Observation for biomaterials by high voltage electron microscopy (HVEM) shows the value for three-dimensional reconstruction of ultrastructure. The technology of the tomography observation is advancing recently, and the application is expected much in addition to the HVEM stereo-observation. So, this technique gives us to a capacity for investigating the ultrastructure not only in the limited, but also the wide area. It is suggested that the HVEM shows a capability to help us for developing researches, with unique good ideas for research strategy. In the paper, the values of HVEM for biological research with our recent scientific data about the morphofunctinal changes in the neurons and the astroglia under different corticosterne conditions using HVEM are discussed.
High voltage electron microscopy (HVEM) is one of powerful tools to be able to visualize the three-dimensional structures of cell organelles. However, this technique cannot be effectively realized without histochemical techniques that give electron densities on various cell organelles selectively. In this section, various staining techniques used for HVEM so far and their applications will be explained showing the examples.
Biological applications of high voltage electron microscopy (HVEM) enable us to examine thick sections embedded in epoxy resin by high energy/accelerating voltage. With this advantage, we can reveal three-dimensional (3D) structure of cell and tissue embedded in the thick section. Recent progress in computer analyses applied to HVEM has created a digital 3D-analysis called “tomography”, with which we can further analyze ultrastructure of neurons and glial cells organizing neural circuits in the brain more accurately. In this article, we would like to show some examples of our recent study by HVEM and discuss possibilities for the future.
The high voltage electron microscopy (HVEM) opens new windows of morphological study in medical and biological science due to improvement in observations of thick sections. Three-dimensional analysis by electron tomography provides us new findings in histopathological observation. As an example, morphological investigation of cerebral edema is reviewed in this chapter.
To clarify the pathophysiology of hypertensive cerebral edema, morphological changes in cerebral cortex of stroke-prone rats were investigated using both usual transmission electron microscope (TEM) and high voltage electron microscopy (HVEM). Although there were distinct limitations to investigating the pathogenesis of cerebral edema with usual TEM pictures, additional pathological findings such as vacuolic changes of dendrite spine in nerve cells and active adhesion of leukocytes to endothelial cells in micro vessels were revealed by observation of thick section with HVEM and electron tomography. Utilization of HVEM seems to be very useful to fill the image gap between the light and electron microscope.
Three-dimensional structural analyses of human hair fibers and comparison of the different fibers were tried by using the High Voltage Electron Microscopy (HVEM).
The analysis condition, sample preparation, and a machine state were adjusted to the suitable condition for tilting observation of from -70 degree to +70 degree, at 2 degrees intervals. The tomography of hair fiber was successfully reconstructed from the different angle pictures with IMODE software in a computer. By using HVEM, the various human hair fibers from Japanese and Caucasians were investigated and discussed about their structures.
Since the electron microscope was first historically developed by von Ardenne in 1930’s, various improvements such as the use of condenser, osmium as a fixative and microtome were achieved to dramatically advance the capability of the electron microscope. Fernandez-Moran (1947) and Tsunoda (1958) are recognized as pioneers of the ultrastructural studies of brain tumors, and various unique ultrastructures of the brain tumors were revealed with the introduction of the electron microscope. Since then, new types of brain tumors were identified one after another. Moreover, electron microscopy contributed in demonstrating the various trends of differentiation of the versatile brain tumors. Finally, electron microscopy can be further applied in evaluating the malignancy level and functional studies of the brain tumors, and further discoveries will be expected in the future.
Raman scattering, coherent anti-Stokes Raman scattering (CARS), harmonic generation can be used to image biological molecules in living cells without labeling. Raman scattering and CARS detect frequencies of molecular vibrations and use them to obtain the molecular distributions in samples. Second-harmonic generation is generated in array structures of noncentrosymmetric molecules and can be used to detect proteins, such as, collagen, myosin and tubulin. Since labeling techniques using chemical and biological reactions may cause a undesirable change in the sample, label-free molecular imaging techniques are significant for observation of living samples.
The anatomical studies are considered to be fundamentals of all research fields in neuroscience. However, it is rather difficult to know the functional significance of specific structural organization by conventional anatomical studies. Therefore, we have tried to apply the spatial pattern analysis to anatomical research. The spatial pattern analysis is a statistical methodology, which helps to understand the mechanisms underlying specific distribution patterns. According to this technique, we have examined the spatial distribution of microglia and astrocytes in the mouse hippocampus. Our results showed the domain structure of microglia and also suggested the attraction between microglia and astrocytes. In this review, I further discuss the potential value of the spatial pattern analysis in modern neuroanatomical research.
