Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira
Edgar Garza-López, Zer Vue, Prasanna Katti, Kit Neikirk, Michelle Biete, Jacob Lam, Heather K. Beasley, Andrea G. Marshall, Taylor Rodman, Trace Christensen, Jeffrey L. Salisbury, Larry Vang, Margaret Mungai, Salma AshShareef, Sandra Murray, Jianqiang Shao, Jennifer Streeter, Brian Glancy, Renata O. Pereira, E. Dale Abel, Antentor Hinton
Abstract
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.