Scanning Microscopy

Online ISSN: 0891-7035
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Article
Fifty-two surgical biopsies from retractile testes of patients in pediatric age (3-14 years), of which 25 were treated with hormonal therapy (RT) and 27 did not undergo therapy before orchidopexy (RNT), were compared with the biopsies of 18 normal (N) and 30 cryptorchid or ectopic (E) testes. A light and electron microscopic morphologic and morphometric study was performed. For the quantitative investigation 4 parameters were selected: a) the mean tubular diameter (on 20 cross-sections); b) the mean spermatogonial number per tubular section; c) the mean nucleolar area of the Sertoli cells; and d) the mean thickness of the tubular basal lamina. The 101 biopsies were collected for statistical evaluation into four age groups: 3-6 years, 7-10, 11-13 without spermatogenesis and 10-14 with signs of early spermatogenesis. In the RT category the mean tubular diameter and the mean spermatogonial number were similar to N in the first two age groups, but were significantly reduced in the RNT categories. The morphometric study of the Sertoli cell nucleolar area confirms the delay of maturation observed in the categories of RT, RNT and E. In normal biopsies, the basal lamina shows a progressive reduction of the thickness, with the lowest values around puberty, while constantly higher values were found in the other categories, although this increase is not statistically significant.
 
Article
Recent work has shown that microscopic wear patterns on teeth may yield insights into variations in diet and tooth use in modern and prehistoric mammals. This paper presents a review of dental microwear and diet in modern mammals, plus a discussion of topics for further research. To date, incisor and molar microwear have been examined, although there are far fewer studies of the former. Facilitated by the use of high-resolution casts and scanning electron microscopy, analyses have ranged from: qualitative to quantitative, low magnification to high magnification, and experimental studies to comparative studies of museum collections. Results are encouraging and may lead to further insights into a variety of topics including food processing and dental microstructure.
 
Article
Human adrenal cortical tumor cells (SW-13) grow into a typical epithelial cell monolayer when seeded onto culture dishes. The cells of the SW-13 population monolayer appear flattened with few conspicuous surface features. The cells are attached to one another at their lateral borders and are arranged in a cobblestone-like manner. Following Triton X-100 extraction, the distribution of the cytoskeletal elements was observed with scanning electron microscopic techniques to correspond to the shape of the non-extracted cell. Changes in the distribution and morphology of projections on the cell surface as well as changes in cell shape were revealed after treatment of the cultures with compounds which bring about microtubular and microfilament disruption. Following 60 minute treatment of the cell population with cytochalasin B (10 micrograms/ml), 90% of the cells became round while remaining attached to neighboring cells and to the substrate by slender cell processes and filopodia. Some bleeding could be seen on the cell surfaces of cytochalasin B treated cultures and an increase in the number of microvilli was evident. When the cytoskeletal elements were observed with scanning electron microscopic techniques after Triton X-100 extraction, the amount of peripheral cytoskeletal elements was decreased and only slender projections of the microfilaments and microtubules were evident. Colchicine (0.06 micrograms/ml) treatment of the SW-13 adrenal cell population resulted in the appearance of surface blebs within 10 minutes of the initiation of treatment. The changes in surface projections are discussed in relationship to the loss of microtubules and microfilaments from the cytoplasm of the cell.
 
Article
The larval development of Wohlfahrtia magnifica (the most important dipteran causing sheep myiasis in the Palearctic region) has been studied by means of scanning electron microscopy. The ultrastructure of mouth-hooks, oral ridges, labial lobes, body spines and anterior and posterior peritremes is described for the first time. Their possible adaptations to a parasitic lifeway are also discussed. Thus, the use of new structures in the Sarcophagidae taxonomy is proposed from the point of view of their ultrastructure and adaptative morphology.
 
Article
The lack of purine salvage enzyme, adenine phosphoribosyltransferase (APRT), leads to 2,8-dihydroxyadenine stone formation and/or crystalluria because it is insoluble in urine. Urolithiasis composed of 2,8-dihydroxyadenine is not only formed in a complete defect of APRT, but also in a partial deficiency of this enzyme. The defect is inherited as an autosomal recessive trait, the homozygous state is associated with high urinary levels of 2,8-dihydroxyadenine and with crystalluria, calculus formation, and potential nephrotoxicity. Determination of the APRT activity will facilitate quantification of the enzyme deficiency and elucidation of the hereditary history. 2,8-dihydroxyadenine excretion in the 24-hour urine and its circadian rhythm were determined using a new method of high performance liquid chromatography determination. By means of a standard case presentation, we illustrate the analysis of urinary sediments and calculi as well as the scanning electron microscopic images of this kind of stone.
 
Article
A short discussion of practical results and theoretical aspects of nuclear magnetic resonance (NMR) on intracellular monovalent cations is presented. 7Li- and 23Na-NMR relaxation studies on human erythrocytes are described which indicate that lithium and sodium ions are essentially free inside the cell. However, there is a contribution to transverse relaxation originating from cations diffusing through electric field gradients imposed by the cytoskeleton, which is reflected in the NMR line shape. 39K-NMR results obtained by other authors for intracellular potassium are compared with the 7Li- and 23Na-NMR results presented here. The few 39K-NMR results obtained so far do not suggest that significant differences in dynamic behavior exist between intracellular potassium and sodium. 39K-NMR studies will, in the future, certainly enhance our understanding of the intracellular state of potassium ions.
 
Article
As a consequence of external and internal ionizing radiation, lysosome-like bodies have been observed to increase both in size and number in some cell types. We investigated this process by morphological methods (electron microscopy, cationized ferritin uptake, acid phosphatase histochemistry, morphometry) in cultured HT-29 cells. In parallel with these studies, we measured the rate of protein degradation on the basis of 14C-valine release from prelabeled cellular proteins. We found that at 2 and 4 Gy doses of X-irradiation the volume of the vacuolar (probably lysosomal) compartment increased without detectable changes of acid phosphatase activity. A 2 Gy irradiation dose did not change protein degradation rate. However, 4 Gy caused a significant inhibition of 14C-valine release from prelabeled proteins. Our results indicate, that the radiation induced expansion of the lysosomal compartment is not necessarily accompanied by increased lytic activity of HT-29 cells.
 
