Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira

• Published in: Cells (Basel, Switzerland) Vol. 11; no. 1; p. 65
• Main Authors: Garza-Lopez, Edgar, Vue, Zer, Katti, Prasanna, Neikirk, Kit, Biete, Michelle, Lam, Jacob, Beasley, Heather, Marshall, Andrea, Rodman, Taylor, Christensen, Trace, Salisbury, Jeffrey, Vang, Larry, Mungai, Margaret, AshShareef, Salma, Murray, Sandra, Shao, Jianqiang, Streeter, Jennifer, Glancy, Brian, Pereira, Renata, Abel, E., Hinton, Antentor
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 27.12.2021; MDPI
• Subjects: 3D reconstruction; Algorithms; Amira; Animals; Apoptosis; Automation; Cancer; Drosophila; Endoplasmic Reticulum; FIB-SEM; GTP Phosphohydrolases - deficiency; GTP Phosphohydrolases - metabolism; Homeostasis; Imaging, Three-Dimensional; Ion beams; Light microscopy; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Scanning; Mitochondria; Mitochondria - ultrastructure; Morphology; Muscle Fibers, Skeletal - metabolism; Muscle Fibers, Skeletal - ultrastructure; Muscle, Skeletal - ultrastructure; Open source software; Organelles; Organelles - ultrastructure; SBF-SEM; Scanning electron microscopy; segmentation; Transmission electron microscopy; Visualization; Windows operating system; FIB-SEM; SBF-SEM; 3D imaging; 3D reconstruction; segmentation; Amira; mitochondrial imaging; organelles; volume analysis
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells11010065

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.