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
, 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.
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.
Abstract
Background and study aim
Magnetic imaging technology is of proven benefit to trainees in colonoscopy, but few studies have examined its benefits in experienced hands. There is evidence that ...colonoscopy is more difficult in women. We set out to investigate (i) associations between the looping configurations in the proximal and distal colon and (ii) differences in the looping prevalence between the sexes. We have examined their significance in terms of segmental intubation times and position changes required for the completion of colonoscopy.
Patients and methods
We analyzed 103 consecutive synchronized luminal and magnetic image videos of diagnostic colonoscopies with normal anatomy undertaken by a single experienced operator.
Results
Deep transverse loops and sigmoid N-loops were more common in females. A deep transverse loop was more likely to be present if a sigmoid alpha-loop or N-loop had formed previously. Patients with sigmoid N-loops were turned more frequently from left lateral to supine before the sigmoid-descending junction was reached, but there was no statistical correlation between completion time and looping pattern.
Conclusions
This study has reexamined the prevalence of the common looping patterns encountered during colonoscopy and has identified differences between the sexes. This finding may offer an explanation as to why colonoscopy has been shown to be more difficult in females. Although a deep transverse loop following a resolved sigmoid alpha-loop was the most commonly encountered pattern, no statistical correlation between completion time and looping pattern could be shown. It is the first study to examine segmental completion times using a magnetic imager in expert hands.
Introduction
This study examines sex differences in the disposition of the sigmoid and transverse segments of the colon in undisturbed cadaveric abdomens and relates these findings to the anecdotal ...observation that colonoscopy is more challenging in females through the formation of tortuous bowel loops.
Materials and Methods
One hundred and twenty two undisturbed cadaveric abdomens were inspected. Three distinct configuration grades were separately assigned to the sigmoid and transverse segments of the colon on the basis of the pattern of the bowel loops observed in situ. Pearson's chi‐squared test was used to analyze sex differences in bowel loop configuration and Spearman's rank correlation coefficient was calculated to identify co‐occurrence of configuration grades in the subjects.
Results
For the transverse segment, females had higher configuration grades corresponding to longer bowel loops with greater redundancy, compared to males (p = .000047). There was no sex difference in the sigmoid segment grade (p = .21636). Sigmoid and transverse segment grades were highly correlated in the subjects (coefficient = 0.9994).
Conclusion
Sex differences in the configuration grades of the sigmoid or transverse colonic segments may be a significant contributing factor to increased difficulty of colonoscopy in females.
With sparse treatment options, cardiac disease remains a significant cause of death among humans. As a person ages, mitochondria breakdown and the heart becomes less efficient. Heart failure is ...linked to many mitochondria-associated processes, including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress. The roles of key mitochondrial complexes that dictate the ultrastructure, such as the mitochondrial contact site and cristae organizing system (MICOS), in aging cardiac muscle are poorly understood. To better understand the cause of age-related alteration in mitochondrial structure in cardiac muscle, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the three-dimensional (3-D) networks in cardiac muscle samples of male mice at aging intervals of 3 mo, 1 yr, and 2 yr. Here, we present the loss of cristae morphology, the inner folds of the mitochondria, across age. In conjunction with this, the three-dimensional (3-D) volume of mitochondria decreased. These findings mimicked observed phenotypes in murine cardiac fibroblasts with CRISPR/Cas9 knockout of
(some members of the MICOS complex), and
, which showed poorer oxidative consumption rate and mitochondria with decreased mitochondrial length and volume. In combination, these data show the need to explore if loss of the MICOS complex in the heart may be involved in age-associated mitochondrial and cristae structural changes.
This article shows how mitochondria in murine cardiac changes, importantly elucidating age-related changes. It also is the first to show that the MICOS complex may play a role in outer membrane mitochondrial structure.
During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of ...mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.
These studies have provided a more comprehensive understanding of the structural and functional changes associated with aging and loss of the MICOS complex. Together, this verifies there is an age‐related loss of both mitochondria structure and the MICOS complex, and shows that the MICOS complex affects the mitochondrial structure, thus suggesting that the MICOS complex plays an evolutionarily conserved role in age‐dependent mitochondrial structural and functional decline.
The sorting and assembly machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex ...connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.
Featured Cover Vue, Zer; Garza‐Lopez, Edgar; Neikirk, Kit ...
Aging cell,
December 2023, Letnik:
22, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Cover legend: The cover image is based on the Research Article 3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex by Zer Vue et al., ...https://doi.org/10.1111/acel.14009
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.
While the role of brown adipose tissue (BAT) in the aging process is known to be important, alterations in mitochondrial and cristae 3D structure, and their related function, remain unclear. Using serial block face‐scanning electron microscopy, the study defines the nature of the mitochondrial and cristae structure in murine BAT across aging.
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