Article
Mouse embryo 3T3 cells were irradiated with 2450 MHz continuous and low frequency (16 Hz) square modulated waves of absorbed energy ranging from 0.0024 to 2.4 mW/g. The low frequency modulated microwave irradiation yielded more morphological cell changes than did the continuous microwave fields of the same intensity. The amount of free negative charges (cationized ferritin binding) on cell surfaces decreased following irradiation by modulated waves but remained unchanged under the effect of a continuous field of the same dose. Modulated waves of 0.024 mW/g dose increased the ruffling activity of the cells, and caused ultrastructural alteration in the cytoplasm. Similar effects were experienced by continuous waves at higher (0.24 and 2.4 mW/g) doses.
 
Article
Cellular alterations of cultured 3T3 cells irradiated with beta-rays from tritiated water were studied by scanning and transmission electron microscopy. We observed decreased negative surface charges, vacuolization of rough endoplasmic reticulum and Golgi-complex, degeneration of mitochondria, increase of lysosomal activity and changes in distribution and amount of microfilaments in the irradiated cells, that parallelled changes in cell shape.
 
Article
Over 400 cases with data from in situ electron microprobe quantitation of non-fibrous inorganic particles (e.g., silica, alumino-silicates, talc, metals) in pulmonary tissue sections, and data from quantitative digestion analyses for fiber content (e.g., asbestos, silica, alumino-silicates, man-made fibers, talc) comprise an extensive microcomputer data set of lung particle burden. When allied with demographic and histopathologic information the result is a comprehensive database of occupational pulmonary pathology. Examples of the kinds of information which can be extracted from the database include: 1) summary information on the types sizes and associations of particles in lungs with a variety of exposures, 2) concentrations of etiologic particle type in cases with recognized pneumoconioses, and 3) correlations between particle type, pathology, occupation and social history. The database provides a powerful tool for assessing such information on statistically meaningful sample sets.
 
Article
Hair cells and nerve fibers inside the organ of Corti of the C57BL/6J mouse, which is known as the precocious presbycusis model, were studied using the scanning electron microscope. For this study, we used thick serial sections cut from celloidin blocks. In the 5-week-old mice, hair cell loss was not seen. The upper tunnel radial fibers crossed the upper part of the tunnel of Corti and entered the Nuel's space between the outer pillar cells. The fibers varied in diameter and showed many varicosities. The basilar fibers emerged into the tunnel of Corti beneath the upper tunnel radial fibers and crossed the floor of tunnel slightly curving basalward. The outer spiral fibers ran along the lateral wall of the Nuel's spaces, sometimes buried in the cytoplasm of Deiters' cells. The nerve endings were clearly seen on the modiolar sides of the outer hair cell bases in the basal turn. In the 30-, 42- and 60-week-old mice, hair cell degeneration was seen both at the basal and apical portions, more pronounced in the former. The outer hair cells were affected more than the inner hair cells. In the basal turn where most of outer hair cells had degenerated, the upper tunnel radial fibers disappeared while the basilar fibers remained. These results suggest that degeneration of the efferent fibers occur earlier than those of the afferent to the outer hair cells.
 
Article
An improved holder has been designed for a cobalt calibrating standard used in an EDS Link 10,000 system attached to a JEOL 840 SEM. This holder allows the calibration of the EDS software for a wide range of samples differing in size, surface and height. These differences usually cause difficulties during quantitative analyses. The new holder allows the position of the cobalt to be adjusted according to the size and height of the sample.
 
Article
Porcine endothelial cells were grown on microcarrier beads and examined by scanning electron microscopy (SEM) at various times after initiation of culture. Total cell coverage on the bead surface varied from mean values of approximately 7% (3h) to 80% (96h). Beam penetration into the subcellular matrix presents a major problem with SEM X-ray microanalysis of microcarrier cultured cells and necessitates the use of an accelerating voltage not exceeding 10kV. At this voltage and below, X-ray contribution from elements present in the microcarrier bead has minimal effect on the determination of cell elemental levels. Washing the cells with 0.15M sucrose was the least perturbing of the rinsing techniques investigated, removing surface culture medium but not internal diffusible ions. X-ray microanalysis revealed detectable levels of Na, P, S, Cl, K and Ca in the cells, with well-marked changes from initial attachment to confluency. The level of K decreased from approximately 1.0% at 3h to 0.4% at 24h, with a corresponding decrease in the K/Na ratio. This unexpectedly low level of K was invariably observed after 24h, and is a genuine feature of established microcarrier culture. The effect of ionophore A23187 was determined at the 3h culture stage, and resulted in significant increases in the concentration of divalent cations (Mg2+, Ca2+), monovalent ions (Na+, Cl-) and a decrease in the level of K+.
 
Article
The amyloid precursor protein (APP) is an integral membrane component of eukaryotic cells. A variety of research approaches have addressed the contribution of the beta amyloid peptide region of the APP to neuritic plaque structure and formation in the Alzheimer disease brain as well as the relationship between beta amyloid accumulation and the occurrence of dementia. However, there is limited information available concerning the cellular consequences of amyloid deposition. The present studies were undertaken to investigate the relationship between beta amyloid and intercellular junctions. Transfected PC12 cell lines, that overexpress the beta amyloid peptide, exhibit structural and functional alterations at the cell surface and tend to form aggregates more readily than normal control cells. Intermediate junctions were the most common intercellular interactions of both normal and transfected cells. However, the control and transfected cells differed since areas of continuous and extensive junctions were readily seen in transfected cells and infrequently seen in control cells. The data suggest that excess accumulation of beta amyloid is associated with the junctional apparatus and may be related to increased intercellular adhesion.
 
Article
Two sets of animal experiments using guinea pigs were planned to evaluate the effect of ascorbic acid supplementation on the lithogenic process. In the first set of experiments, 10, 40, and 60 mg doses of ascorbic acid/100g body weight/day were given for 105 days. Neither of the ascorbic acid doses given induced crystalluria, calcification or stone formation, thereby confirming our previous findings that ascorbic acid in the doses used by clinicians does not cause urolith formation. In the second set of experiments, ascorbic acid was supplemented in hypercalciuric (induced by calcium carbonate feeding) and hyperoxaluric (induced by sodium oxalate feeding) animals for 45 days. The results indicated that it exacerbated the calcification process in renal and bladder tissue.
 