By improving coherence and brightness of electron beam, high-angle annular dark field scanning transmission electron microscopy (HAADF STEM) enables us to obtain incoherent images with comparable resolution of conventional high-resolution transmission electron microscopy. Recently, developments in correcting the aberration of the lens have pushed achievable spatial resolution into sub-ångstrom, thus providing a new level of analysis for local structures as well as electric states in areas such as nanotechnology. This review mainly shows how bight-field STEM images and Cs-corrected STEM images are understood through several key points.
The three-dimensional ultrastructure of the Golgi apparatus in different cells－epithelial principal cells in the epididymal duct, gonadotrophs in the pituitary gland, and dorsal root ganglion cells－was observed by scanning electron microscopy (SEM) of osmium-macerated tissues. The Golgi apparatus in the epididymal principal cells was shaped like a candle flame or cup with the cis-most cistern outside. In the gonadotrophs, flat cisterns of the Golgi apparatus were piled up concentrically to form a spherical structure with the cis-most cistern outside. The dorsal root ganglion cells had numerous small Golgi stacks, each of which was composed of several cisterns piled up in layers. At a higher magnification, the cis-most cistern was rather uniform in structure and was composed of a flattened cistern with numerous small fenestrations on its surface. On the other hand, the shape of the trans-most cisterns depended on the cell type, although it was basically composed of tubules and/or flattened plates which were sometimes connected with each other. The trans-most cistern was often associated with rER, but no direct connection was observed between them.
The gate dielectrics separate gate electrode and channel, and it is one of the key components of the metal-oxide-semiconductor field effect transistors (MOSFETs). The microscopic properties of gate dielectrics considerably affect the device performance of the nano-meter scaled MOSFETs. Nowadays, high permittivity (high-k) metal oxides are expected to replace conventional silicon dioxides (SiO2) as the gate dielectrics for the next-generation MOSFETs. In this paper, we introduce the results of the observation of the charge distributions within high-k films through scanning capacitance microscopy (SCM) measurements.
Positron interactions with matter provide different signals with electron ones and the positron microbeam enables us to obtain information on the arrangement of atoms in the first few atomic layer and to the two-dimensional mapping of vacancies. A novel method for the positron microbeam has been recently designed and developed in the connection with a high intense linac-based positron source. It consists of the extraction of the positron beam from the magnetic field and the brightness enhancement by Ni(100) thin foil. In this paper, transmission positron microscopy and positron probe microanalyzer are described for the structural analysis and the defect mapping, respectively.
Synaptic organization contributes important role in exerting output of neuron. Here we present the 3-D reconstructed image of the synaptic organization in cerebellar Purkinje cell (PC) from transmission electron microscopic analysis. The serial ultrathin sections in the horizontal plane were prepared from the basal pole of cell soma to the distal end of PC dendrites. Purkinje cell dendrite is categorized into three domains according to synapse input: the proximal domain innervated by climbing fibers (CF), but not by parallel fibers fibers (PF), the intermediate domain with mixed CF/PF innervation, and the distal domain innervated by PF, but not by CF. In addition, single climbing fiber stem, which climb up along Purkinje cell dendrite from soma-dendrite border, divides into many branches with associating dendritic branches. Thus, single climbing fiber stem exclusively innervates single Purkinje cell. Recently, the molecules; Glutamate receptor δ2 subunit, Cbln1, and P/Q type Ca2+ channel α1A subunit, are reported to perform formation and maintenance of the characteristic synaptic organization, as well as the existence of parallel fibers and the electrical activity of climbing fiber.
To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell-types such as inflammation and atherosclerosis, real-time and three-dimensional analysis of cell dynamics in vivo is required. We developed a visualization technique based on confocal laser microscopy that enabled us to analyze the three-dimensional structures and cellular dynamics in vivo with high spatiotemporal resolution (2007 Diabetes, 2008 JCI). We applied this technique to visualize cellular interplay in adipose tissue obesity to elucidate the mechanism of metabolic syndrome, a major cause of cardiovascular disease.
We found close spatial and temporal interrelationships between angiogenesis and adipogenesis, and both were augmented in obese animals. In the microcirculation of obese visceral adipose tissue, we also found increased leukocyte-platelet-endothelial cell interactions, which were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Platelets were also activated locally in obese visceral adipose. Our results clearly demonstrated the power of our imaging technique to analyze complex cellular interplays in vivo and to evaluate new therapeutic interventions against them. Results also indicate that visceral adipose tissue obesity is an inflammatory disease.