Subepithelial fibroblasts of intestinal villi form contractile cellular networks via gap junctions. They sheathe lamina propria and are in close contact with epithelium, neural and capillary networks, smooth muscles, and immune cells. Subepithelial fibroblasts have unique characteristics such as: cAMP-dependent rapid reversal changes in cell-shape, cell-shape dependent mechanosensitivity that induces ATP release as a paracrine mediator, contractile ability, and expression of various receptors for endothelins, substance-P, ATP etc. They serve as mechanosensor and signal transduction machinery in the intestinal villi, which are regulated locally and dynamically by rapid cell-shape conversion.
The mechanism of angiogenesis and the origin of vascular endothelial cells (VECs) of newly-formed blood vessels (NBVs) remain to be elucidated. Here we present a new mechanism of angiogenesis and a new source of VECs. We cultured dermal fibroblasts from a rabbit, labeled them with various markers, and autologously injected them into the rabbit ear chamber. Labeled VECs were observed in NBVs in the chamber. Then we cultured corneal stromal cells (CSCs) from Flk1-lacZ mice and Tie2-GFP mice. CSCs from Flk1-lacZ mice were transplanted via a tail vein into wild type (WT) mice in which angiogenesis had previously been induced in the cornea. lacZ positive VECs were found in the NBVs of the corneas of recipient mice. Corneal stromal tissue grafts from Tie2-GFP mice were transplanted into the corneas of WT mice where afterward angiogenesis was induced. GFP fluorescence was observed in the NBVs in the mice which received tissue grafts from Tg mice. These results suggest that fibroblasts can transform into VECs. We propose a new mechanism of angiogenesis, in which blood vessels may elongate by incorporation of tissue-resident fibroblasts into the endothelia and usefulness of fibroblasts as a source of VECs for a gene and cell therapy for vascular diseases.
Myofibroblasts, which emerged in the tubulointerstitium of diabetic nephropathy, were called activated fibroblasts and gave rise to accumulation of extracellular matrices including collagen fibers, i.e. fibrosis. Also, it has been considered due to the morphological characteristics that they caused cell contraction following by contraction of fibrosis tissue, and they were regarded to be an intermediate type of cell between a fibroblast and a smooth muscle cell. Myofibroblasts have been demonstrated to emerge via transdifferentiation that specific cells such as fibroblasts expressed αSMA (αsmooth muscle actin) by exposure to TGF-β1. Significance of αSMA expression which was the major morphological feature of myofibroblasts was examined in the in vitro study using renal fibroblasts. It was revealed that expression of αSMA was not always necessary for contraction since fibroblasts formed stress fibers and contracted without expression of αSMA, and αSMA expression evoked contraction. Also, the intracellular signaling pathway of αSMA expression was distinct from that of contraction.
Corneal fibroblasts are responsible for the homeostasis of the transparent cornea through synthesis and degradation of collagen fibrils. Under the pathological conditions, such as infection, corneal fibroblasts play roles as a control tower to recognize the microbial pathogen and to modulate the secretion and activation of MMPs.
“Cancer” consists of cancer cells and surrounding stromal cells. The main constituents of cancer stroma are inflammatory cells, including lymphocytes, granulocytes and macrophages, the endothelial cells of blood and lymph vessels, pericytes, and fibroblasts. Recruited fibroblasts produce collagens and extracellular matrix proteins in response to several extracellular stimuli. This step is called the “desmoplastic reaction” and is a kinetic sequence of events in the invasion process. Investigators of the biology of this cancer-induced stroma have found evidence that biological activity of stromal fibroblasts is closely linked to tumor progression. To analyze the biology of fibroblast within cancer-induced stroma as well as cancer cell will allow us to better understand cancer microenvironment. In this article, we summarize the origin of fibroblast recruited into cancer stroma.
Mg-based alloys containing rare earth elements show remarkable precipitation hardening by aging of supersaturated solid solutions at low temperatures, and so examinations about microstructures of precipitates and precipitation process have been important subjects by TEM and HRTEM. We have studied structures of fine precipitates in Mg-Gd, Mg-Y and Mg-Gd-Zn alloys by the combination of HRTEM and HAADF-STEM, and obtained some valuable results, which can not be accomplished by TEM and HRTEM. In this paper, we review recent results about crystal structures and microstructures of fine coherent precipitates and GP-zones, and demonstrate the power of HAADF-STEM for the examination of fine precipitates in Mg-RE alloys.
Observation of viruses by phase contrast electron microscopy which was recently developed in Japan has been performed. In this review, the results of Zernike phase contrast electron microscopy of ice-embedded influenza A virus are described in detail. Influenza A virions were 100-120 nm in diameter and consisted of core, envelope, and glycoprotein spikes. In addition to spherical and elongated virions, three other classes of virions were distinguished on the basis of the features of their viral envelope: virions with a complete matrix layer, which were the most predominant, virions with a partial matrix layer, and virions with no matrix layer under the lipid bilayer. Eight ribonucleoprotein complexes, that is, a central one surrounded by seven others, were distinguished. About 450 glycoprotein spikes were present in an average-sized spherical virion. Thus, phase contrast electron microscopy is a powerful tool for resolving the ultrastructure of viruses, because it enables the generation of high-contrast images of ice-embedded particles. It will enable 3D structural analysis of enveloped viruses at the nanometer level that was difficult to achieve by conventional electron microscopy.
