Scanning electron microscopic study of the freeze-fractured pineal body of the rat

Summary The three-dimensional ultrastructure of the pineal body of the rat is described on the basis of freeze-fractured preparations. The pineal capsule consists of irregular cells with very flat and perforated processes. Through these openings, extremely branched canaliculi, extending to almost every pineal cell, communicate with the tissue compartment outside the organ. The pericapillary spaces contain, in juxtaposition with capillaries of the fenestrated type, nerve fibers as well as a flocculent granular and filamentous material of unknown origin and chemical nature.With the technical assistance of Mr. P.-A. Milliquet     

Posthodiplostomum minimum: Examination of cyst wall and metacercaria containing calcarious concretions with scanning electron microscopy and X-ray microanalysis

Scanning electron micrographs show a smoothly fibrous structure of the cyst wall of the trematode Posthodiplostomum minimum, whereas the enclosed metacercaria posses a porous and papillate surface with occasional spines. Numerous excretory concretions fill the body of the metacercaria. The concretions appear layered from a central core and show an abundance of calcium, and, in some cases, magnesium.     

A correlated scanning and transmission electron microscopic study of maturation ameloblasts in developing molar teeth of rats

Summary Maturation ameloblasts of developing molar teeth of the rat were studied by both scanning and transmission electron microscopy. After fixation, teeth were frozen and split. One face of the fractured tooth was used for SEM, the other for TEM.It was found that in some regions proximal junctional complexes separate the interameloblast space from the intercellular space of the papillary layer. Thereby an intercellular ameloblastic compartment is delineated which in some specimens contains a substance interpreted to be colloidal. Elsewhere the proximal junctions of ameloblasts are not present and free communication between the extracellular spaces is evident. The apical pole of ameloblasts varies in structure. Over some areas there is a distinct distal border zone with membranous infoldings which in some regions resembles a striated or ruffled border, but in other regions the membranes show whorl configurations. The distal border zone also contains granules with flocculent material. Elsewhere the ameloblasts display no distal border zone and the cells show a smooth membrane (except for pinocytotic vesicles and hemidesmosomes) facing the enamel surface. The lateral surface of ameloblasts exhibits a variety of surface configurations similar to but not as pronounced as those reported previously in rat incisor maturation ameloblasts.The authors wish to thank Pauletta Sanders and Helen Ruane for technical assistance. This project was supported in part by USPHS NIH Grant DE04059-03 and by the Medical Research Council of Great Britain     

Transmission and scanning electron microscopic study of adepidermal granules of teleosts and amphibia

Summary Granules in the adepidermal space of larvae of Salmo irideus, Hynobius tokyoensis and Rhacophorus buergeri, were observed by transmission and scanning electron microscopy. Adepidermal granules of S. irideus were smooth and spherical structures, those of H. tokyoensis were smooth and spherical, or oval, while in R. buergeri these granules appeared as single or grouped tangled strand-like or starfish-like structures under the scanning electron microscope. These adepidermal granules were spread all over the basal lamina in every animal investigated. The different sizes of adepidermal granules of S. irideus and H. tokyoensis seen under the transmission electron microscope are not the result of differently sectioned faces of granules, but the granules themselves exhibit different sizes. The probable functions of these granules are discussed.     

The rat pituitary cleft: A correlated study by scanning and transmission electron microscopy

Summary SEM reveals that the inner surface of the pituitary cleft is lined by a continuous layer of marginal cells possessing microvillous and ciliated apical surfaces. The ciliated cells are more numerous on the posterior side (toward the pars intermedia) than on the anterior side of the cleft (toward the pars distalis). In contrast small infoldings (crypts) were occasionally noted only on the marginal layer covering the distal part of the hypophysis. In some areas of the cleft the surface features of the marginal cells are rather similar to the epithelial cells populating the upper parts of the respiratory tract in their topography and distribution. In other regions they also show striking similarities with the ependymal cells (tanycytes) lining the lateral recesses of the 3rd ventricle and the infundibular process with which the pituitary cleft has a very close topographical relationship.The parenchymal cells of the pars distalis are closely related to the flattened marginal cells of the cleft. The intercellular spaces of the pars distalis form a three-dimensional labyrinthic series of cavities continuous with the submarginal spaces of the cleft. Further SEM and TEM results demonstrate that the majority of the microvillous marginal cells lining both sides of the cleft possess surface features such as bulbous protrusions, laminar evaginations and large cytoplasmatic vacuoles, which are very likely the expression of an active transport of fluids.On the basis of these results it is concluded that the fluid-like material (colloid) present in the pituitary cleft is mainly derived from the fluids contained in the lacunar spaces of the pars distalis. Thus, marginal cells by absorbing fluids from the cleft by active endocytosis, may transport to the pars intermedia material (or hormones) produced in the distal part of the gland and vice versa.The cilia present on many marginal cells, based on their 9+2 tubular pattern, possess a kynetic role. This is very similar to that shown by the ciliated cells of the ependyma lining the brain ventricles. The occurrence of ciliated cells within the pituitary parenchyma (mainly in the follicles) suggests that they probably arise from the ciliated cells populating the marginal layer of the cleft and with which the parenchyma cells are closely related.     

