Three-dimensional fine structures in deep fascia revealed by combined use of cryo-fixed histochemistry and low-vacuum scanning microscopy.

Recent physiological studies have shown that the deep fascia has received much attention concerning clinical medicine; however, histological examination of the deep fascia has not been well established. In this study, we aimed to clarify and visualize the structure of the deep fascia by taking advantage of cryofixation techniques and low-vacuum scanning electron microscopy. As a result, the ultrastructural observations revealed three-dimensional stratification of the deep fascia composed of three layers: the first superficial layer consisting of collagen fibers extending in various directions with blood vessels and peripheral nerves; the second intermediate layer formed by single straight and thick collagen fibers with flexibility; and the third deepest layer, consisting of relatively straight and thin collagen fibers. We explored the use of two hooks to hold a piece of deep fascia in place through the course of cryo-fixation. A comparative observation with or without the hook-holding procedure would indicate the morphological adaptation to physiological stretch and contraction of the deep fascia. The present morphological approach paves the way to visualize three-dimensional ultrastructures for future biomedical studies including clinical pathophysiology.

Insertion sites of the muscles attached to the clavicle: a cadaveric study of the clavicle.

BACKGROUND: Clavicle fractures are common injuries, especially in young, active individuals. Operative treatment is recommended for completely displaced clavicle shaft fractures, and plate fixation is stronger than the use of intramedullary nails. Few studies have reported on iatrogenic injuries to the muscle attached to the clavicle during fracture surgery. The aim of this study was to clarify the area of the insertion sites of muscles attached to the clavicle in Japanese cadavers using gross anatomy and three-dimensional (3D) analysis. We also aimed to compare the effects of anterior plate templating and superior plate templating on clavicle shaft fractures using 3D images. METHODS: Thirty-eight clavicles from Japanese cadavers were analyzed. We removed all clavicles to identify the insertion sites and measured the size of the insertion area of each muscle. Three-dimensional templating was performed on both the superior and anterior plates of the clavicle using data obtained from computed tomography. The areas covered by these plates on the muscles attached to the clavicle were compared. Histological examination was performed on four randomly selected specimens. RESULTS: The sternocleidomastoid muscle was attached proximally and superiorly; the trapezius muscle was attached posteriorly and partly superiorly; and the pectoralis major muscle and deltoid muscles were attached anteriorly and partially superiorly. The non-attachment area was located mainly in the posterosuperior part of the clavicle. It was difficult to distinguish the borders of the periosteum and pectoralis major muscles. The anterior plate covered a significantly broader area (mean 6.94 ± 1.36 cm2) of the muscles attached to the clavicle than did the superior plate (mean 4.11 ± 1.52 cm2) (p < 0.0001). On microscopy, these muscles were inserted directly into the periosteum. CONCLUSION: Most of the pectoralis major and deltoid muscles were attached anteriorly. The non-attachment area was located mainly from the superior to posterior part of the clavicle midshaft. Both macroscopically and microscopically, the boundaries between the periosteum and these muscles were difficult to demarcate. The anterior plate covered a significantly broader area of the muscles attached to the clavicle than that by the superior plate.

In situ strategy for biomedical target localization via nanogold nucleation and secondary growth.

Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. However, conventional nanogold labelling requires time-consuming nanogold probe preparation and ultrathin sectioning of cell/tissue samples. Here, we introduce an in situ strategy involving nanogold nucleation in immunoenzymatic products on universal paraffin/cryostat sections and provide unique insight into nanogold development under hot-humid air conditions. Nanogold particles were specifically localized on kidney podocytes to target synaptopodin. Transmission electron microscopy revealed secondary growth and self-assembly that could be experimentally controlled by bovine serum albumin stabilization and phosphate-buffered saline acceleration. Valuable retrospective nanogold labelling for gastric H+/K+-ATPase was achieved on vintage immunoenzymatic deposits after a long lapse of 15 years (i.e., 15-year-old deposits). The present in situ nanogold labelling is anticipated to fill the gap between light and electron microscopy to correlate cell/tissue structure and function.