Loss of mitochondrial membrane potential (ΔΨm) triggers dramatic structural changes in mitochondria from a tubular to globular shape, referred to as mitochondrial fragmentation; the resulting globular mitochondria are called swelled or ring/doughnut mitochondria. We evaluated the early period of structural changes during the ΔΨm loss-induced transformation after carbonyl cyanide m-chlorophenyl hydrazine (CCCP) administration using a newly developed correlative microscopic method combined with fluorescence microscopic live imaging and volume electron microscopy. We found that most mitochondria changed from a tubular shape to a globular shape without fusion or fission and typically showed ring shapes within 10 min after CCCP exposure. In contrast, most ring mitochondria did not have a true through hole; rather, they had various indents, and 47% showed stomatocyte shapes with vase-shaped cavities, which is the most stable physical structure without any structural support if the long tubular shape shortens into a sphere. Our results suggested that loss of ΔΨm triggered collapse of mitochondrial structural support mechanisms.

Previous investigators have reported that transplanted demineralised dentin matrix (DDM) influences bone formation in vivo. However, the specific mechanism of how dentinal tubules contribute to bone formation has not been determined with regard to DDM transplantation therapy. In this study, we ultrastructurally investigated how DDM contacted the surrounding newly formed bone using a scanning electron microscopy (SEM) three-dimensional reconstruction method that is based on focused ion beam slicing and SEM (FIB/SEM). A pulverised and processed DDM derived from human teeth was implanted into rat calvarial bone defects, and a series of X-ray computed tomographic images were obtained over 12 weeks. Implants with surrounding new bone were removed and histologically examined using FIB/SEM. After obtaining objective block-face images, the target boundary face was reconstructed three-dimensionally. The osteocytes of the new bone tissue surrounding the DDM formed a network connected by their cellular processes and formed bone tissue. It is also interesting that the cellular processes of the osteocytes extended into the dentinal tubules, and that bone tissue with canaliculi had formed and filled the DDM surface.

These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

We report a case of smoking-related idiopathic nodular glomerulosclerosis (ING) with overexpression of glomerular advanced glycation end products (AGEs) and their receptor (RAGE). A 59-year-old Japanese man with nephrotic syndrome, who had a smoking history of one pack of cigarettes per day for approximately 40 years, presented with a 3-year history of urinalysis abnormalities without clinical evidence of diabetic mellitus. The patient's leg edema progressively worsened over the previous 2 years, and he was admitted to our hospital. Renal biopsy showed mesangial expansion with diabetic Kimmelstiel-Wilson-like nodular lesions, glomerular basement thickening, and arteriosclerosis. No electron-dense deposits, fibrils, or microtubule deposits were seen in the glomeruli on electron microscopy. Skin AGE level measured using AGE reader was higher in this case than the average level in age-matched Caucasians. In addition, immunohistochemical analysis revealed that N-carboxymethyl lysine, one of the major AGEs, and RAGE were overexpressed and podocin expression was decreased in the peripheral area of the glomerular nodular lesions. These observations suggest that AGEs-RAGE system may be activated in smoking-related ING, possibly leading to the progression of renal dysfunction.

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The ultrastructure of the nuclear envelope (NE) and redundant NE (RNE) of the spermatozoon cannot be observed in detail using conventional electron microscopy. Thus, this study aimed to employ transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography to fill this research gap. Male mice aged 13 weeks were deeply anesthetized, and the testes and vas deferens were extracted and processed for electron microscopy. In round spermatids, the acrosomal vesicle compressed the nucleus, and the acrosomal center was depressed. The nucleoli concentrated on the contralateral side of the acrosome formation site. In mature spermatozoa, the RNE accumulated in the neck with the residual bodies. The NE pores exhibited a hexagonal pattern. The body surface area and volume of the nuclei of spermatids and spermatozoa in each maturation phase were analyzed using FIB/SEM tomography. The body surface area and volume of the nuclei decreased during spermatid maturation into spermatozoa. The RNE converged at the sperm neck and possessed a honeycomb structure. The method used revealed that the nuclei of spermatids gradually condense as they mature into spermatozoa. This method may be used to analyze small tissues, such as RNE, and detect morphological abnormalities in microtissues, such as spermatozoa.

Conventional imaging for three-dimensional (3D) ultra-architectural analysis of collagen fibers and fibroblasts is time-consuming and requires numerous ultrathin sections to search the target area. Currently, no method allows 3D ultra-architectural analysis of predetermined areas including spatial relationships between collagen fibers and fibroblasts in vitro. Herein, we developed a new method for in vitro analysis of the 3D ultrastructure of fibroblasts and collagen fibers using CLEM optimized for picrosirius red staining and FIB/SEM tomography. Collagen fibers were observed between, rather than on top of, stacked cells. This method offers the advantage of mesoscopic and ultrastructural analysis, thus minimizing bias and ensuring accurate observation.