Genetically encoded Ca2+ sensor (GECS) is a Ca sensor coded by DNA and synthesized in the cell as protein molecule. There are two types of GECS used in biological field. One is a chemiluminescence sensor and the other is a fluorescent sensor. The fluorescent GECS was developed in 1990's. However, recent advancement of the fluorescent GECS is remarkable. Since the GECS is coded by DNA, it is a promising tool for monitoring cellular function in vivo in living animals. In this review, I will discuss characteristic of the GECS and show some application of the fluorescent GECS.
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 conventional high resolution STEM images are understood through several key points.
For obtaining useful information from TEM observations, both the performance of the TEM system and the sample preparation technique are important. The preparation process for a high-definition TEM sample must include high precision sample positioning and a cleaning process of the damage layer formed by FIB.
FIB has been used for TEM sample preparation, because it can determine accurately the position from where a TEM lamella is picked up. As an alternative, argon ion milling has also been used for sample preparation causing less damage.
These two systems (argon ion-beam system and FIB) and SEM are combined, forming the ‘triple-beam system’. In this system, all beams are orientated to the same point at which the sample is placed on. Because of this arrangement, in-situ SEM monitoring can be performed during FIB and Argon processing.
Using the triple-beam system, both very high precision and low damage finishing of the sample can be achieved. A TEM sample preparation case of an actual semiconductor device is introduced in this paper. This case illustrates the effectiveness of the triple-beam system.
Shallow junction technology within depth of a few tens of nm plays a key role in recent Si-based devices of high integration. This paper reports Raman scattering studies of lattice damages induced by shallow ion implantation and recovery by thermal treatment. Penetration depth of the probe laser is greatly different between visible and UV lasers: for example, a deep UV laser at wavelength 266 nm can probe surface layers of Si with depth ～5 nm. We can make a thorough analysis of Si top layers by cooperating with TEM cross-sectional observation.
We have developed a real-time stereo transmission electron microscope (TEM) with tilting illumination. This system allows us to observe three-dimensional (3-D) images directly with a video-rate of 1/30 sec. The system comprises two electrostatic deflectors. The first, included in the illumination system, is able to illuminate a specimen at two oblique stereoscopic angles (left and right of the optical axis) up to ±2.3°. The second deflector, in the imaging system, is used to correct the image separation caused by defocusing of the tilted illumination. These deflectors are operated in synchronization with an NTSC video signal output from a CCD camera to record left projections on odd fields and right projections on even fields. The time series of stereo pairs is transferred to a 3-D display that enables viewing of the 3-D images without special glasses. Real-time observations of ZnO particles and dislocations in an Al thin film are demonstrated.
This technical note describes tips and techniques for light and electron microscopy using ultrathin cryosections (50-100 nm in thickness) in immunocytochemical studies. A positive contrast enhancement method described here provides excellent contrast in ultrathin cryosections at the electron microscopic level. Immunofluorescence microscopy of ultrathin cryosections is important since these physical sections minimize the potential for false co-localization in the z-dimension at the light microscopic level. The use of ultrathin cryosections is a powerful approach for the investigation of the in situ localization of antigens in the complex structure of tissue and organ systems.
Identification of functionally differentiated structural specializations in the plasma membrane and precise localization of functional molecules in the specialization is crucial in understanding the intercellular signaling mechanisms. SDS-digested freeze-fracture replica labeling (SDS-FRL) was innovated as a suitable technique to reveal the fine structure of plasma membrane and quantitative localization of biomolecules over the membrane structure. However, it was difficult to apply this technique to the tissues having multiple types of cell populations due to two reasons. One reason is the poor morphological clues in the replicated membrane in order to identify the cellular origin of observed profiles. Another is the difficulty in reproducibility of the quantification results due to uneven removal of tissue by SDS treatment from the replica membrane. Here, we introduced our current SDS-FRL protocol, which is optimized for the precise localization and quantitative investigation of the plasma membrane molecules on neural tissue. We also elaborated several technical clues to overcome above mentioned problems. Hopefully, this information can provide help to those who are interested in employing this technique.
Photo-induced charge transfer is essential process in the wide range of scientific and technological issues including optoelectronic devices, light harvest, photocatalysis, and electrophotography. Although the dynamics of photoexcited charge are seriously influenced by local structure and electronic properties with nanometer scale, there is no effective method to observe the dynamics with nanometer scale resolution. This paper describes a development of time-resolved electrostatic force detection based on dynamic mode atomic force microscopy. In the dynamic-mode operation, the tip interacts with the surface only at the moment when the tip closes to the sample surface. The interactive duration can be estimated 1μs or less. Taking advantage of this short duration, submicrosecond-class time-resolved force detection has been achieved by the coincidence between photo-irradiation and cantilever motion. The oscillation amplitude is changed clearly as a function of bias voltage indicating the polarity of photo-excited charges. These results suggest that the transient charges generated by pulsed laser irradiation can be detected with μs-class resolution.