A scanning electron microscopic study of larval development in the marine polychaete,Galeolaria caespitosa Lamarck (Serpulidae)

Summary Gametes and developing larvae of the polychaete Galeolaria caespitosa were examined by scanning electron microscopy. The sperm display a primitive morphology. When treated with 0.33 M CaCl₂, they release a branched acrosomal process. At spawning, the polygonal oocytes have a granular surface made up of spherules and the tips of microvilli. The oocyte coat develops a ridged appearance as the oocyte rounds up. At fertilization, the microvilli are withdrawn from the coat surface. Microvilli again appear on the coat surface during the trochophore stage, but the egg coat appears to be retained as the larval cuticle until the demersal stage. The surface of the larva now shows many microvilli. Details of the organization of several ciliary structures are clarified. Moreover, the present study shows rapid, sequential development of paired setal sacs, with the most anterior pair appearing first.     

High-resolution transmission electron microscopy of adult human peritubular dentine

Summary Single crystals from adult human peritubular dentine were studied by high-resolution transmission electron microscopy. Periodic fringe patterns were obtained from which the exact shape of the inorganic crystals were deduced. The crystals were found to have a mean length of 36.00±1.87 nm, a mean width of 25.57±1.37 nm, and a mean thickness of 9.76±0.69 nm. They consisted of platelets with a mean width-to-thickness ratio of 2.61, each being a flattened hexagonal prism of hydroxyapatite. Such conclusions are based upon a) the electron diffraction patterns that we obtained, and b) our comparison of the values of the periodic, equidistant fringes seen along different planes of sectioning with the corresponding theoretical values for hydroxyapatite.     

A scanning electron microscopic survey of the brain ventricular system of the female armadillo

Summary The scanning electron microscope was used to survey the brain ventricular system of the female armadillo (Dasypus novemcinctus) with emphasis on the third ventricle. The walls of the lateral ventricles, aqueduct, and fourth ventricle are covered by long cilia. In the lateral ventricle, the cilia are arranged in groups; but in the aqueduct and fourth ventricle, they are evenly placed over the cellular surfaces. The ependymal cells of the third ventricle are densely ciliated except for the organum vasculosum and infundibular recess. The non-ciliated luminal surface of these areas has a pebblestone appearance punctuated by numerous microvilli and two types of supraependymal cells.Supported by Edward G. Schlieder Foundation GrantThe authors would like to thank Jacqueline Skaggs for her secretarial assistance and Garbis Kerimian for his photographic work     

Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L.

Various combinations of intermorph, selfing and intramorph pollinations were carried out in Averrhoa carambola and the pollinated pistils were observed under fluorescence and scanning electron microscopes in time-course experiments. In both compatible and incompatible pollinations, similar behavior of pollen germination and penetration was observed in the first 4 h after pollination. In compatible intermorph pollination, pollen tubes were found at the base of the transmitting tract of the style at 8 h and 24 h after pollination in both the pin and thrum morphs. With thrum flowers, selfing resulted in pollen tubes being uniformly arrested at the junction between the stigmatic and stylar tissues. Penetration of pollen tubes into the upper portion of style was observed in thrum intramorph pollination and, when the second member was treated as the gynoecial tissue under the same pollination, penetration of tubes was further enhanced. Pin flowers, on selfing, resulted in pollen tube penetration farther down the style than was the case with thrum selfing. Intramorph pollination of pin morph behaved in a similar manner to selfing and was not affected by genotypes. Beside the stamen-style dimorphism, the receptive surface of the cob stigma was larger in pin than thrum flowers. While pin pollen was round, thrum pollen was oblong in shape with pin to thrum ratio on the polar axis being 1.2 and on the equatorial axis 0.8. The stigma of pin morph belonged to the dry type, while that of the thrum resembled the wet type.     

