Combination Of Light Microscopy Research Articles

Non-uniform fine structural alteration in the sinus node after cessation or reduction of blood flow

The sinus node of the dog was examined, using a large-specimen resin-embedding method for combined light and electron microscopy, after 30-240 min of total in vitro ischaemia and after 15-180 min of reduced blood flow in hearts with 2:1 AV block. Although progressive fine structural alteration was observed in the in vitro series, not all cells developed changes at the same rate. Complete loss of glycogen stores and swelling of organelles could be seen in many after only 30 min, whilst others retained their glycogen and showed minimal alteration even after 90 min. Despite relatively severe alteration throughout the node after 120 or more min, some groups of cells showed more extreme degrees of swelling and surface bleb formation than others. In the partially ischaemic in vivo series focal alteration, consisting chiefly of mitochondrial swelling and/or gross dilatation of sarcoplasmic reticulum vesicles, was detectable after 20 min and increased little in extent or degree during longer periods of reduced blood flow. This non-uniform response of sinus nodal cells to ischaemic injury parallels that of contractile atrial muscle cells.

Pulmonary endothelial and alveolar epithelial lesions induced by O,O,S-trimethyl phosphorothioate in rats.

The morphogenesis of pulmonary injury induced by an impurity present in a commercially important organophosphorus insecticide, O,O,S-trimethyl phosphorothioate (OOS-TMP), was studied by combined light and transmission electron microscopy. Weanling female WAG/Rij rats received OOS-TMP dissolved in corn oil by gavage and were studied at intervals from 6 to 168 h after treatment. Sequestration of neutrophils was initially observed at 12 h after treatment and was accompanied by interstitial oedema. Plasmalemma alterations in endothelium lining capillaries and small arteries and veins were observed from 12 to 120 h after treatment and were accompanied by endothelial cell detachment and separation from the basal lamina. Abundant aggregates of fibrin were sequentially observed in intravascular, interstitial, and alveolar spaces. Platelet aggregation and degranulation were occasionally observed in capillaries as early as 12 h after treatment, and frequently observed in capillaries and small vessels from 24 to 96 h after treatment. Significant increases in wet lung weight and lung water content occurred at the same time that morphologic changes were observed in pulmonary endothelium. Alterations in type I alveolar epithelial cells were initially observed at 24 h after treatment. Cell swelling, fragmentation, and necrosis were observed in both type I and type II cells and resulted in a bare basal lamina. Marked attenuation, hypertrophy, and proliferation of type II epithelial cells followed alveolar epithelial cell injury and loss. Minimal changes were observed in non-ciliated bronchiolar epithelial (Clara) cells; predominant changes included the loss of surface microvilli and apical cytoplasmic bulge. The results of this study indicate that the endothelium and alveolar epithelium are the predominant cell types in the rat lung injured following OOS-TMP administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature.

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.

Synaptic organization of the nucleus of the optic tract in the rabbit: a combined Golgi-electron microscopic study.

The organization of the nucleus of the optic tract was investigated with light and electron microscopy in combination with Golgi impregnation. In Golgi material, neurons ranged in size from 10 to 25 microns with three to seven principal dendrites extending predominantly parallel to the fibres of the optic tract, irrespective of their location within the nucleus. In some areas dendrites extended into the neuropil of the adjacent dorsal terminal nucleus of the accessory optic system and the posterior pretectal nucleus. Occasionally spines and appendages were observed. The fine structure of the nuclei, perikarya and the dendritic arborization did not allow a well-defined distinction between interneurons and projection neurons. The synaptic organization of the nucleus of the optic tract showed great resemblance to the neuropil of the lateral geniculate nucleus and the superior colliculus. Similar types of presynaptic terminals were noticed: (i) R-terminals were either large and scalloped or small and regular in outline with spherical vesicles and electron-lucent mitochondria, and showed asymmetric contact zones; (ii) F-terminals with flattened vesicles, opaque mitochondria and symmetric contact zones; (iii) RLD-terminals with spherical vesicles and electron-dense mitochondria and asymmetric contact zones; (iv) P-terminals with pleomorphic vesicles and electron-lucent or opaque mitochondria and asymmetric synaptic thickenings. These different types of terminal were found isolated in the neuropil or in clusters of synapses. The most striking differences between the nucleus of the optic tract and the lateral geniculate nucleus were the relative scarcity of F-terminals in the clusters, the paucity of triadic arrangements and the relatively small size of the R-terminals. The differences in ultrastructure may be related to retinal W-type ganglion cells, which form the main retinal input to the nucleus of the optic tract and could also be related to the physiologically identified direction-selective units within the nucleus of the optic tract.

