Demonstration of neurosecretory substance in previously scanned specimens: adaptation of the Gomori aldehyde-fuchsin method for correlative SEM/TEM/LM histochemistry.

The Gomori aldehyde-fuchsin (AF) method for selective staining of neurosecretory substance (NSS) has been adapted to tissue previously prepared for both scanning and transmission electron microscopy (SEM/TEM). The procedure results in precise correlation of light microscopic (LM) histochemistry with SEM/TEM of the same tissue.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Innervation of the renal cortex.

Morphologic studies of renal innervation have utilized the methods of histochemistry and electron microscopy. Much information has been derived from examination of the renal cortex in monkey and rat. Fluorescence histochemistry shows a rich adrenergic innervation. Acetylcholinesterase can be demonstrated histochemically in the renal nerves by light and electron microscopy. Studies in the rat using 6-hydroxydopamine, a drug that selectively destroys adrenergic nerves, indicate that the glomerular arterioles and surrounding tubules are innervated by adrenergic nerves containing acetylcholinesterase. Distinct neurovascular and neurotubular junctions are observed the electron microscope. They are anatomically consistent with being the sites of synaptic transmission. Ultrastructural analysis of serial sections reveals that single individual axons contact multiple vascular cells and renal tubules. We now have a considerable body of information concerning the morphology of renal innervation are are beginning to appreciate the role of the renal nerves in kidney function.     

The leucocytes of the elasmobranch Scyliorhinus canicula L.—a morphological study

The leucocytes of the peripheral blood of the elasmobranch Scyliorhinus canicula L. were examined by light, transmission and scanning electron microscopy and histochemistry. Seven distinct leucocytes were identified including lymphocytes, thrombocytes, monocytes and granulocytes. The major characteristics of these cells and their relative distribution is described.     

A calcium oxalate-secreting tissue in branchlets of the Casuarinaceae

Summary Deciduous branchlets of casuarina trees have an unusual calcium oxalate-secreting system in which the epidermal tissue deposits calcium oxalate crystals in cell walls of the branchlet surface. These prismatic crystals were identified by light and electron microscopy, histochemistry, and elemental X-ray analysis. This calcium oxalate-secreting tissue was found in all species of casuarinas examined, including three of the four genera of the Casuarinaceae:Allocasuarina sp.,Casuarina sp., andGymnostoma papuanum. Because crystals were present throughout the epidermis soon after it formed, the mechanism for their induction was likely to be different than that for calcium oxalate crystal idioblasts. Secreting cells had a complex endoplasmic reticulum that may be involved in the secretory process.Abbreviations EDS energy-dispersive X-ray spectroscopy - HPF/FS high pressure-frozen/freeze-substituted - SEM scanning electron microscopy - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Histochemistry of carbohydrates as performed by physical development procedures

Summary The histochemistry of carbohydrates demonstrated by means of physical development procedures has been reviewed in terms of the use and reliability of the procedures, physical developers, practice of the procedures, a fundamental series of light and electron microscopic methods and certain other promising aspects of this area of histochemistry. A line of fundamental light- and electron-microscopic histochemical methods for carbohydrates using physical development procedures such as periodic acid thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD), high- or low-iron diamine (HID or LID)-TCH-SP-PD and lectin-gold (LT-G)-PD and related methods has been found to be more efficient, compared with those without physical development procedures. Since a series of other promising histochemical methods for carbohydrates using physical development procedures have been derived or are now being introduced, these procedures could be regarded as an unusually potent vehicle for effectively advancing carbohydrate histochemistry in both light and electron microscopy.     

Possibilities for the histochemical study of the nuclei of normal brain cells and in pathology

Possibilities of application of some histochemical methods to studying cell nuclei of brain are reviewed considering the following techniques: hybridization histochemistry on the light and electron microscope levels, immunohistochemistry and immunoelectron microscopy, absorbtion and fluorescence histochemistry of nucleic acids, histones, non-histone proteins of chromatin, and of cell nucleus lipids, electron histochemistry. Besides, some physicochemical and molecular-biological methods are considered. Data on human brain research in the norm and upon various brain disorders are particularly provided.     

The structure and function of the bursa copulatrix of the monarch butterfly (Danaus plexippus)

The bursa compulatrix of the Monarch butterfly was investigated utilizing light microscopy, histochemistry, and scanning and transmission electron microscopy in order to relate its morphology to the release of sperm from the spermatophore. The bursa has a row of large chitinous teeth on either side of the organ. The dorsal and ventral surfaces are covered with chitinous plates, the plates having bristles on one side. A single layer of cells lies under both the plates and teeth, one columnar cell under each plate, one cuboidal cell under each tooth. The toothed area has no muscle cells. However, the dorsal and ventral hemispheres of the bursa each have a crescent-shaped packet of muscle fibers that traverse the organ; there are no longitudinal fibers. Spermatophores with thick walls were found in the bursal lumen. Morphological evidence suggests that the presence of the spermatophores is sensed by the bristles and that the packets are opened by contraction of the muscles bringing the large teeth into contact with the spermatophore wall.     

Applications of immunogold and lectin-gold labeling in tumor research and diagnosis

Immunohistochemistry and carbohydrate histochemistry have had an enormous impact on both tumor research and diagnosis. In particular, immunogold labeling has provided significant advantages over classical fluorescence and enzyme-based techniques. In light microscopy, the silver-intensified gold labeling has proven highly sensitive and precise in localization. In electron microscopy, the gold particle marker was a prerequisite for succesful and unequivocal antigen detection in electron-dense cellular structures such as secretory granules. In this review we demonstrate the usefulness of light and electron microscopiaal gold labeling techniques as applied in tumor research and diagnosis. The examples include expression of β-1,6 branches and specific sialoglycoconjugates in colon carcinoma, b-12 carbohydrate epitope in breast carcinoma, polysialic acid in neuroendocrine tumors of lung, adrenal and thyroid, as well as studies on proinsulin to insulin conversion in insulinomas. In addition, practical hints for prevention of background taining, tissue fixation, and silver intensification of gold labeling are given.     

