathological processes. Therefore, a three-dimensional ultrastructural understanding of this arrangement is key to developing modern diagnostic and therapeutic applications in disease. Herein, we discuss our application of transmission and scanning electron microscopy techniques such as Cryo-Electron Microscopy (Cryo-EM), Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) alone and in combination with light microscopy, to develop an understanding of biological processes at the nanoscale. Cryo-EM is a now established technique for the elucidation of protein structures at atomic resolutions. A key advantage of cryo-EM methods over other structural techniques is its ability to generate 3D maps of proteins in solution, in their native environments. Here, we apply cryo-EM techniques to mutant structures of the protein p97 towards a molecular understanding of the mechanisms of p97-related disease. We solved eight nucleotide-bound cryo-EM structures of four full-length hexameric mutants (R155H, A232E, D592N and E470D) implicated in p97-related neurodegenerative disease. 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 developed a semi-automated segmentation method that enables rapid, large-scale acquisition of data from tissue specimens. 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. Correlated Light Electron Microscopy (CLEM) combines the high-resolution isotropic resolving power of electron microscopies with the discriminatory power of fluorescent light microscopy. Here, we present a CLEM study of molecular interactions between nanoparticles and cells, enabling us to describe the path of a model nanoparticle through the cell and identify key stages of nanoparticle uptake in greater detail than either methodology used independently.

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