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.
Highlights
Research on the physiology and metabolism of organelles is critical to understanding vital cellular processes such as apoptosis, respiration, and mitosis, and metabolic organelles such as mitochondria and endoplasmic reticulum (ER) have been well characterized
Research on cardiovascular health and diabetes demonstrates that ultrastructural imaging is essential to understanding how genes, proteins, and other macromolecules alter organelle morphology [51,52,53], and several methods have been used to quantify the morphology of organellar structures [52,53,54]
Reconstruction using serial block-face scanning electron microscopy (SBF-Scanning electron microscopy (SEM)), which is common in biomedical research, and a protocol for using Amira software that may be printed for lab use and reference (Supplemental Material S1)
Summary
Research on the physiology and metabolism of organelles is critical to understanding vital cellular processes such as apoptosis, respiration, and mitosis, and metabolic organelles such as mitochondria and endoplasmic reticulum (ER) have been well characterized Because these organelles play major roles in regulating homeostasis and ensuring organism survival, it is important to study the relationships between their structure and function. Volume electron microscopy, which refers to SEM techniques that can analyze relatively large volume structures, generates near-TEM resolution images using backscatter detection from a block face rather than from an ultrathin section. We describe the use of Amira for segmentation and 3D reconstruction of images acquired through SBF-SEM that can increase our understanding of structure–function relationships in the context of (1) mutations that alter cellular components, and (2) the therapeutic target potential of cellular components. Our paper outlines how Amira can be used for 3D reconstruction of mitochondria in SBF-SEM, Amira can potentially be used to 3D reconstruct any volumetric raw data from any equipment
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