Article
Dental hard tissue abnormalities have never been described as part of the symptoms associated with hyperphosphatasemia. Fourteen teeth obtained from a young man, who had a mild form of hyperphosphatasemia, were analyzed using scanning electron microscopy (SEM), secondary ion mass spectroscopy (SIMS), X-ray diffraction (XRD), and infrared (IR) spectroscopy. SEM revealed a thin enamel, presenting a prismatic structure with many pits, and atypical cementum and dentin showing numerous resorption areas. The X-ray diffractograms revealed poorly crystalline hydroxyapatite associated with alpha-tricalcium phosphate and magnesium hydroxide phases. SIMS data showed high Ca concentrations: 40.5 weight % ¿wt%; standard deviation (SD = 0.13) and 42.5 wt% (SD = 1.03) in enamel and dentin respectively, and high Ca/P weight ratios: 2.28 in the enamel, 2.65 in the dentin. The lack of crystallinity may be linked to the high content of proteins and magnesium adsorbed onto apatite. This study demonstrates the need for thorough radiographical and biological investigations for skeletal abnormalities, even in the absence of systemic symptoms, when generalized dental abnormalities of both enamel and dentin are observed.
 
Article
The aim of this study was to test the hypothesis that deficiency of vitamin B6 would produce morphological characteristics of osteochondral lathyrism. To accomplish this goal, morphological characteristics of chick cartilage in which lathyrism was produced by two separate dietary regimens was compared to morphological changes encountered in vitamin B6 deficiency. Vitamin B6 deficiency should reduce activity of lysyloxidase needed for producing intermolecular cross-links. The question to be addressed was: would this latter deficiency impair collagen morphological features and secondarily other structures indirectly by reducing collagen molecular assembly? Failure of cross-linking of collagen in the positive controls was related to a lack of functional aldehyde cross-link intermediates which are blocked by homocysteine and aminoacetonitrile. Day-old-male Lohmann chicks were fed adequate (6 mg/kg) or vitamin B6-deficient diets. Cross-link defects were induced by homocysteine-rich diets (0.6% w/w) or a diet containing aminoacetonitrile (0.1% w/w). Animals were sacrificed at 6 weeks of age and Ossa tarsalia articular cartilage specimens, as well as the proximal end of tarsometatarsus were dissected from the tibial metatarsal joint, a major weight-bearing site. Light microscopic observations revealed reduction of subarticular trabecular bone formation, concurrent with overexpansion of the hypertrophic cell zone. Ultrastructural electron microscopy observation of articular fibro-cartilage indicated significant thickening of collagen fibers in vitamin B6 deficient birds, as well as the positive controls in comparison to that of cage-matched control birds. It was concluded that vitamin B6 deficient cross-linking may be responsible for the observed delay in bone development and aforementioned cartilage histological alterations.
 
Article
A single 4.7 x 3.3 x 1.5 cm solid nodule was removed from the bladder of a 24 years old white female who had lost an intrauterine contraceptive device (IUD) installed approximately four years ago. The nodule showed no external evidence of an IUD or its string. An examination of the nodular surface by scanning electron microscopy (SEM) showed mostly amorphous material with some adherent filamentous structures. Its energy dispersive x-ray microanalysis revealed the presence of calcium and phosphorus suggesting that the nodule was actually a urolith. Fracturing the nodule exposed an embedded entity consistent with being a copper IUD. Apparently, the lost IUD had migrated from the uterus into the bladder where it became mineralized. Thus the solid nodule was actually a foreign body stone.
 
Article
Automatic brushing with and without commercial dentifrice containing calcium hydrogen phosphate as an abrasive, commonly available in Japan, was performed on the tangential ground surfaces of sound enamel and dentin in human young premolar teeth with loading of about 120 g for 10 minutes in vitro. Their brushed surfaces were observed with a scanning electron and a scanning laser microscope. Brushing with abrasive dentifrice caused rough surfaces to appear and the abrasion of the enamel and the dentin was measured to be about 0.1 and 1.5 microns/min in mean depth, respectively. Under brushing with only distilled water, however, the enamel surfaces remained intact while the dentin surfaces became smoother.
 
Article
Transepithelial oxalate transport across polarized monolayers of LLC-PK1 cells, grown on collagen-coated microporous membranes in Transwell culture chambers, was studied in double-label experiments using [14C]-oxalate together with [3H]-D-mannitol as an extracellular marker. The [14C]-labeled glucose analog alpha-methyl-glucoside (alpha-MG) was used as functional marker for active proximal tubular sugar transport. Cellular uptake of oxalate and alpha-MG at both the apical and basolateral plasma membrane was determined. When added to the upper compartment, alpha-MG was actively taken up at the apical membrane, directed through the cells to the basolateral membrane and transported to the lower compartment, indicating functional epithelial sugar transport by LLC-PK1 cells. In LLC-PK1 cells, the uptake of alpha-MG at the apical membrane was approximately 50 times higher than that at the basolateral membrane. In contrast to this active transport of sugar, LLC-PK1 cells did not demonstrate oxalate uptake either at the apical or basolateral plasma membrane. The apical-to-basolateral (A- > B) flux of oxalate in LLC-PK1 cells was identical to the basolateral-to-apical (B- > A) oxalate flux in these cells. Moreover these flux characteristics were similar to those found for D-mannitol, indicating paracellular movement for both compounds. From these data, it is concluded that, under the experimental conditions used, LLC-PK1 cells do not exhibit a specific transcellular transport system for oxalate.
 
