Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1.

Autophagosomes and lysosomes work in tandem to conduct autophagy, an intracellular degradation system which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, specific identification of the various components of the cellular degradation machinery remains difficult. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in autophagy. We provide methods that avoid common pitfalls, including a detailed explanation for how to distinguish lysosomes and lipid droplets and discuss the differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. In addition to TEM, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling, which can be utilized for the reliable and accurate classification of cellular organelles. Our results show how these methods may be employed to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or the ablation of dynamin-related protein 1.

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high‐resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria are essential for the production of ATP, which occurs in the cristae, the folds of the inner mitochondrial membrane. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging are unknown. Further, we do not understand how 3D mitochondrial networks and the specialization of mitochondria alter during aging. Methods: We measure changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels are also measured through 3D reconstruction. CRISPR/Cas9 KD is performed to examine changes in mitochondria upon loss of MICOS complex and OPA-1. Metabolomics are used to find key metabolite pathways changed upon MICOS complex loss. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, and cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle across aging. Cardiac muscle showed similar differences but less fragmentation and wide-spread changes across 2-year aaging. We also found an association of optic atrophy 1 (OPA-1) and the MICOS complex in the gastrocnemius with mitochondrial aging, decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: We are the first to examine mitochondria changes in skeletal muscle and cardiac muscle across aging. 3D reconstructions of nanotunnels elucidated novel patterns in skeletal muscle. Notably, we noticed differences in skeletal and cardiac muscle that suggests a differential response to mitochondrial aging in cardiac and skeletal muscle. Importantly, we found similar changes in mitochondrial morphology were observed in aging skeletal muscles and for loss of MICOS proteins in mouse skeletal muscle. Furthermore, MICOS proteins decreased with age. In tandem, this suggests a relationship between the MICOS complex and aging, which further 3D reconstruction could potentially further link to disease states.