A workflow for visualizing human cancer biopsies using large-format electron microscopy

Abstract

Recent developments in large format electron microscopy have enabled generation of images that provide detailed ultrastructural information on normal and diseased cells and tissues. Analyses of these images increase our understanding of cellular organization and interactions and disease-related changes therein. In this manuscript, we describe a workflow for two-dimensional (2D) and three-dimensional (3D) imaging, including both optical and scanning electron microscopy (SEM) methods, that allow pathologists and cancer biology researchers to identify areas of interest from human cancer biopsies. The protocols and mounting strategies described in this workflow are compatible with 2D large format EM 2 mapping, 3D focused ion beam-SEM and serial block face-SEM. The flexibility to use diverse imaging technologies available at most academic institutions makes this workflow useful and applicable for most life science samples. Volumetric analysis of the biopsies studied here revealed morphological, organizational and ultrastructural aspects of the tumor cells and surrounding environment that cannot be revealed by conventional 2D EM imaging. Our results indicate that although 2D EM is still an important tool in many areas of diagnostic pathology, 3D images of ultrastructural relationships between both normal and cancerous cells, in combination with their extracellular matrix, enables cancer researchers and pathologists to better understand the progression of the disease and identify potential therapeutic targets.

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

“…This creates its own set of issues as detailed above, but there is little doubt that FIB-SEM has the potential to reveal new biology in a variety of systems. One area to look forward to is the speeding up of FIB-SEM protocols in order to make it more practical in the diagnostic laboratory [ 111 ], and another is the application of ever more powerful deep learning and other computational approaches to extract meaningful insights from these giant datasets. At the same time, it behooves virologists to familiarize themselves with these methods and think imaginatively about research questions that fully exploit the potential of FIB-SEM, while being realistic and careful about their drawbacks.…”

Noise Reconstruction & Removal Network: A New Architecture to Denoise FIB-SEM Images

“…In addition, we look at the interquartile range (IQR) of the signal across a straight line in an area with flat signal. In the FIB-SEM case, the entire workflow from tissue collection to final image harvesting takes roughly two weeks for 1500 images (Riesterer, López, Stempinski, Williams, Loftis, Stoltz, Thibault, Lanicault, Williams & Gray 2020). During this process the clinical specimen undertakes several epoxy resin infiltration steps to fill the space between the cellular structures.…”

An Omic and Multidimensional Spatial Atlas from Serial Biopsies of an Evolving Metastatic Breast Cancer

“…Figures 4 and S4 show changes between the primary tumor and three metastases in the composition and functional status of individual leukocyte lineages using multiplex immunohistochemistry (mIHC; (Banik et al, 2020;Tsujikawa et al, 2017;Tsujikawa et al, 2019)). Figures 5 and S5 assess tumor and microenvironment cellular composition, functional state, and spatial organization using cyclic immunofluorescence (CycIF; (Eng et al, 2020;Lin et al, 2015)) and focused ion beam-scanning electron microscopy (FIB-SEM; (Riesterer et al, 2020)), highlighting differences between the metastatic lesions. Figures 6 and S6A show additional two-dimensional (2D) and three-dimensional (3D) cellular and subcellular features, with potential therapeutic relevance revealed using FIB-SEM.…”

“…We explored the ultrastructure of inter-and intracellular interactions in large format 2D and targeted 3D images acquired using FIB-SEM as previously described (Riesterer et al, 2020), with emphasis on aspects of nanobiology that might affect therapeutic response. Computational renderings of the 3D images were particularly informative in Bx1 (Movies S1, S2) and Bx2 (Movie S3).…”

“…A more detailed description of the fixation procedure, sample preparation protocol, and imaging workflow can be found in a recently published open access book chapter (Riesterer et al, 2020) and via protocols.io: "Sample fixation of biopsy tissue for Electron Microscopy (EM)" dx.doi.org/10.17504/protocols.io.4bigske "Post-Fixation Heavy Metal Staining and Resin Embedding for Electron Microscopy (EM)" dx.doi.org/10.17504/protocols.io.36vgre6 "2D and 3D Electron Microscopy (EM) Imaging of Tissue Biopsies and Resections" dx.doi.org/10.17504/protocols.io.bg58jy9w…”