Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4–5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques. In vivo T1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring. In vivo MRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting that in vivo MRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use of in vivo MRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

An energy dispersive x-ray microanalysis study was performed throughout the total length of cementum on five impacted human teeth. Mineral content of calcium, phosphorous, and magnesium were determined with an electron probe from the cemento-enamel junction to the root apex on the external surface of the cementum. The concentration profiles for calcium, phosphorous, and magnesium were compared by using Ca/P and Mg/Ca atomic percent ratio. Our findings demonstrated that the Ca/P ratio at the cemento-enamel junction showed the highest values (1.8–2.2). However, the area corresponding to the acellular extrinsic fiber cementum (AEFC) usually located on the coronal one-third of the root surface showed a Ca/P media value of 1.65. Nevertheless, on the area representing the fulcrum of the root there is an abrupt change in the Ca/P ratio, which decreases to 1.3. Our results revealed that Mg2+ distribution throughout the length of human cementum reached its maximum Mg/Ca ratio value of 1.3–1.4 at.% around the fulcrum of the root and an average value of 0.03%. A remarkable finding was that the Mg/Ca ratio pattern distribution showed that in the region where the Ca/P ratio showed a decreasing tendency, the Mg/Ca ratio reached its maximum value, showing a negative correlation. In conclusion, this study has established that clear compositional differences exist between AEFC and cellular mixed stratified cementum varieties and adds new knowledge about Mg2+ distribution and suggests its provocative role regulating human cementum metabolism.

The mammalian tongue has evolved for specialized functions in different species. The structure of its papillae tells about the animal's diet, habit, and taxonomy. The opossum has four kinds of lingual papillae (filiform, conical, fungiform, vallate). Scanning electron microscopy of the external features, connective tissue cores, and corrosion casts of the microvasculature show the filiform papillae have a spearhead-like main process and spiny accessory processes around the apical part of the main process. The shape and number of both processes depend on their position on the tongue. On the apex, the main processes have shovel-like capillary networks and the accessory processes have small conical networks. On the lingual radix, the processes have small capillary loops. In the patch region, conical papillae have capillaries arranged as a full sail curving posteriorly. The fungiform papillae are scattered among the filiform papillae and have capillary baskets beneath each taste bud. Giant fungiform papillae on the tongue tip are three to four times larger than the ones on the lingual body. Capillaries of giant papillae form a fan-shaped network. The opossum has three vallate papillae arranged in a triangle. Their tops have secondary capillary loops but not their lateral surfaces. Mucosal folds on the posterolateral border have irregular, fingerlike projections with cylindrical capillary networks. These findings and the structure of the rest of the masticatory apparatus suggest the lingual papillae of opossum have kept their ancestral carnivorous features but also developed the herbivore characteristics of other marsupials.

A comparative study of scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging of the healthy human optic nerve was carried out to determine the similarities and the differences. In this study we compared the fine optic nerve structures as observed by SEM and AFM. The fibers of the right optic nerve of a 61-year-old man show different arrangements in transverse sections taken from the same individual 5 mm central to the optic canal and 5 mm peripheral to the optic chiasma; this difference can be recognized by light microscopy (LM), SEM, and AFM. AFM revealed such typical optic nerve fibers (taken from a point 5 mm central to the optic canal) with annular and longitudinal orientations, which were not visible by SEM in this form. By contrast, LM and SEM visualized other structures, such as pia mater and optic nerve fibers loosely arranged in bundles, none of which was visualized by AFM. The images, however, taken 5 mm peripheral from the optic chiasma show shapeless nerve fibers having a wavy course. Our results reveal that more detailed information on optic nerve morphology is obtained by exploiting the advantages of both SEM and AFM. These are the first SEM and AFM images of healthy human optic nerve fibers, containing clear representations of the three dimensions of the optic nerve.

We have explored the possibility of measuring small changes of orientation within grains by electron backscattering diffraction (EBSD), in the scanning electron microscope. Conventional orientation maps (using EBSD) index the orientation of each position on the sample separately. This does not give accurate results for small differences of orientation. We have studied methods of measuring small changes in orientation by measuring the change from one EBSD pattern to the next directly, without indexing either. Previous workers have measured the change of position of a zone axis in the EBSD pattern. We have compared this with an alternative method, which we show to be superior, of measuring the shift of the peaks in the Hough transform from one diffraction pattern to the next. This means that we are measuring the change of orientation of sets of crystal planes within the grain, rather than measuring the change of orientation of zone axes. We show that it is possible, with a standard EBSD configuration, to measure the shift of the Kikuchi bands to a precision of about a 10th of a pixel, which corresponds to a change of orientation in the sample of about 0.1 mrad (0.006°).

Physical, chemical, and isotopic analyses of individual radioactive and other particles in the micron-size range, key tools in environmental research and in nuclear forensics, require the ability to precisely relocate particles of interest (POIs) in the secondary ion mass spectrometer (SIMS) or in another instrument, after having been located, identified, and characterized in the scanning electron microscope (SEM). This article describes the implementation, testing, and evaluation of the triangulation POIs re-location method, based on microscopic reference marks imprinted on or attached to the sample holder, serving as an inherent coordinate system. In SEM-to-SEM and SEM-to-SIMS experiments re-location precision better than 10 μm and 20 μm, respectively, is readily attainable for instruments using standard specimen stages. The method is fast, easy to apply, and facilitates repeated analyses of individual particles in different instruments and laboratories.

A method for automatic classification of the shape of graphite particles in cast iron is proposed. In a typical supervised classification procedure, the standard charts from the ISO-945 standard are used as a training and validation population. Several shape and size parameters are described and used as discriminants. A new parameter, the average internal angle, is proposed and is shown to be relevant for accurate classification. The ideal parameter sets are determined, leading to validation success rates above 90%. The classifier is then applied to real cast iron samples and provides results that are consistent with visual examination. The method provides classification results per particle, different from the traditional per field chart comparison methods. The full procedure can run automatically without user interference.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at http://www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to JoAn Hudson, [email protected] or Nestor Zaluzec, [email protected].