Steedman's polyester wax embedment and de‐embedment for combined light and scanning electron microscopy

AbstractSteedman's polyester wax mixture is a good, general‐purpose histological embedding medium that is suitable and convenient to use when it is desirable to combine light microscopy with scanning electron microscopy (SEM). A range of properties recommend this wax: it has a low melting temperature (37°C), is readily soluble in most dehydrating agents, results in negligible tissue shrinkage, preserves tissue antigenicity, and may even be used as a solvent for fixative agents. We prepare and embed tissues in polyester for light microscopy much as they would be for paraffin wax. For SEM, the block surface is micro‐ or ultraplaned, utilizing, respectively, a standard rotary microtome with razor blade knives or an ultramicrotome with glass knives. The block is de‐waxed in absolute alcohol and then taken to critical point drying. Similarly, sections mounted on coverslips or glass slides may be brought to the SEM after removing the wax. This enables one to bring to the SEM relatively large block faces or sections with good control over orientation. We find the results to be superior to similar procedures employing paraffin. We believe it to be more versatile and equivalent or superior to a variety of other techniques designed to gain access to the interior of tissues with SEM.

Intrahepatic cholangiectases and large-duct obliteration in primary sclerosing cholangitis.

We studied intrahepatic bile ducts of five patients with chronic ulcerative colitis and primary sclerosing cholangitis. The livers had been obtained at the time of orthotopic liver transplantation. After specimen cholangiography and perfusion fixation, sequential blocks and sections from portal tracts were studied, combining light microscopy with scanning electron microscopy. In vivo cholangiograms were studied also. The specimens revealed: absence of normal bile ducts; presence of thin-walled tubular or saccular cholangiectases with semicircular and annular fibrous crests, without evidence of superinfection; cholangiectases with secondary acute or chronic-cellular cholangitis, with or without cholangitic abscesses; fibrous cholangitis without ductal dilatation; transformation of bile ducts into fibrous cords which were either solid or contained remnants of bile duct epithelium, and complete loss of bile ducts. The shape and distribution of the cholangiectases suggested that these lesions were manifestations of the disease process and not passively dilated normal ducts. Fibrous-obliterative cholangitis with formation of fibrous cords was found not only at the level of interlobular and adjacent septal bile ducts but also at the level of segmental bile ducts that normally would have been demonstrable in cholangiograms. The "pruned-tree" appearance in cholangiograms appears to result from the transition between patent and often cholangiectatic ducts, and duct obliteration. At present, intrahepatic cholangiectases in association with duct obliteration can be considered pathognomonic morphologic features of primary sclerosing cholangitis.

Cytology of Self Fusions in Hyphae of Phanerochaete velutina

SUMMARY: Hyphal fusions forming in secondary mycelia of Phanerochaete velutina were examined using combined light and electron microscopy. Only ‘self’ fusions, occurring between hyphal compartments derived from the same colony, were studied. At points of contact, fusions formed from a single opening which expanded radially by highly localized lysis of the surrounding cell wall. Fusion pore enlargement failed to reach the full hyphal diameter and a rim of undissolved cell wall remained marking the original point of contact. Within 2 h of this, the compartments became re-partitioned by septum formation, synthesis being associated with mitotic division and beginning precisely at the site of fusion pore expansion. Septa formed at fusions showed identical structure and development to those occurring in unfused compartments.

Cytology of Non-self Hyphal Fusions and Somatic Incompatibility in Phanerochaete velutina

SUMMARY: The somatic incompatibility reaction occurring at sites of fusion between hyphae of genetically different secondary mycelia of Phanerochaete velutina has been examined using combined light and electron microscopy. Hyphal compartments affected by incompatibility rapidly showed increased vacuolation and the development of autophagic bodies throughout the cytoplasm. Dense osmiophilic spherical bodies that developed within the vacuole lumen characterized the early and highly regulated phase of the reaction. Eventually, as expansion of the vacuolar system proceeded, more widespread degeneration began in the remaining cytoplasm, nuclei and mitochondria. The large microtubule bundles present within this species showed variable behaviour during degeneration of the cell compartments, either breaking down early on in the process or persisting intact during breakdown of the other cell components. The incompatibility finally caused extensive disruption and death of the compartments engaged in fusion and often contiguous cells. Plugging of dolipore septa apparently restricted spread of the incompatibility response along the fused hyphae.

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy.

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.

Culture and Characterization of Rat Middle-ear Epithelium

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.