Article
Dietary oxalate is currently believed to make only a minor contribution (< 20%) to urinary oxalate excretion. A recent prospective study of stone disease suggested that dietary oxalate may be a significant risk factor. This observation led us to re-evaluate the contribution of dietary oxalate to urinary oxalate excretion. Previous studies have been hampered by inaccurate food composition tables for oxalate and inadequate methods for studying intestinal oxalate absorption. This evidence as well as factors that modify oxalate absorption are reviewed. New approaches to measure food oxalate and intestinal oxalate absorption have been examined. Capillary electrophoresis appears to be well suited for the analysis of the oxalate content of food. Two individuals consumed an oxalate-free formula diet for 7 days. This diet decreased urinary oxalate excretion by an average of 67% (18.6 mg per 24 hours) compared to oxalate excretion on self-selected diets. The absence of detectable oxalate in feces by day 6 of the diet suggested that the intestinal absorption was minimal. However, an effect of the formula diet on endogenous oxalate synthesis cannot be excluded. Restoring oxalate to the formula diet increased urinary oxalate excretion and illustrates that this experimental protocol may be well-suited for studying oxalate absorption and factors that modify it. Our results suggest that the intestinal absorption of dietary oxalate makes a substantial contribution to urinary oxalate excretion and that this absorption can be modified by decreasing oxalate intake or increasing the intakes of calcium, magnesium, and fiber.
 
Article
X-ray microanalysis, neutron activation analysis and atomic absorption spectrometry were performed on normal and injured skeletal muscle. X-ray microanalysis of tenotomized rat soleus muscle showed significantly elevated levels of sodium and chlorine and lower potassium compared with normal muscle. Similar ion shifts could be demonstrated by neutron activation analysis and atomic absorption spectrometry. The concentrations of sodium and chlorine obtained by these techniques were somewhat higher and that of potassium lower than the values obtained by X-ray microanalysis. This can probably be attributed to the fact that in atomic absorption spectrometry and in neutron activation analysis the entire muscle biopsy contents are measured while in X-ray microanalysis only the content of muscle cells unaffected by extracellular, non-muscular components are determined. It can be concluded that X-ray microanalysis is a reliable technique to study the elemental content of biological tissue, especially tissue undergoing pathological changes affecting the extracellular spaces. Other types of analysis should be used when elements not detectable by X-ray microanalysis are of interest.
 
Article
Reduced citrate in urine and increased fasting excretion of calcium are abnormalities frequently reported in stone forming (SF) patients. Increased dietary acid (or reduced alkali) introduction or absorption may be a potential cause of both these pathological findings. To test this hypothesis, we studied 64 SF patients (32 with fasting hypercalciuria (FH) and 32 without FH (NFH)). After a basal evaluation for nephrolithiasis, while on a 500 mg calcium diet, they were evaluated for: (1) daily intestinal alkali absorption (IAA), by urinary electrolyte excretion; (2) basal concentrations of PTH, calcitonin (CT) and 1,25(OH)2-VitD; (3) oral calcium load for evaluation of changes in calcium and hydroxyproline urinary excretions; (4) intestinal calcium absorption (18 patients), with double curve analysis (stable Sr as tracer); and (5) changes in citrate excretion after an alkali load (50 mEq of a mixture of calcium gluconate, lactate and carbonate) in 10 patients. The results demonstrated: (1) FH stone formers had reduced citrate excretion and lower mean IAA levels than NFH stone formers; (2) FH stone formers also had higher bone resorption levels with lower PTH and higher CT levels; (3) IAA levels were related to both citrate excretion and bone turnover indices; and (4) the increases in citrate excretion after oral alkali load were strictly related to basal IAA values (index of alkali absorption and/or generation after oral load), demonstrating that a different absorptive capacity of alkali rather than a different dietary content may underlie these metabolic abnormalities.
 
Article
In contrast to fixation of tissue in externally heated fixative, microwave-irradiation can generate uniform internal heat, which is of utmost importance for successful fixation of biological tissue. To evaluate the effectiveness of microwave-accelerated chemical fixation, we compared the structure of rat kidney fixed by a conventional method and a microwave-accelerated method, by scanning and transmission electron microscopy. Following perfusion, rat kidney pieces 1-2 mm in size were irradiated in Karnovsky's fixative in a domestic Amana microwave oven, till the temperature of the fixative reached 45-50 degrees C. For conventional fixation, tissue pieces were fixed overnight at room temperature in the same fixative. Both types of samples were processed further for electron microscopy using identical protocols. The microwave fixed samples showed excellent preservation of structure comparable to the samples fixed by the conventional method. Glomeruli and the renal tubules showed normal morphology with no cellular swelling. The cytoplasm and nuclear matrix of the epithelial cells was uniformly dense. Other fixation sensitive organelles like mitochondria and Golgi apparatus showed superior preservation with continuous membranes. These results demonstrate that microwave accelerated chemical fixation results in excellent preservation of tissue structure, reduces processing time significantly and is therefore a practical alternative to conventional protocols.
 
Article
A systematic, comparative study of the accessory sex glands of the adult male rat after androgen withdrawal was carried out. The changes were investigated by using scanning electron microscopy at different intervals after surgical castration. The main common signs of epithelial cell involution were flattening of the cell surface, reduction of the size and number of microvilli, some blurring of the cell borders, cessation of secretory activity and diminution of the luminal volume of the glands. Overall, confident signs of atrophy were evident after one week, and complete epithelial involution was reached by the third week. The epithelial cell atrophy was accompanied by a relative stromal hyperplasia. The new observation seems to be that the process of stroma consolidation is progressing for a considerable time subsequent to the completion of the epithelial involution. This phenomenon is particularly evident in the dorsal prostate, the seminal vesicle and the coagulating gland.
 
Article
This study describes the morphology of the accessory sex glands of the adult male rat as observed by scanning electron microscopy (SEM). The purpose was to obtain a systematic and comparative SEM description of these glands and to evaluate different preparation techniques. A common morphological feature is polyhedral delineation of the cells, which exhibited a variable convexity of their apical surface. The cell apices were more or less studded with microvilli. Nevertheless, it was possible to distinguish the glands by their surface morphology. In the ventral prostate, there was a considerable heterogeneity in cell surface appearance. The lateral lobe had a characteristic brush border, and in the dorsal lobe, surface blebbing and intracellular cisternae were observed. The cells of the seminal vesicle were covered by long microvilli, while particularly distinct, elevated cell borders and intracellular cisternae were typical for the coagulating gland. The secretory mechanism was merocrine in the ventral and lateral prostate and the seminal vesicle, and was mainly apocrine in the dorsal prostate. Surprisingly, only merocrine secretion was obvious in the coagulating gland. The most controversial observation, which needs further investigation, was the discovery of large orifices in the apical surface of individual seminal vesicle cells. These orifices may be indicative of an additional apocrine secretion in this gland. In studying this organ system, SEM provides information that adds to previous transmission electron microscopical investigations.
 