High-resolution imaging of proteins in human teeth by scanning probe microscopy

High-resolution studies of dental tissues are of considerable interest for biomedical engineering and clinical applications. In this paper, we demonstrate the application of piezoresponse force microscopy (PFM) to nanoscale imaging of internal structure of human teeth by monitoring the local mechanical response to an electrical bias applied via a conductive tip. It is shown that PFM is capable of detecting dissimilar components of dental tissues, namely, proteins and calcified matrix, which have resembling morphology but different piezoelectric properties. It is demonstrated that collagen fibrils revealed in chemically treated intertubular dentin exhibit high piezoelectric activity and can be visualized in PFM with spatial resolution of 10 nm. Evidence of the presence of protein inclusions of 100-200 nm wide and several micrometers long in tooth enamel has been obtained. Furthermore, it is found that the peritubular dentin and intertubular dentin exhibit different piezoelectric behavior suggesting different concentration of collagen fibrils. The obtained results demonstrate a high potential of PFM in providing an additional insight into the structure of dental tissues. It is suggested that the PFM approach can be used to study the structure of a wide range of biological materials by monitoring their electromechanical behavior at the nanoscale.     

A comparative scanning electron microscopic analysis of the human cerebral ventricular system

Summary A scanning electron microscopic analysis of the adult human third ventricular wall revealed ultra-architectural differences between dorsal and ventral portions. In the brains of thirteen and sixteen week old human fetuses regional differences in the surface organization of lining ependyma were more sharply defined than those of the adult. Alterations in the luminal surfaces of ependyma may reflect differences in the functional capacity of various ventricular areas. The potential role of certain ependyma (tanycytes) and their putative participation in neuroendocrine events is discussed.Supported by U.S.P.H.S. Grant NS 08171.U.S.P.H.S. Career Development Awardee K 04 GM 70001.     

Light and scanning electron microscopic study of cerebellar cortex of teleost fishes

Summary The teleostean cerebellar cortex has been studied with respect to its cytoarchitectonic arrangement and intracortical neuronal circuits. Samples of fish cerebellum were fixed either by immersion or vascular perfusion in 5% glutaraldehyde solution and processed for light and scanning electron microscopy. The cerebellar cortex shows four distinct layers: granular; fibrous stratum; Purkinje cell; and molecular layers. In the granular layer, mossy and climbing fiber glomeruli were characterized. The mossy glomerular region appeared as polygonal, round or ovoid clews formed by the convergence of up to 17 dendritic profiles upon a thick mossy fiber branch. The en passant nature of mossy fiber-granule cell dendrite synaptic relationship was clearly appreciated. The climbing fibers showed tendril and glomerular collaterals. The latter form thin, elongated glomeruli. Remnants of a neuroglial envelope were observed in the mossy fiber glomeruli but are apparently absent from the climbing fiber glomeruli. The beaded-shape Golgi cell axonal ramifications were observed participating in the formation of both glomerular types. Velate protoplasmic astrocytes and oligodendrocytes were also identified. The fibrous stratum appeared to be formed by compact bundles of thick and thin myelinated axons, running horizontally beneath the Purkinje cell layer and apparently belonging to ascending climbing fibers and descending Purkinje cell axons. At the Purkinje cell layer a selective removal of Bergmann glial cells was observed allowing the visualization of the pericellular basket and the pinceaux. Climbing fiber stems and their tendril collaterals were seen on their way to the molecular layer ascending parallel to the Purkinje dendritic ramifications. Stellate neuron processes were found passing through the fan-like arborescence of Purkinje cell dendrites.     

A scanning electron microscope study of the papilla basilaris of Gekko gecko

Summary The sensory hair cells of the ventral 2/3 of the papilla basilaris of Gekko gecko are divided into anterior (pre-axial) and posterior (post-axial) portions by a mid-axial gap or hiatus where there are no hair cells. There is no separation of the hair cells in the dorsal third of the papilla. There are three tectorial membrane modifications: an attached thickened membrane covering the pre-axial hair cells, sallets covering the post-axial hair cells, and an attached filamentous membrane covering the dorsal hair cells. The number of hair cells is greatest ventrally and decreases dorsally. There are approximately 2000 to 2100 hair cells. The kinocilia of the hair cells of the anterior halves of both the pre- and the post-axial vertical hair-cell rows are oriented posteriorly, while the kinocilia of the posterior halves are oriented anteriorly. The kinocilia of the hair cells of the dorsal third of the papilla are mostly oriented posteriorly. Thus, kinocilial orientation of the ventral 2/3 of the papilla is doubly bidirectional, and the dorsal 1/3, largely unidirectional.I would like to thank Ms. Maria Maglio for her skill in handling the technical aspects of the scanning electron microscopy as well as her artistry in achieving photographic excellence on the scope, David Akers for expert photographic assistance, and Wayne Emery for the drawings. Research sponsored by United States Public Health Service Grant NS-09231.     