Structuro-functional organization of the fibrillar component and ground substance of human rib cartilage

A complex morphological investigation (histology, histochemistry, scanning and transmissive electron microscopy, electron histochemistry) has been performed to study the intercellular substance of the costal hyalinous cartilage. It has been demonstrated that the fibrillar framework of the costal cartilage consists of branching collagenous fibrillae, chaotically scattering. The fibrillae are surrounded with the ground substance; one of its components is the reticular ruthenium-positive structure.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Functional morphology of the subgenual organ of the carpenter ant

Using light microscopy, confocal microscopy, electron microscopy and histochemistry, the subgenual organ (SGO) of an ant, Camponutas ligniperda, is investigated. Sensory units and attachment cells together enclose a large extracellular cavity, which is filled by acid mucopolysaccharides, as revealed by staining with ruthenium red. Due to this cavity, the whole SGO has the shape of a deformed sphere and the scolopidia exhibit a distribution of angles between 0 degrees and 60 degrees with the tibial long axis (as is shown by phalloidin-rhodamin staining of the actin filaments of the scolopale, viewed in situ by laser scanning confocal microscopy). The subgenual organ is innervated by a branch of the tibial nerve, which splits within or shortly distal to the femur-tibia joint. The other features of the SGO of Camponotus ligniperda are similar as in other insects: the SGO of Camponotus ligniperda contains about 35 scolopidial sensilla; it is fixed to the subgenual nerve on its proximal end, by its attachment cells to the opposite part of the cuticle; the fixation by the attachment cells is accomplished by a vast quantity of cytoplasmic microtubules; the construction of the sensory units is the same as in other mononematic scolopidial organs. The role of the extracellular lumen inside the organ and the special shape of the SGO of Camponotus ligniperda in mechanical transmission is discussed.     

Versatile Method for Evaluating a Cell Culture by Various Morphological Techniques

Cellulose acetate is a versatile material for evaluating cells grown under identical conditions by various morphological techniques. This inexpensive material is transparent, easily cut to size and shape, nontoxic to cell cultures, and resistant to most chemicals used in histochemistry and in scanning and transmission electron microscopy. Samples may be obtained during and after the culture process. Cellulose acetate slides can be mounted directly over glass slides for direct observation and are easily peeled off plastic blocks for electron microscopy, leaving the cells behind. Relative disadvantages include its autofluorescence and a tendency to soften in strong acids or pure solutions of organic solvents such as xylene and propylene oxide.     

The Histochemistry and Fine Structure of the Nutritional Reserves in the Fin Rays of a Lancelet,Branchiostoma lanceolatum (Cephalochordata = Acrania)

Abstract Adults of the European lancelet were collected at Banyuls-sur-Mer (Mediterranean France) in mid-spring, shortly before the onset of the breeding season. The dorsal and ventral fin rays were studied by light microscopic histochemistry and by transmission electron microscopy (TEM). Each fin ray is a mass of extracellular material that accumulates beneath the mesothelium of a fin box coelom. The fin ray material is rich in lipids, proteins, and neutral mucopolysaccharides. TEM reveals no lipid droplets in this material, which consists entirely of a packed mass of 15–20 nm granules of medium electron density. It is likely that these granules consist of glycoproteins or glycolipoproteins. Our results are consistent with the proposal of Azariah (1965, Journal of the Marine Biological Association of India 7: 459–661) that lancelet fin rays are nutritional reserves supporting gametogenesis during the breeding season.     

Localization of Hydrogen Peroxide Production in Pisum sativum L. Using Epi-Polarization Microscopy to Follow Cerium Perhydroxide Deposition

Cerium is becoming an increasingly popular reagent for histochemical localization of oxidases and phosphatases because it combines directly with reaction products to form fine precipitates of electron-dense materials that can be easily detected using transmission electron microscopy or laser confocal scanning microscopy. We used epi-polarization microscopy to detect cerium perhydroxide deposits formed when H2O2 was produced by diamine oxidase in pea (Pisum sativum L.) epicotyls exposed to exogenous putrescine. Diamine oxidase activity was abundant in cortical cell walls but showed little, if any, association with vascular tissues. Maps of cerium deposition generated using scanning electron microscopy/x-ray microanalysis verified these observations. This study demonstrates the use of epi-polarization microscopy to follow cerium deposition, and the ready accessibility of this microscopy technique should facilitate more widespread use of cerium for plant histochemistry and cytochemistry.     

Pollen Development in Actinidia deliciosa var. deliciosa: Histochemistry of the Microspore Mother Cell Walls

The development of the microspore mother cell walls in Actinidiadeliciosa (kiwifruit) has been studied using light and electronmicroscopy. The microspore mother cell wall is similar, histochemically,and structurally in anthers from both functionally staminateand functionally pistillate flowers. Deposition, which beginsduring early prophase I, produces an electron-dense multilaminatedwall layer (layer a) and by the end of meiosis I a thick electron-lucentlayer (layer b) to the inside of this multilayered wall. Thereasons for histochemical differences and similarities betweenthese layers are discussed. The original primary wall persistsuntil the late uninucleate microspore stage. Layer (b), whichis probably mainly callose, dissolves at the late tetrad/earlymicrospore stage while layer (a), which probably also containsother polysaccharides, persists and dissolves concurrently withthe primary wall. Actinidia deliciosa, kiwifruit, microspore mother cell wall, callose, histochemistry, light microscopy, electron microscopy, male sterility