Vang, L. | bioRxiv Science

bioRxiv2021

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/461807v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c40041org.highwire.dtl.DTLVardef@ecde92org.highwire.dtl.DTLVardef@10870daorg.highwire.dtl.DTLVardef@1290c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Systematic Transmission Electron Microscopy-Based Identification of Cellular Degradation Machinery

Many interconnected degradation machineries including autophagosomes, lysosomes, and endosomes work in tandem to conduct autophagy, an intracellular degradation system that 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, identification of specific components of the autophagy machinery has been challenging. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in macro autophagy. We provide methods that help avoid common pitfalls, including a detailed explanation for distinguishing lysosomes and lipid droplets and discuss 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. We also utilize serial block face-scanning electron microscopy (SBF-SEM) to offer a protocol for visualizing 3D morphology of degradation machinery. In addition to TEM and 3D reconstruction, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling that can be utilized to reliably and accurately classify cellular organelles. Our results show how these methods may be used to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

biophysics↗