Article
Epithelial cysts may develop in virtually any epithelium. All cysts, regardless of their origins, are characterized by epithelial hyperplasia and fluid accumulation. Additional features may include tubular atrophy, basement membrane alterations and association with inflammatory cells. In spite of the intense research effort in recent years directed at uncovering the cellular mechanisms of cyst development and growth, we still do not know the primary events that lead to cyst formation. However, there are at least three candidate mechanisms. These include: 1) increased cell proliferation (epithelial hyperplasia) in the cyst wall, 2) net fluid accumulation in the cyst cavity and 3) alterations of extracellular matrix components linked to cyst formation and growth. This review discusses the evidence to support the role of each mechanism as a possible primary event necessary for cyst initiation and continued enlargement. Present data on the pathogenesis of epithelial cyst formation strongly suggests that no single mechanism, as yet described, can adequately account for all situations of cyst occurrence.
 
Article
Radiographic iodine-containing contrast media (meglumine calcium metrizoate, iohexol and meglumine sodium ioxaglate) were injected intravenously in rats. At various intervals after exposure, in situ cryofixation of kidneys was performed. Thin, freeze-dried cryosections were examined by electron microscopy and X-ray microanalysis. In endothelial cells, erythrocytes and tubular cells high dry weight concentrations of iodine were found. Twenty-four hours after iohexol was injected, no trace of iodine was found in the plasma, microvilli or the nuclei of the tubular cells. Small organelle-like compartments in the cytoplasm of the proximal tubular cells contained high concentrations of iodine, whereas no iodine was found in the surrounding cytoplasm. Since no metabolism of contrast medium has been demonstrated, the iodine signals must be emitted from contrast medium molecules. Other elements were also measured, with the concentrations being always within the ranges found in tubular cells of control animals. The detection of intracellular contrast thus does not seem to be an artifact due to cell injury, but rather represents a physiological event in healthy cells in the rat kidney. Our results are in contradiction to the prevailing opinion that contrast media do not enter healthy cells. However, previous conclusions have been based on the use of conventional preparation methods, and the highly water soluble contrast molecules may have been lost during the different steps of fixation and processing.
 
Article
The skin of the skh-1 mouse after ultraviolet B (280-320 nm, UVB) irradiation shows the pathological changes typical of sunburn damage: spongiosis (edematous spaces) around some cells, necrosis of keratinocytes, giving rise to sunburn cells, inflammatory infiltration of polymorphonuclear leucocytes, etc. In our previous study, these were accompanied by erythema, increased skin sensitivity, and edematous swelling. The topical application of tocopherol acetate (TA) immediately after the UVB exposure decreased these changes. In this paper, multiple measurements of the skin thickness were made at different locations along the magnetic resonance imaging (MRI) cross-sectional image of the skin. This permits effects to be quantified with (if desired) the contralateral half of the back serving as an internal control, either exposed (positive control) or unexposed (negative control). Topical application of TA resulted in an increase in the concentration of free tocopherol in the skin. No qualitative differences in ultrastructural appearance of the UVB-irradiated, TA-treated skin could be discerned by careful examination. In vivo high resolution video microscopy of blood flow in venules of the irradiated mouse ear revealed a large (tenfold) but not statistically significant decrease in stationary lymphocytes adhering to the venule walls. The delaying of the application of TA up to 8 hours after the termination of UVB irradiation still offered statistically significant protection as did immediate application of 5% TA in diluent Myritol 318 (Delios S, Henkel).
 
Article
Lesions developing in the gastric mucosa of the rat after exposure to different gastric damaging agents (100 mg/kg aspirin, and 70% or 100% ethanol) were assessed by scanning electron microscopy. The severity of the lesions was quantified according to morphological criteria. Modifications in the severity of these lesions induced by pretreatment with zinc acexamate were also analyzed. The scanning electron microscope revealed that with the exception of absolute ethanol, which caused distinctive morphological features, lesions found under the different experimental agents shared a common pattern of progression. Ultrastructural lesions on surface epithelial cells preceded further alterations of parietal cells. After the integrity of the epithelial cells was lost, detachment of the parietal cells occurred, probably, through peptic digestion of the connections between cells and their extracellular matrices. Pretreatment of animals with zinc acexamate increased the presence of mucus on the gastric surface and significantly prevented the progression of lesions towards the severest stages. Ultrastructural damage of surface epithelial cells was not influenced by this treatment, but detachment of damaged cells was clearly diminished. These data confirm the protective effect of zinc acexamate against gastric aggressions. Moreover, our studies confirm the notion that mucus secretion and maintenance of continuity on the gastric lumen by surface epithelial cells is of critical importance in preventing the gastric damage induced in these experimental models.
 
Article
Reconstituted type I collagen was processed into fibers which were subsequently severely dehydrated and cyanamide cross-linked. Fibers prepared by this method were stronger and more resistant to degradation than uncrosslinked fibers. When used as a tendon replacement prosthesis, morphological events occurred which were observed by light, scanning, transmission electron microscopy and electron histochemistry. Resorption was the initial host response to the prosthesis and involved gradual biodegradation. Formation of a host-replacement tendon was the second response. Increased collagen fibril diameters and a transition in the proteoglycan/collagen fibril interactions occurred in the newly developing connective tissue between 3 and 10 weeks postimplantation. These extracellular matrix transitions were major events occurring during wound healing and led to the assembly of a mature connective tissue. When used as a tendon prosthesis, these collagen fibers rapidly resorb while allowing simultaneous formation of aligned connective tissue. The fibers may have other applications in the fields of Orthopaedic Surgery, Neurosurgery and Biomaterials Research.
 
