Microscopic tomography with ultra-HVEM and applications

Takaoka, Akio; Hasegawa, Toshiaki; Yoshida, Kengo; Mori, Hirotarô

doi:10.1016/j.ultramic.2007.06.008

Cited by 45 publications

(26 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For observation by transmission electron microscope, 500-nmthick sections of the 2-month-old mouse retinas of either sex were cut with a diamond knife (Nanotome) using a Reichert Ultracut E ultramicrotome (Leica), and mounted on Formvarcoated 50 mesh square grids. Sections were stained as described previously (Takaoka et al, 2008). Colloidal gold beads (20 nm; BB International), used as fiducial markers for alignment, were applied to the section surface.…”

Section: Methodsmentioning

confidence: 99%

Presynaptic Dystroglycan–Pikachurin Complex Regulates the Proper Synaptic Connection between Retinal Photoreceptor and Bipolar Cells

Omori¹,

Araki²,

Chaya³

et al. 2012

J. Neurosci.

104

View full text Add to dashboard Cite

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC)at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin Ϫ/Ϫ retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.

show abstract

Section: Methodsmentioning

confidence: 99%

Presynaptic Dystroglycan–Pikachurin Complex Regulates the Proper Synaptic Connection between Retinal Photoreceptor and Bipolar Cells

Omori¹,

Araki²,

Chaya³

et al. 2012

J. Neurosci.

104

View full text Add to dashboard Cite

show abstract

“…We employed UHVEM (3 MeV) to reconstruct the osteocyte network in 3-lm-thick sections. The most important advantage of using this microscope was the marked increase (about 15 times as thick as that of the 0.1-MeV electron microscope) in the maximum observable thickness of specimens [5]. We succeeded in acquiring transmission electron micrographs from 3-lm-thick sections of bone at a resolution of 8 nm/pixel (Fig.…”

Section: Ultra-high Voltage Electron Microscopy For Osteocyte Morphologymentioning

confidence: 99%

“…Therefore, it is of critical importance to obtain precise morphological and/or morphometrical data from osteocytes. In the first chapter, we introduce our application of confocal laser scanning microscopy [4] and ultra-high voltage electron microscopy [5] to reveal the morphology and cytoskeleton of osteocytes. In the second chapter, we describe osteocyte biology, especially mechanosensitivity of osteocytes in vitro and in vivo analyzed by calcium imaging [6], mechanical properties of osteocytes measured by atomic force microscopy (AFM) [7], and cell-cell communication among osteocytes in vitro and in vivo analyzed by fluorescence replacement after photobleaching (FRAP) analysis [8] and a calcium imaging technique.…”

Section: Introductionmentioning

confidence: 99%

Application of Bioimaging to Osteocyte Biology

Kamioka

Adachi

2010

Clinic Rev Bone Miner Metab

View full text Add to dashboard Cite

In a variety of scientific fields, it is helpful to visualize natural phenomena. Newly developed methods of visualization often lead to breakthroughs in scientific fields. Especially, in the bioscience field, it is significant to reveal temporal-spatial responses in cells while visualizing molecular phenomena. Such visualization may provide information to help understand cellular behavior in response to an extracellular stimulus. Although osteocytes are the most abundant cells in bone, it has been difficult to study their biological nature because they are embedded in hard bone tissue. So, the real 3D structure of osteocytes was not clarified until recently. On the other hand, a newly developed technique of visualization was recently introduced into bone cell biology. In this review, we introduce and review our application of a bioimaging technique to reveal the morphology of osteocytes as well as part of their biological nature.

show abstract

“…Due to this high acceleration voltage, this technology allows us to employ thicker sections than before [6]. Thus, it is possible to observe a specimen of several micrometers thickness at a nano-level resolution using UHVEM with electron tomography [7].…”

Section: Introductionmentioning

confidence: 99%

Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy

et al. 2015

Self Cite

View full text Add to dashboard Cite

In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.

show abstract

Microscopic tomography with ultra-HVEM and applications

Cited by 45 publications

References 14 publications

Presynaptic Dystroglycan–Pikachurin Complex Regulates the Proper Synaptic Connection between Retinal Photoreceptor and Bipolar Cells

Presynaptic Dystroglycan–Pikachurin Complex Regulates the Proper Synaptic Connection between Retinal Photoreceptor and Bipolar Cells

Application of Bioimaging to Osteocyte Biology

Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy

Contact Info

Product

Resources

About