Light and electron microscopy of contacts between primary afferent fibres and neurones with axons ascending the dorsal columns of the feline spinal cord

In addition to primary afferent fibres, the dorsal columns of the cat spinal cord contain ascending second-order axons which project to the dorsal column nuclei. The aim of the present study was to obtain morphological evidence that certain primary afferent axons form monosynaptic contacts with cells of origin of this postsynaptic dorsal column pathway. In ten adult cats, neurones with axons ascending the dorsal columns were retrogradely labelled with horseradish peroxidase using a pellet implantation method in the thoracic dorsal columns. In the lumbosacral regions of the same animals, primary afferent fibres were labelled intra-axonally with ionophoretic application of horseradish peroxidase. Tissue containing labelled axons was prepared for light and combined light and electron microscopy. Ultrastructural examination demonstrated that slowly adapting (Type I), hair follicle, Pacinian corpuscle and group Ia muscle spindle afferents formed monosynaptic contacts with labelled cells and light microscopical analysis suggested that they also received monosynaptic input from rapidly adapting (Krause) afferents. This evidence suggests that sensory information from large-diameter cutaneous and muscle spindle afferent fibres is conveyed disynaptically via the postsynaptic dorsal column pathway to the dorsal column nuclei. Some of the input to this pathway is probably modified in the spinal cord as the majority of primary afferent boutons forming monosynaptic contacts were postsynaptic to other axon terminals. The postsynaptic dorsal column system appears to constitute a major somatosensory pathway in the cat.

Fuchsia Pollen from the Early Miocene of New Zealand

Fossil pollen of Diporites aspis Pocknall & Mildenhall, from the early Miocene of New Zealand, was examined by combined light, scanning, and transmission electron microscopy, in order to determine its relationship to extant Fuchsia pollen. Based on overall morphology, exine structure, exine surface sculpture, viscin thread morphology, and apertural features, the fossil pollen definitely can be considered to represent Fuchsia. Parallel studies of extant Fuchsia pollen indicate that a more precise identification of the fossils is not possible. With the examination of additional fossil material, it should be possible to learn a great deal about the timing of origin and migration of the genus Fuchsia, but not of its constituent parts.

FUCHSIA POLLEN FROM THE EARLY MIOCENE OF NEW ZEALAND

Fossil pollen of Diporites aspis Pocknall & Mildenhall, from the early Miocene of New Zealand, was examined by combined light, scanning, and transmission electron microscopy, in order to determine its relationship to extant Fuchsia pollen. Based on overall morphology, exine structure, exine surface sculpture, viscin thread morphology, and apertural features, the fossil pollen definitely can be considered to represent Fuchsia. Parallel studies of extant Fuchsia pollen indicate that a more precise identification of the fossils is not possible. With the examination of additional fossil material, it should be possible to learn a great deal about the timing of origin and migration of the genus Fuchsia, but not of its constituent parts.

Classification and Phylogenetic Significance of Molluscan Shell Microstructure

Like most classifications of molluscan shell microstructure published during the past 25 years (e.g., MacClintock, 1967; Kobayashi, 1964, 1971; Taylor, Kennedy and Hall, 1969, 1973; Grégoire, 1972a), the present one is based largely on Bøggild's (1930) monographic work, redefined from a modern perspective of combined light and scanning electron microscopy. However, this is the first attempt to integrate shell microstructure terminology for mollusks with that employed by students of bryozoan and brachiopod shell microstructure (e.g., Williams, 1968a,b, 1970, 1973; Williams and Wright, 1970; Armstrong 1968, 1969; Sandberg, 1971, 1977; Brunton, 1972; MacKinnon, 1974, 1977; MacKinnon and Williams, 1974; Iwata, 1981, 1982). An integration of nomenclatorial schemes is desirable for purposes of interphylum comparison, and is presently needed because there is considerable overlap and inconsistency in the application of microstructural terminology even within single molluscan classes. The present synthesis of shell microstructure nomenclature is possible primarily because of the extensive data base of invertebrate shell mineralogy, microstructure and especially ultrastructure published in more than 300 references in the past 15 years. To these data, the authors have contributed original information of shell mineralogy and microstructure for scores of Recent and fossil mollusks, brachiopods and bryozoans, with a clear emphasis on bivalved mollusks. Many inadequately described microstructure terms have been reanalyzed during the course of this study, either by examining species cited in the literature, or by using closely related species. Perhaps because they are better studied, but probably for other reasons as well, the diversity of molluscan shell microstructures is considerably greater than that of brachiopods and bryozoans combined (Carter, 1979). Consequently, most of the present nomenclature is based on mollusks, and only three of the major microstructural arrangements described in this guide (crossed bladed, semi-nacreous and semi-foliated) were known first in brachiopods or bryozoans and later recognized in molluscs.