Article
Neonatal chicks were exposed to an octave band noise with a center frequency of 1.5 kHz at 116 dB SPL for 4 hours. Seven days following overstimulation, the birds were sacrificed. Their basilar papillae were removed, fixed in 4% paraformaldehyde, and processed in two steps. First, the ears were immunostained with a supernatant of mouse anti-tectorial membrane antibodies, followed by a diaminobenzidine process. Examinations of the papillae under an optical stereo microscope revealed a patch site with a partially regenerated tectorial membrane (referred to as the honeycomb). After the optical studies, the same ears were post-fixed in 1% osmium tetroxide, dehydrated in ethanol, and processed for scanning electron microscopy (SEM). SEM examinations demonstrated a honeycomb-covered patch lesion in the papilla. Patch lesion perimeters were traced from both the optical and SEM images, and patch areas were calculated. Also, papilla height was measured at the midpoint of the inner ear in both groups. These calculations showed that the patch area and papilla height had shrunk by approximately 37% and 33%, respectively, following the SEM methodology. The decrease in these dimensions may be attributed to several steps required for the SEM specimen preparation, such as critical point drying.
 
Article
The development of ideas on mechanotransduction in acousticolateral hair cells is described, leading to the current idea that transduction depends on deflection of the bundle of stereocilia by a force parallel to the plane of the sensory epithelium. Electrophysiological experiments are summarised, suggesting that transduction depends on a shear between the different rows of stereocilia, and that the transducer channels are situated towards the tips of the stereocilia. Analysis of the ways that shear between the rows of stereocilia could be detected suggests that tip links are the structures which are most likely to transmit the stimulus-induced forces to the transducer channels on the membrane. The directional selectivity of mechanotransduction is associated with the position of the kinocilium and gradation in heights of the stereocilia; evidence is presented suggesting that in the lateral line these are partly determined by the mitosis giving rise to the hair cell. Tip links differentiate out of links which initially join the stereocilia in all directions, with their final spatial organisation, which sets the directional selectivity of mechanotransduction, probably being determined by the gradient in growth of the stereocilia.
 
Article
Confocal Scanning Laser Microscopy (CSLM) is particularly well suited for the acquisition of 3-dimensional data of microscopic objects. In the CSLM a specific volume in the object is sampled during the imaging process and the result is stored in a digital computer as a three-dimensional memory array. Optimal use of these data requires both the development of effective visual representations as well as analysis methods. In addition to the well known stereoscopic representation method a number of alternatives for various purposes are presented. When rendering in terms of solid-looking or semitransparent objects is required, an algorithm based on a simulated process of excitation and fluorescence is very suitable. Graphic techniques can be used to examine the 3-dimensional shape of surfaces. For (near-)real time applications a representation method should not require extensive previous data-processing or analysis. From the very extensive field of 3-D image analysis two examples are given.
 
Article
This review presents the theory of solute transport across frog skin, epithelial cells of the intestine, the kidney tubules, and other similar systems. This theory is a part of a broader theory of the living cell, called the association-induction hypothesis. The central pumping mechanism is the cyclic activity of a sponge-like cytoplasmic protein(s), which alternately sop-up (by adsorption) the solute being transported and squeeze it out again (by desorption) into the cytoplasmic water at a high concentration level. The uptake phase begins with the adsorption of ATP on key cardinal sites of the protein involved; the release phase is triggered by the desorption of ATP through its dephosphorylation during a transitory activation of an ATPase. The theory recognizes the different nature of the two surfaces of the epithelial cells and assigns to each a key role in the active transport. The surface facing the "source solution" has a higher permeability to the solute being transported, while the surface facing the "sink solution" has a low permeability to the solute. This asymmetry in permeability insures that the solute sopped up by the cytoplasmic protein(s) comes primarily from the source solution. Depolarization of the water of the cell surface facing the sink solution (but not that facing the source solution) insures that the solute released into the cytoplasmic water during the squeezing phase leaves the cell only through the opposite surface as that where the solute has entered the cell.
 
Article
Video-enhanced differential interference contrast (VDIC) light microscopy in conjunction with fibrinogen labelled colloidal gold was employed as a probe to follow the mobility of the fibrinogen receptor on platelets. Correlative studies by both high voltage and scanning electron microscopy confirms localization of labels relative to platelet ultrastructural and surface characteristics, respectively. Treatment of platelets with trifluoperazine prior to and after incubation with fibrinogen-gold labels results in a concentration dependent inhibition of receptor movement. The results obtained from this study suggest that phosphorylation of myosin by the Ca++-calmodulin dependent enzyme, myosin-light chain kinase, is important in the fibrinogen redistribution that occurs during platelet activation.
 
Article
It has been generally accepted for over twenty years that epinephrine stimulates platelet aggregation without inducing shape change. However, it has been recently reported that discoid platelets are not recruited into ADP- or epinephrine-stimulated aggregates. Previous work in our laboratory has suggested that platelet shape change is necessary for the binding of fibrinogen to its surface receptor, which is a prerequisite for platelet aggregation. These studies seem to indicate that epinephrine-induced platelet aggregation does involve shape change. To investigate this possibility, the extent of shape change and fibrinogen binding in suspensions of epinephrine- and ADP-activated and control platelets was assessed by scanning electron microscopy (SEM). Platelets were incubated with 20 microM epinephrine, 20 microM ADP, or vehicle and labelled with 18 nm gold beads conjugated to fibrinogen or to a monoclonal antibody directed against the glycoprotein IIb/IIIa complex which comprises the fibrinogen receptor. Results indicate that shape change does occur in epinephrine-activated platelets as well as ADP-activated platelets. Although GP IIb/IIIa was shown to be present on both discoid and shape-changed, pseudopodial platelets, a significant degree of fibrinogen binding did not occur earlier than the pseudopodial stage in either activated or control suspensions. Platelet aggregation studies showed that the majority of platelets involved in aggregates had changed shape in both ADP- and epinephrine-treated platelet suspensions. These studies suggest that epinephrine- and ADP-induced platelet aggregation occurs via the exposure of fibrinogen receptors on shape-changed platelets.
 
