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.
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Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira
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.
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- Award ID(s):
- 2011577
- NSF-PAR ID:
- 10326356
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Date Published:
- Journal Name:
- Cells
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2073-4409
- Page Range / eLocation ID:
- 65
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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.
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Abstract Serial block face scanning electron microscopy (SBF‐SEM), also referred to as serial block‐face electron microscopy, is an advanced ultrastructural imaging technique that enables three‐dimensional visualization that provides larger
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The equine hoof wall has a complex, hierarchical structure that can inspire designs of impactresistant materials. In this study, we utilized micro-computed tomography (µ-CT) and serial blockface scanning electron microscopy (SBF-SEM) to image the microstructure and nanostructure of the hoof wall. We quantified the morphology of tubular medullary cavities by measuring equivalent diameter, surface area, volume, and sphericity. High-resolution µ-CT revealed that tubules are partially or fully filled with tissue near the exterior surface and become progressively empty towards the inner part of the hoof wall. Thin bridges were detected within the medullary cavity, starting in the middle section of the hoof wall and increasing in density and thickness towards the inner part. Porosity was measured using three-dimensional (3D) µ-CT, twodimensional (2D) µ-CT, and a helium pycnometer, with the highest porosity obtained using the helium pycnometer (8.07%), followed by 3D (3.47%) and 2D (2.98%) µ-CT. SBF-SEM captured the 3D structure of the hoof wall at the nanoscale, showing that the tubule wall is not solid, but has nano-sized pores, which explains the higher porosity obtained using the helium pycnometer. The results of this investigation provide morphological information on the hoof wall for the future development of hoof-inspired materials and offer a novel perspective on how various measurement methods can influence the quantification of porosity.more » « less
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Abstract Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner‐membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, it is hypothesized that significant morphological changes in BAT mitochondria and cristae will be present with aging. A quantitative 3D electron microscopy approach is developed to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, the 3D morphology of mitochondrial cristae is investigated in adult (3‐month) and aged (2‐year) murine BAT tissue via serial block face‐scanning electron microscopy (SBF‐SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, an increase is found in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/doi.org/10.3390/cells11010065
