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

Edgar Garza-Lopez, Zer Vue, Prasanna Katti, Kit Neikirk, Michelle Biete, Jacob Lam, Heather K. Beasley, Andrea G. Marshall, Taylor A. Rodman, Trace A. Christensen, Jeffrey L. Salisbury, Larry Vang, Margaret Mungai, Salma Ash Shareef, Sandra A. Murray, Jianqiang Shao, Jennifer Streeter, Brian Glancy, Renata O. Pereira1, E. Dale Abel, and Antentor Hinton, Jr. - Hinton and Garza Lopez Family Consulting Company, Iowa City ; Department of Molecular Physiology and Biophysics, Vanderbilt University ; National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda ; Department of Biology, University of Hawaii at Hilo ; Department of Internal Medicine, Carver College of Medicine, University of Iowa ; Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, School of Graduate Studies and Research, Meharry Medical College, Nashville ; Microscopy and Cell Analysis Core Facility, Mayo Clinic, Rochester ; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester ; Central Microscopy Research Facility, University of Iowa ; Fraternal Order of Eagles Diabetes Research Center, Iowa City

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.

How Amira-Avizo Software is used

High-quality images obtained by SBF-SEM or other data acquisition methods can be reconstructed in 3D using Amira. Amira is a user-friendly application that allows 2D-image slices, known as orthos, to be digitally analyzed, segmented, color-coded, and rendered for 3D reconstruction.