Article
Na+/H+ antiporter activation in human Chang liver cells produces a flat-to-round (FTR) change in cell shape with gross reduction in cell profile area. Scanning electron microscopy (SEM) vividly displays a third phenomenon, viz., the development of focal microvillus anchors. Reduction in cell profile area concomitant with the development of this microvillus form of focal anchorage is quantitated by on-line image analysis during SEM examination. The reduction in profile area is corroborated by spectrophotometric digitization in light microscopy. Transmission electron microscopy (TEM) of rounded cells shows large endocytic channels and endosomes consistent with the observation of internalization of fluoresceinated-dextrans (FDx) of a diverse range of sizes, from 4,400 to 2,000,000 molecular weight, with cell rounding. Concomitant endocytosis of this magnitude indicates massive plasma membrane internalizations which could explain the very considerable profile area reduction and suggest that the microvillus anchors are probably traction processes. Antiporter mediated rounding (AMR) provides a highly reproducible and simple model for the production of anchoring microvilli ('filopodia') whereby they can be further explored.
 
Article
We analyzed the distribution patterns of nonspecific acid esterase and acid phosphatase activities with cytochemistry-scanning electron microscopy in backscattered and secondary electron imaging modes in isolated normal human peripheral blood lymphocytes. The analysis of non-specific acid esterase activity in the backscattered electron imaging mode showed, in some cells, focal distribution with a well-defined, homogenous deposit. Two patterns of acid phosphatase activity were evident with the backscattered electron imaging mode, i.e., focal and granular. Peripheral blood lymphocytes showing focal activity of both enzymes presented a smooth surface with few scattered microvilli as seen with the secondary electron imaging mode; while lymphocytes with a granular pattern of acid phosphatase activity had abundant microvilli. The correlation between patterns of enzymatic activity as seen in backscattering electron imaging mode, and surface morphological features as seen with secondary electron imaging mode, distinguished a subpopulation of lymphocytes of T lineage in human peripheral blood.
 
Article
We have identified an airway epithelial response following acute injury that cannot be termed 'repair' or 'regeneration'. It precedes these well characterized events and it is termed the 'response phase'. We tested the hypothesis that for the first 6 h following acute injury to the tracheal mucosa, the initial cellular events of the response phase will continue as in vivo even if the tissue is maintained in vitro in an Ussing chamber. The tracheal mucosa of anesthetized, intubated mongrel dogs was injured by the inhalation of SO2 500 ppm for 1 h (7 dogs); controls (3 dogs) breathed filtered, compressed air for 1 h. 4 dogs were killed, in pairs, at 1 and 6 h after 500ppm of SO2; their tracheas were removed and fixed for microscopic examination. 3 dogs were killed immediately after the SO2 exposure, their tracheas were removed and epithelium isolated from the posterior-membranous sheath was mounted in Ussing chambers in oxygenated, Krebs-Henseleit buffer (8 per dog with aperature area of 1.5 cm2). These tissues (and those from control dogs prepared identically) were fixed after 1 and 6 h incubation for microscopic examination. Epithelial damage was not observed in any controls but was in all tissues exposed to SO2. A wide spectrum of mucosal cell injury during the response phase was observed. The patterns of exfoliation noted were: individual cells, rows (several cells wide) of mucosal cells and entire regions (several hundred microns 2). At 1 h after exposure, in some lesions, the injury is difficult to assess because the tracheal surface was either blanketed in exfoliated cells or appeared in total disarray. By 6 h, the lesions were well defined and large flattened cells (130 microns 2 in surface area) covered the basement membrane in areas where mucosal cells had exfoliated. Some ciliated cells still remained attached at their base in these areas. These were the findings whether the tissues were taken fresh from the animal or have been maintained in Ussing chambers for up to 6 h. These results show that cellular repair of the tracheal epithelium can be studied in vitro during the first 6 h after injury, even if the injury has occurred in situ.
 
Article
Rats were given strontium chloride (SrCl2) intraperitoneally at a dose of 500 mg/kg. The upper incisors were removed after injection of strontium. These incisors were studied by scanning electron microscopy-energy dispersive spectroscopy analysis (SEM-EDS) and light microscopy to examine the calciotraumatic lines of strontium in the rat incisor labial dentin. At 24 hours after injection of strontium, the calciotraumatic response was observed in the predentin using hematoxylin and eosin (H-E) staining. At 5 days, three layers of calciotraumatic lines were present in the labial dentin using an H-E staining and backscattered electron imaging in the SEM. The external layer consisted of unmineralized dentin, the intermediate layer of relatively unmineralized dentin, and the internal layer of unmineralized dentin. By SEM-EDS analysis, strontium was detected in these layers. The elemental dot map showed that the external and internal unmineralized layers had a low calcium content. The magnesium concentration was higher in the internal unmineralized layer than the external unmineralized layer.
 
Article
The vas deferens of the salamander Rhyacotriton olympicus is composed of (1) a peritoneal epithelium, (2) connective tissue with fibroblasts, melanophores, circular smooth muscle, capillaries, and unmyelinated nerves within a collagenous matrix, and (3) an inner layer of cuboidal epithelium partially covered by ciliated squamous cells at the lumen. The lumen and apical cytoplasm of both epithelial cell types contain strongly PAS-positive granules. The cuboidal cells contained numerous swollen rough endoplasmic reticulum cisternae, mitochondria, and apical dense granules suggesting a high degree of secretory activity possibly involved in sperm maintenance. Fewer mitochondria, rough endoplasmic reticula, and granules in squamous cells suggest less secretory activity. Squamous cells may protect the cuboidal cells from possible abrasion by sperm masses and/or their cilia may aid in distributing secretory products in the lumen.
 
Article
In order to investigate the effect of various factors on urinary crystallization processes, a series of five experiments was carried out using an artificial urine (AU) in a rotary evaporator. The influence of ageing, pH and organic, inorganic and potential inhibitory additives formed the basis of the study. Precipitates were characterized by X-ray powder diffraction, scanning electron microscopy and energy dispersive X-ray analysis. In the ageing experiment, AU aliquots, adjusted to various pH values, were allowed to stand for several days and were not evaporated. Calcium oxalate monohydrate (COM) was formed at low pH, while whitlockite, apatite and struvite occurred at pH greater than 7. In the second experiment, AU aliquots at various pH values, were evaporated. Similar results to those of series 1 were recorded but, in addition, calcium oxalate trihydrate (COT) precipitated in the pH range 3 to 6.5 and brushite at pH greater than 5.5. In series 3, uric acid, creatinine and urea were included in AU aliquots (pH 5.5) which were subjected to evaporation. Uric acid promoted the formation of uric acid dihydrate; however, when present with creatinine, dihydrate formation was inhibited. Urea appeared to inhibit precipitation. In the fourth experiment, MgO, methylene blue and chondroitin sulphate A were independently included in the AU (pH 5.5). Precipitates of calcium oxalate mono-, di- and trihydrates were obtained. In the final experiment fluoride aliquots of variable concentrations were included in the AU (pH 5.5 and 6.5). COT crystals of superior quality to those observed in control solutions were obtained.
 
