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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 largerx‐ andy‐axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF‐SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF‐SEM. Beyond this, the applications of SBF‐SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence‐based segmentation which may contribute to devising a feasible workflow involving SBF‐SEM, are also considered. 

Abstract 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 inSam50‐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 andSam50‐deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation inSam50‐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 inSam50‐deficient myotubes. Furthermore, impairment of oxidative capacity was observed uponSam50ablation 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 ofSam50‐deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle. 

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. 

Abstract Chronic infection withHelicobacter pyloriincreases risk of gastric diseases including gastric cancer. Despite development of a robust immune response,H. pyloripersists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection withH. pyloristrains which encode thecagType IV Secretion System (cag T4SS). ThecagT4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro‐inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response toH. pyloriinfection, the host produces a variety of antimicrobial molecules, including the iron‐binding glycoprotein, lactoferrin. Our work shows that apo‐lactoferrin exerts antimicrobial activity againstH. pyloriunder iron‐limited conditions, while holo‐lactoferrin enhances bacterial growth. CulturingH. pyloriin the presence of holo‐lactoferrin prior to co‐culture with gastric epithelial cells, results in repression of thecag T4SS activity. Concomitantly, a decrease in biogenesis ofcag T4SS pili at the host‐pathogen interface was observed under these culture conditions by high‐resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro‐inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment ofH. pylori‐related disease.