Septal Sealing in the Basidiomycete Coriolus versicolor

The way in which the dolipore apparatus contains hyphal damage, and the process of septal sealing have been studied in Coriolus versicolor using combined light and electron microscopy. The technique used allows the structure of septa in adjacent damaged and undamaged hyphae to be compared. The results show that septal sealing, following damage, is a two stage process. The first is the instantaneous plugging of the pore channel with electron-dense material. The second, beginning several minutes later, involves the detachment of the septal apparatus present in the ruptured compartment and a re-modelling of the septal swelling on the other side of the wall to give a permanent seal. The parenthesomes play no part in the plugging response.

Stereological estimation of average cell volume in monolayer culture by combined light and electron microscopy.

In order to convert stereological ratio estimates into 'absolute' values ('per cell' values) the average cell volume must be estimated. The present paper describes a stereological method based on well-known point counting procedures for the estimation of average cell volumes in monolayer cultures fixed in situ. This method involves estimation of the average attachment area per cell by light microscopy combined with estimation of the attachment membrane surface density by electron microscopy. There is no need for any assumption as to cellular or nuclear shape. The method has been tested on an established cell line, NHIK 3025, and shows a good accuracy. It has also been used to analyse the volume changes that take place in human monocytes during monolayer culture, demonstrating a 28-fold increase of the average cell volume over 10 days.

A microculture chamber and improved method for combined light and electron microscopy of filamentous fungi

A microculture chamber has been developed which enables specific fungal hyphae to be followed in their living state before being processed and longitudinally sectioned for electron microscopy. Photomicrographs and their corresponding electron micrographs are presented which illustrate the effectiveness of this approach. Examples of the application of the method are presented.

Bacterial infection of the common bile duct in chronic fascioliasis in the rat

Bile taken from rats infected with the liver fluke, Fasciola hepatica contained spiral bacteria whereas bile from uninfected rats was free from spiral bacteria. The bacterium and its relationship to the bile duct epithelium and the liver fluke was studied with a combination of light microscopy, scanning and transmission electron microscopy. Its morphological characteristics suggest that the bacterium belongs to the genus Spirillum. In contrast to many other co-infections of bacteria and helminths, the present one seems to be a fairly passive relationship so that neither the helminth nor the rat suffers from the presence of bacteria. The presence of the bacteria is thought to be due to changes in the biliary environment, produced as a result of the fluke infection; these changes subsequently allow a multiplication of bacteria normally present in the uninfected animal.

The architecture of rat lymph nodes

Non-lymphoid cells in rat lymph nodes are described as seen by combined light and electron microscopy of normal adult, congenitally athymic, germ-free, irradiated, or newborn rats. The cells are divided into stromal and non-stromal. The latter category consists of a variety of morphologically distinct cell types with characteristic distribution patterns. The presence of paracortical interdigitating cells in lymph nodes of germ-free rats, athymic rats, and newborn euthymic and athymic rats, refutes the ideas that interdigitating cells differentiate from macrophages under immune stimulation or T cell influences. Follicular dendritic cells are more clearly visualized and appear to be polynucleated after emptying the follicles of lymphoid cells by irradiation. Follicular dendritic cells and tingible body macrophages are found in conventionally raised euthymic and athymic rats, but not in germ-free rats. The interrelationships of these and other types of non-lymphoid cells are discussed.

Distribution of electrotonic synapses on identified lamprey neurons: a comparison of a model prediction with an electron microscopic analysis

1. Intracellular recordings of the monosynaptic excitatory postsynaptic potential (EPSP) were made from an identified giant interneuron in the lamprey spinal cord following stimulation of a single identified presynaptic giant axon. The EPSP had both electrotonic and chemical components. 2. The giant interneuron and sometimes the giant axon were labeled with horseradish peroxidase (HRP). Using combined light and electron microscopy (EM), gap junctions formed by the axon on the HRP-labeled dendritic trees of seven interneurons were identified and their position marked on dendritic diagrams made from tracings of the cells. 3. The total number of gap junctions on a giant interneuron varied from a minimum of 9 to a maximum of 23. The number of gap junctions on a branch of a dendritic tree varied from 1 to 9. For all seven neurons the closest gap junction was located approximately 65 microns from the soma and the furthest 280 microns from the soma. 4. When gap junction position was plotted as a function of electrotonic distance from the soma, the variability in their distribution decreased significantly. Synaptic location was found to be on the average about 0.5 space constants from the soma, and this value compared favorably with that determined from an analysis of half-width and rise time of the electrotonic synaptic potential for these neurons. 5. These results suggest that the presynaptic axon tends to make, on the average, synaptic contact at the same electrical distance from the soma.