Article
The scanning tunneling microscope has been used to obtain images of DNA that reveal its major and minor grooves and the direction of helical coiling, but sufficient resolution has not yet been achieved to identify its bases. To determine if this technology is capable of identifying individual DNA bases, we have examined the molecular arrangements of adenine and thymine attached to the basal plane of highly oriented pyrolytic graphite. Both molecules form highly organized lattices following deposition on heated graphite. Lattice dimensions, structural periodicities, and the epitaxy of adenine and thymine molecules with respect to the basal plane of graphite have been determined. Images of these molecules at atomic resolution reveal that the aromatic regions are strongly detected in both molecules while the various side-groups are not well-resolved. These studies provide the first evidence that tunneling microscopy can be used to discriminate between purines and pyramidines.
 
Article
The aim of this study was to investigate the localization of ecto-Ca-adenosine-triphosphatase (ecto-Ca-ATPase) in different parenchymal cells of the human pituitary in tissue culture. The distribution of ecto-ATPases on the surface membrane of a particular parenchymal cell varied with the type of cells in contact with this parenchymal cell; the membrane portions immediately exposed to the medium showed low if any ecto-ATPase activity. These results suggest that ecto-Ca-ATPases of the parenchymal cells may be involved in cell adhesion processes and may be of crucial importance in the organization (in vivo) and reorganization (in vitro) of human adenohypophyseal tissue.
 
Article
The three-dimensional configuration of isolated crypts of normal human colonic mucosa and colorectal adenomas was examined by scanning electron microscopy. For isolation of the crypts, the digestion method with HC1 was used for formalin fixed tissues, and the separation method with ethylenediaminetetraacetic acid (EDTA) following ultrasonication was applied to fresh tissues. In a comparative study, the NaOH cell-maceration method, which visualized the sub-basal laminal collagen sheath, was applied. The isolated crypts from the normal colon were visualized as a single straight tubule resembling a test tube. Most isolated crypts of the tubular adenomas were visualized as elongated fan-like structures with several protuberances and a few short branchings. Their average length was more than twice that of the normal colonic mucosa crypts. Most crypts of the villous adenomas were visualized as slender tubules without protuberances and short branchings, and their average length was three times that of the tubular adenoma crypts. Most crypts of the tubulovillous adenomas were long and triangular with several longitudinal folds and protuberances, and the average length was about three times that of the tubular adenoma crypts.
 
Article
Ionizing radiation provokes an increase of the cAMP level in several organs and body fluids. After reviewing the relevant literature we present the results of our own experiments on primary human fibroblasts. X-irradiation at doses of 0.5 and 2.5 Gy in vitro evoked a rapid and reversible increase of adenylate cyclase enzyme activity. A significant increase in cAMP level of these cells was also observed. Adenylate cyclase was usually localized basolaterally on the surface of unirradiated cells, while irradiation resulted in a modification of distribution, i.e., the enzyme activity also appeared in apical localization.
 
Article
Pseudomonas aeruginosa adheres to respiratory epithelial cells in a highly specific fashion. In order to study the role of P. aeruginosa polar pili in the adherence process we conducted a quantitative morphological electron microscopic examination of P. aeruginosa adherence to SO2 injured canine tracheal cells in vitro. A pilin lacking background strain of P. aeruginosa PAK (BLP2) was constructed using a gene replacement and it in turn was engineered to express either the pilin gene of P. aeruginosa PAO, PAK, or no pilin gene. After 30 minutes incubation of these bacterial strains with injured canine tracheal rings the P. aeruginosa strains expressing pili adhered quantitatively more to the injured tracheal cells than did the pili lacking strains. PAO bearing strains adhered in greater numbers than PAK bearing strains. Healthy tracheal cells did not have any bacteria bound to their surfaces. The bacteria bound to the cilia and lateral edge of the exfoliating tracheal cells. Invasion of tracheal cells by piliated P. aeruginosa bacteria and penetration into the submucosa was also demonstrated. These data confirm the role of pili as important adhesins to injured tracheal cells. The difference in the adherence characteristics of pilin types PAK versus PAO may relate to the differences in the primary structure of these two pilin molecules.
 
Article
As part of a feasibility phase of an investigator-initiated multicenter NIH supported study on the Pathobiological Determinants of Atherosclerosis in Youth (PDAY), we report observations on microthrombi and adherent platelets on the intima of the aorta and left anterior descending coronary artery. The long-term objective of this cooperative study is to define more precisely the pathogenesis of atherosclerosis during late childhood and early adulthood and to investigate the influence of selected risk factors known to be associated with clinically manifest disease in later life. Scanning electron microscopy (SEM) was utilized to survey broad areas of arterial intima. Of 109 specimens studied from 52 cases, microthrombi composed of a mixture of aggregated platelets and fibrin and measuring approximately 30-70 micron in size were observed in about 4% [corrected] of the specimens and in about 6% of the cases, while individually adherent platelets were observed in approximately 7% of the specimens and about 10% of the cases. Microthrombi and adherent platelets may be important in atherogenesis by stimulating proliferation of intimal smooth muscle cells through the release of a growth factor from platelets. This feasibility study has shown that SEM is a rapid and effective method for surveying large areas of arterial intima for the study of adherent platelets and microthrombi.
 
Top-cited authors
Alois Lametschwandtner
  • University of Salzburg
Mark F Teaford
  • Touro University, California, College of Osteopathic Medicine
Carolin Bäder
  • Hochschule der Medien Stuttgart
Michel Daudon
  • Assistance Publique – Hôpitaux de Paris
Paul Jungers
  • Hôpital Universitaire Necker