How cells settle on glass: A study by light and scanning electron microscopy of some properties of normal and stimulated macrophages

Summary Some properties of normal and stimulated peritoneal macrophages have been studied using light microscopy, cinemicroscopy, and scanning electron microscopy. No difference in the overall rate of translational movement was found between normal and stimulated cells. Macrophages were found to settle on glass by a process involving initial protrusion of very fine finger-like processes, followed by veils. Full extension occurred sooner in stimulated cells.We are grateful to Professor R. Barer for his criticisms, to Miss Anne Edwards for technical help, to Mr. G. Tuck for help with cinemicroscopy, and to the Science Research Council and the Medical Research Council for grants.     

Scanning electron microscopic study of spermatozoa from gossypol-treated rats

Summary Spermatozoa from the cauda epididymidis of gossypol-treated rats exhibit distinctive departures from the morphology of spermatozoa from control rats: wrinkled and disorganized cell membrane in the head and tail regions, cell membrane missing from segments of the tail midpiece and principal piece regions, malformed heads, decapitate spermatozoa, retention of a cytoplasmic droplet at variable loci along tail midpieces, and looped tails. The observations suggest that gossypol exerts its contraceptive effect during spermatocytogenesis and spermiogenesis, including the posttesticular development and maturation of spermatozoa in the epididymis.     

Photoreceptor-like outer segments in the pineal organ of the lovebird,Uroloncha domestica (Aves: Passeriformes)

Summary In the pineal organ of the lovebird, Uroloncha domestica, bulbous, cup-shaped and elongated outer segments of photoreceptor-like pinealocytes are demonstrated by scanning electron microscopy. These scarce outer segments, 4–11 m in length, extend into the pineal lumen. The present structural observations speak in favor of photosensitive pinealocytes in the pineal organ of Uroloncha domestica. The relation of the photoreceptor-like pinealocytes to acetylcholinesterase-positive nerve cells and a nervous connection between the pineal and the brain indicate that the pineal organ of this passeriform species may be the site of neuroendocrine and photoreceptive functions.Supported by a fellowship from the Japan Society for the Promotion of Science to M. UeckSupported by a grant from the Ministry of Education of Japan to K. Wake and by a grant of the Deutsche Forschungsgemeinschaft to M. Ueck     

Scanning electron microscopic study on the structure of the lingual papillae of the Saanen goat

The morphology of lingual papillae of the ten male mature Saanen goats (11 months old, approximately 42 kg in weight and of a known pedigree) was examined by scanning electron microscopy. Tissues were taken from the dorsal and ventral surfaces of the apex, body and root of the tongue, and were prepared accordingly and observed under the scanning electron microscope. On the dorsal and ventro-lateral surfaces of the lingual mucosa, three types of mechanical papillae (filiform, lenticular, and conical) and two types of gustatory papillae (vallate and fungiform) were observed. The structure and density of the filiform papillae differentiated on the anterior, posterior and ventro-lateral aspects of the tongue. Two types of lenticular papillae, both possessing a prominent surrounding papillary groove, were determined. The pyramidal-shaped type I lenticular papilla had a pointed apex while the round-shaped type II lenticular papilla possessed a blunt apex. Certain number of the type I lenticular papillae had double apices. The larger conical papillae were hollow structures, differing structurally from the filiform papillae with their larger size, a tip without projections and lack of the secondary papillae. The vallate papillae were present on both rims of the torus linguae, were encircled by a prominent gustatory furrow which was also surrounded by a thick annular fold. The fungiform papillae were scattered among the filiform papillae in the anterior two-thirds of the dorsal and lateral surfaces, and each of them was highly protected by surrounding filiform papillae, yet encircled by a papillary groove. Our findings indicate that Saanen goat have profuse distribution of papillae on the tongue displaying morphological features characteristic of mechanical function.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Microvasculature of the pineal organ of the rainbow trout (Salmo gairdneri)

Summary The angioarchitecture of the pineal organ of the rainbow trout (Salmo gairdneri) was investigated by means of the corrosion-cast preparation method and scanning electron microscopy. Two main arteries (aa. epiphyseales) supply the pineal parenchyma. They emerge from the aa. cerebri anteriores and run in the fissure between the prosencephalon and the mesencephalon. After entering the pineal stalk, the aa. epiphyseales branch off into several arterioles, most of which extend to the pineal end-vesicle where they give rise to a lobular, bilaterally symmetric capillary network. Capillaries establishing the main portion of the pineal vessels appear widened in comparison to those supplying other portions of the brain and resemble capillaries in other endocrine organs. In Salmo gairdneri, no specialized system of portal vessels appears to exist between the pineal organ and other portions of the brain.     

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2ⁿ×2ⁿ pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.