In this article, the effects of the transmission electron microscopy (TEM) specimen preparation techniques, such as ion milling and tripod polishing on perovskite oxides for high-resolution TEM investigation, are compared. Conventional and liquid nitrogen cooled ion milling induce a new domain orientation in thin films of SrRuO(3) and LaFeO(3) grown on (001)-oriented SrTiO(3) substrates. This is not observed in tripod-polished specimens. Different ion milling rates for thin films and substrates in cross-section specimens lead to artefacts in the interface region, degrading the specimen quality. This is illustrated by SrRuO(3) and PbTiO(3) thin films grown on (001)-oriented SrTiO(3). By applying tripod polishing and gentle low-angle, low-energy ion milling while cooling the sample, the effects from specimen preparation are reduced resulting in higher quality of the TEM study. In the process of making face-to-face cross-section specimens by tripod polishing, it is crucial that the glue layer attaching the slabs of material is very thin (<50 nm).

Electron energy-loss spectra of single double-walled carbon nanotubes (DWCNTs) were compared with calculated joint density of states (jDOSs) obtained by a simple tight-binding (STB) and an extended tight-binding (ETB) method. From the comparisons, interband transition energies of ETB calculations show better agreement with peak positions of the experimental spectra than those of STB results. From a further comparison among calculated jDOS, real and imaginary parts of a dielectric function and a loss function Im[-1/epsilon], it was confirmed that the peak energies in a spectrum of single DWCNTs are almost equal to those of the optical absorption spectrum epsilon(2).

Vitrification is a morphological and physiological disorder affecting plants during their in vitro vegetative propagation. Vitrified plants have a poor survival rate when transferred from in vitro to greenhouse conditions, a fact mainly due to water loss and dissecation. It has been shown that normal and vitreous leaves of Datura insignis differ in the frequency of normal and abnormal stomata. The purpose of this work was to compare the surface of normal and vitreous leaves of D. insignis, using a modification of the platinum/carbon replica method. Adaxial and abaxial leaf surfaces of normal plantlets have a smooth and homogenous cuticle. A granular aspect, probably due to leaf age, rarely occurs at the periphery of the epidermal cells. Both adaxial and abaxial leaf surfaces of vitrified plantlets show discontinuities in the cuticle, occurring at several regions of the outer periclinal cell walls. However, such discontinuities are most noticeable in the region between adjacent epidermal cells. Fibrils 20-30 nm thick show a random arrangement or an oriented pattern in cuticular discontinuities. In D. insignis vitrified plantlets, adaxial and abaxial leaf cuticle has discontinuities or gaps which may cause an increase in cuticular transpiration contributing to the low survival rate of vitrified plantlets.

We describe techniques for electropolishing irradiated ferritic specimens for examination under the TEM in situations where the foil quality is of utmost importance. First, we describe some modifications to the standard technique for making plan-view specimens aimed at optimizing the foil quality. Second, we describe a technique for making plan-view specimens from a region of buried damage in a specimen irradiated with 2 MeV Fe(+) ions.

The subcellular localization of biomolecules at high resolution has traditionally been investigated by combining transmission electron microscopy (TEM) and chemical staining with heavy metals or immuno-based labeling with gold-conjugated antibodies. Here, we employ genetically encoded tags to examine the localization of proteins in transfected cultured cells by TEM. We purified a fusion protein of postsynaptic density-95 (PSD-95) coupled to three tandem repeats of metallothionein (MT) (PDS-95-3MT) from COS7 cells grown in the presence of Cd2+. PSD-95-3MT was detected as black particles by TEM. To visualize the subcellular localization of PSD-95-3MT, expression constructs encoding this fusion protein were transfected into primary hippocampal neurons cultured in medium containing Cd2+. The subcellular accumulation of PSD-95-3MT and Cd2+ provided excellent contrast in TEM micrographs. To address if genetically encoded tags affect the function of the target proteins, we found that the conjugation of 3MT to PSD-95 did not alter its association with known binding partners. These results demonstrate that 3MT coordinating Cd2+ is a valuable genetically encoded tag to study the localization of proteins by TEM.

The contrast-to-gradient (CG) resolution has been defined as a weighted harmonic mean of the local resolution, i.e. the length defining the object's local fine structure at each pixel position. The newly defined resolution Res is defined as 2 sigma/ sqrt 2, where 2 sigma is a sharpness factor of the image pattern obtained by the conversion 2 sigma = ARCG + B using default constants A and B. In the present study, we have extended the algorithm to change the constants from default values to calibrated ones using standard images that are same in both pattern and contrast-to-noise ratio (CNR) as the original SEM image to be evaluated. The calibrated image resolution with a weak noise dependency is evaluated with the CNR as a given parameter.

Gadolinium (Gd) has been identified as a possible causative agent of an emerging cutaneous and systemic fibrosing disorder, nephrogenic systemic fibrosis (NSF), which can cause serious disability and even death. To date, there are only two known associations with this disorder--renal insufficiency and Gd enhanced magnetic resonance imaging (MRI). We developed an automated quantitative scanning electron microscopy (SEM) and Energy dispersive x-ray spectroscopy (EDS) method for Gd in tissue of NSF patients. Freshly cut paraffin block surfaces examined using the variable pressure mode under standardized conditions and random search of the tissue area allow in situ detection and semiquantitative morphometric (volumetric) analysis of insoluble higher atomic number features using backscattered electron imaging. We detected Gd ranging from 1 to 2270 cps/mm2 in 57 cutaneous biopsies of NSF. Gd was associated with P, Ca, and usually Na in tissue deposits. Our method reproducibly determines the elemental composition, relative concentration, and spatial distribution of detected features within the tissue. However, we cannot detect features below our spatial resolution, nor concentrations below the detection limit of our SEM/EDS system. The findings confirm transmetallation and release of toxic Gd ions in NSF and allow dose-response analysis at the histologic level.

We examined muscle spindles in the posterior cricoarytenoid (PCA) muscle of the marmoset. The spindle capsule contained only one intrafusal muscle fiber which did not form a typical aggregation of equatorial nuclei such as bags and chains. The intrafusal fiber was innervated by sensory endings which branched and arranged irregularly. These sensory endings lay on the surface of the fiber in shallow grooves or deeply penetrated into it. These findings suggest that the muscle spindles in the marmoset PCA muscle are strikingly different in the structure of intrafusal fibers in the arrangement of sensory endings from those in common mammalian skeletal muscles.

A case of elastofibroma in a middle-aged Japanese woman was examined by the quick-freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient.

In this study, X-ray micro-computed tomography (CT) was used to reconstruct the fine structure macro- and microvasculature in three dimensions in contrast-enhanced rat liver samples. The subsequent application in the experimental CC531s colorectal cancer model was concurrent with results obtained from confocal microscopy in earlier studies. The en bloc stains osmium tetroxide in combination with uranyl acetate provided an excellent contrasting result for hepatic tissue after a trial of several contrasting agents. X-ray micro-CT allowed us to image the large blood vessels together with the branching sinusoids of hepatic tissue in three dimensions. Furthermore, interruption of the microvasculature was noted when rats were injected with CC531s colorectal cancer cells indicating the presence of hepatic metastases.