Blood vessels help to establish the circulatory system, which is essential for exchanging gas, nutrients, and waste. The blood vessels also play a vital role in developing embryo. A whole-embryo culture method allows quail embryo to develop outside the eggshell by keeping the circulatory system intact. To take advantage of the easy accessibility to developing quail embryos, a transgenic line Tie1:H2B::EYFP that expresses H2B-tagged EYFP in endothelial cells under the control of the Tie1 promoter gene was generated. The Tie1:H2B::EYFP transgenic quail enables us to image the nuclei of every blood vessel endothelial cell in the living embryo with the use of confocal laser microscopes. EYFP signals in the individual endothelial cells are automatically tracked by computer analysis to determine their behaviors and their collective morphometric changes during the blood vessel formation. This manuscript introduces endothelial cell behaviors in higher vertebrates revealed by time-lapse imaging of the Tie1:H2B::EYFP transgenic quail embryo.
Across species, to out-do competitors, during sperm competition between sperm from multiple males to fertilize limited number of eggs, great variations in sperm morphology have evolved. Sperm tail of Drosophilidae elongate up to 6 cm as a result of sexual selection for longer sperm by female. Sperm elongation takes place post meiotically and can proceed in the absence of an axoneme. We used primary cultures of spermatids of D. melanogaster to demonstrate that sperm elongation is driven by interdependent extension of giant mitochondria and cytoplasmic microtubules that is organized around the mitochondrial surface. Defects in sperm elongation in mutants of Milton-dMiro complex, a linker for mitochondria and microtubule motor Kinesin, suggests mitochondria-microtubule interaction is essential for the elongation. It is demonstrated that, in addition to the function as energy source, mitochondria can serve as internal skeleton for shaping sperm morphology.
Recent advances of light microscopy and tissue culturing allow us to observe cellular behavior in an intact tissue. In this article, such methods and novel concept of cellular migration are presented by introducing following study, analyzing the apical-basal nuclear oscillation in polarized neural progenitor cells in the developing brain. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1 phase and apically during G2 phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Our study demonstrates that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. The microtubule-associated protein Tpx2 links cell cycle progression and autonomous apical nuclear migration by promoting nuclear migration by altering microtubule organization during G2 phase. In contrast, in vivo observations of implanted microbeads and computational modeling imply that the basal migration of G1 phase nuclei depends on a “crowding effect” by G2 phase nuclei migrating apically. The proposed model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.
Development of bioimaging enables us to obtain various image and movie data on the dynamics of biological phenomena. However, the evaluation of these data largely depends on the visual inspection by our eyes, and therefore the quantitative information that these data contain has not yet been maximally exploited. In addition, the growing amount of image data makes visual inspection unfeasible. Bioimage information provides us with technology to overcome these problems by enabling quantitative analysis of image data and automated screening of huge amount of image data. In this article, I review the background and basic information on the bioimage informatics with a plenty of the latest references. More specific problems on object detection, boundary identification, and quantification of molecule abundance are illustrated. Finally, I discuss a couple of yet-to-be-solved issues in this field.
Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-releveant (µM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules bound, or occupancy of the ribosome, in real time. The methods outlined here have broad application to the study of mRNA sequences, and the mechnanism and regulation of translation.
Recent developments of aberration-correction electron optics have successfully generated coherent electron beam with large convergent angles, which are now capable for annular-bright-filed (ABF) imaging in a scanning transmission electron microscope (STEM) and enable to detect extremely light atoms such as lithium and hydrogen. In this short article, based on reciprocity we attempt to interpret the origin of high-sensitivity of ABF imaging in relation with a hollow-cone illumination transmission electron microscopy. We also demonstrate that the hydrogen atom columns in a hydride crystal can be distinctly imaged as dark-dot, as anticipated from phase-contrast of a weak-phase object.
We have developed and applied the tissue-engineered periosteal sheets in periodontal regenerative therapy. Periosteal sheets are simply prepared by explant culture and characterized as a highly integrated, tissue-engineered tissues composed mainly of cell-multilayers, abundant deposition of extracellular matrices. The osteogenic induction drastically up-regulates alkaline phosphatase activity and mineral deposit formation. This indicates that periosteal sheets contain many immature osteogenic progenitor cells, but the possible contamination of tissue-specific stem cells, which could eventually contribute to the osteogenic capacity, is not yet ruled out. In addition, periosteal sheets could be expected to function as “a living drug-delivery system (DDS)” to provide major growth factors involved in bone metabolism. Therefore, we believe the autologous periosteal sheet is a promising grafting material that is alternative to the autologous bone grafting in periodontal regenerative therapy. To expand this cell-based therapeutic methodology from periodontal field to other related fields, such as oral and maxillofacial surgery, plastic surgery, and orthopedic surgery, we are now investigating to improve the tissue-processing procedure, for example, by optimization of culture media and scaffolding materials.
Recently, one of the dental treatments for the loss of teeth has been prosthetic treatment with implants. The environment surrounding implants has entered a new phase following Prof. Branemark’s osseointegration proposed approximately 40 years ago. There has been considerable interest in finding novel applications and functions for existing dental implant materials. Recent implant fixtures stabilize osseointegration quicker by controlling the titanium surface through various methods. This acquiring of good clinical initial fixation is very important. The modification of the surface property of the titanium is conducted in many ways. From the viewpoint of cell adhesion, the construction of foundation influences cellular behavior leading to subsequent differentiation. We reported on an approach using various microscopes in examining the surface property of the new modified nanostructure of the titanium.
The present paper describes a method to analyze anisotropy of unoccupied states above the Fermi level by using inelastic scattering patterns of fast electrons accompanied by inner-shell excitation. First, we describe how inelastic scattering patterns are observed by the energy-selected diffraction technique by showing examples of the |1s>→|π*> transition of graphite, then, we introduce our method to visualize partial density of states (pDOS) of the unoccupied states. In the present method, a series of inelastic scattering patterns obtained at successive energy-losses are quantitatively decomposed by using basis patterns calculated from orbitals which are possible as unoccupied states above the Fermi level. As a result, we can obtain the energy-loss dependence of the magnitudes of the components, that is, partial EELS (pEELS) spectra, which are closely related to pDOS of materials. The present method is applied to the derivation of pEELS spectra of carbon nanotube and superconductor MgB2, and to the decomposition of the d hole state of a high-Tc superconductor of Bi2Sr2CaCu2O8.
Conventional microscopes have the resolution limit of approximately 200 nm due to the diffraction limit. To break this limit, a lot of approaches have been suggested. Recently, several approaches are applied into the microscopes by manufactures and became available in the market.“Structured Illumination Microscope (SIM) ” is the one of those approaches that enables us to get super resolution fluorescent images with the resolution of approximately 100nm laterally and 300nm axially. In SIM microscope, the illumination pattern is “stripe patterned” illumination which is called “structured illumination”. With this pattern of illumination, the small structure information is acquired as “moiré effect”. By analyzing these acquired image data, a super resolution image is generated.“Localization Method” is another approach to get super resolution image. This is based on single-molecule-imaging. Using photo-activatable dyes, molecules are stochastically activated and dots localizations are recorded. Repeating this process, super resolution image is generated by accumulating dots. This approach gives the resolution of approx. 20 nm laterally, 50 nm axially.
Positron probe microanalyzer (PPMA) which is a positron lifetime measurement system using a pulsed slow-positron microbeam with a minimum diameter of 30 micrometers has been developed in AIST, with the intense slow-positron beam generated by an electron accelerator. Three dimensional distributions of atomic sized defects can be visually evaluated through positron lifetime mapping obtained by the PPMA.