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PubMed1977

Cryo-ultramicrotomy and myofibrillar fine structure: a review.

In the past, the techniques of electron microscopy and X-ray diffraction have both been very informative about the ultrastructure of the muscle myofibril But X-ray diffraction patterns are difficult to interpret unambiguously and until now specimen preservation in plastic embedded muscle has been sufficiently poor to make it difficult to use electron micrographs of muscle as a means of interpreting the available X-ray diffraction evidence. The possibility of using ultrathin sections of frozen muscle, in which the disruptive steps of chemical dehydration and plastic embedding can be avoided, promises to help to bridge the information gap between present X-ray and electron microscope results. For this reason we here review the application of the cryosectioning technique to muscle, we assess the technique in terms of the improvements in preservation which have so far been obtained and which might be expected and we discuss some of the many potential advantages and uses of this technique for studies of muscle ultrastructure and function. It is concluded that this technique should be developed vigorously since it promises to play a very important role in muscle research in the future.

Animals↗
PubMed1979

Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality.

The cytoplasmic ground substance of cultured cells prepared for high voltage transmission electron microscopy (glutaraldehyde/osmium fixed, alcohol or acetone dehydrated, critical-point dried) consists of slender (3-6 nm Diam) strands--the microtrabeculae (55)--that form an irregular three-dimensional lattice (the microtrabecular lattice). The microtrabeculae interconnect the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The lattice is found in all portions of the cytoplast of all cultured cells examined. The possibility that the lattice structure is an artifact of specimen preparation has been tested by (a) subjecting whole cultured cells (WI-38, NRK, chick embryo fibroblasts) to various chemical (aldehydes, osmium tetroxide) and nonchemical (freezing) fixation schedules, (b) examination of model systems (erythrocytes, protein solutions), (c) substantiating the relaibility of critical-point drying, and (d) comparing images of whole cells with conventionally prepared (plastic-embedded) cells. The lattice structure is preserved by chemical and nonchemical fixation, though alterations in ultrastructure can occur especially after prolonged exposure to osmium tetroxide. The critical-point method for drying specimens appears to be reliable as is the freeze-drying method. The discrepancies between images of plastic-embedded and sectioned cells, and images of whole, critical-point dried cells appear to be related, in part, to the electron-scattering properties of the embedding resin. The described observations indicate that the microtrabecular lattice seen in electron micrographs closely represents the nonrandom structure of the cytoplasmic ground substance of living cultured cells.

Animals↗
PubMed1990

Immunocytochemical localisation of collagens (I-V) in the human iris.

In this study we investigated the distribution of collagen types I-V in the human iris at the fine-structural level using cryoultramicrotomy and London Resin White plastic embedding. Collagen type I was shown to be present in the basement membrane of iris vessels, in contrast to type III, which was absent; both types I and III were present in the iris stroma. Collagen type IV was a major component of basement membranes of vascular cells, myoepithelial cells, fibroblasts and epithelial cells. Types II and V were absent. Both cryo and plastic embedding techniques produced closely comparable results.

Adult↗
PubMed1995

[The new Hannover method of synthetic embedding of bone marrow. Cold polymerization of methylmethacrylate].

A patented low-temperature polymerization method for methylmethacrylate (MMA) infiltrated bone marrow biopsies is described: it has been developed from our previous MMA technique and is a patented procedure. Differences from the previous method are (1) removal of stabilizer from the MMA monomer before its application, (2) the use of a different starter, (3) avoidance of O2 influence during polymerization by means of vacuum exchange with N2, and (4) polymerisation in a water bath to draw off residual heat. After this procedure, all immunohistochemical reactions are possible provided that the previous fixation is adequate. The effects of different fixatives are reviewed briefly without detailed analysis. Technically, this plastic embedding can be performed at least as rapidly as the classic paraffin embedding after decalcification. The advantages over the latter method are: (1) the cells can be better differentiated because semi-thin sections can be made; (2) the immunoreactions can also be performed on the basis of semithin sections, which means they can be interpreted more easily; (3) morphometric analyses yield more reliable results because of the constant thickness of sections; (4) osteological examination of bone trabeculae, especially the search for mineralisation deficiencies, is possible; (5) the plastic embedding procedure is less dependent on individual instabilities in the quality of performance of the staff members involved. Furthermore, it is worth mentioning that the costs for additional equipment necessary remain below DM 100,000 including an excellent microtome.

Biomarkers, Tumor↗
PubMed2006

Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers.

High-pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze-substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high-pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high-pressure freeze, freeze-substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high-pressure freezing, freeze-substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.

Animals↗
PubMed1992

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.

Animals↗
PubMed1979

Histochemical demonstration of enzyme activities in plastic and paraffin embedded tissue sections.

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, beta-galactosidase, and cytochrome oxidase in plastic embedded and ruotine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 micrometers, were far superior to frozen sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.

Acid Phosphatase↗
PubMed1975

[Light and electron microscopic studies for classification of pituitary adenomas (author's transl)].

85 surgically removed pituitary adenomas were studied by light and electron microscopical and in part immunohistochemical methods. The tumors were histogically classified and reexamined by the ultrastructure. Histochemically the adenomas could be differentiated in acidophil adenomas (1. group, 41%), mucoid cell adenomas (2. group, 6%), and chromophobe adenomas (3. group, 37%) whereas oncocytic adenomas (4. group, 16%) could be identified only in plastic-embedded sections. About half of the acidophil adenomas were highly differentiated and showed structures which correspond to those of normal STH cells (subgroup 1.1). 1 adenoma consisted of cells of prolactin type (subgroup 1.2). The other acidophil adenomas were differentiated to a lower degree and showed no resemblance to the structures of normal acidophil cells. The 5 mucoid cell adenomas were proved to be with all methods highly differentiated adenomas of ACTH-cell type (subgroup 2.1). TSH-cell adenomas (subgroup 2.2) and lower differentiated mucoid cell adenomas (subgroup 2.0) were lacking in our collection. More than one third of the chromophobe adenomas showed well developed endoplasmic reticulum and Golgi complexes. The other had little and small organellas that resemblances to immature stem cells were evident. The oncocytic adenomas were identified in plastic-embedded sections by their fine-granular structures which were based ultrastructurally not on small secretory granules but on closely arranged mitochondrias.

Adenoma↗
PubMed1989

A versatile new mineralized bone stain for simultaneous assessment of tetracycline and osteoid seams.

A versatile mineralized bone stain (MIBS) for demonstrating osteoid seams and tetracycline fluorescence simultaneously in thin or thick undecalcified sections has been developed. Bone specimens are fixed in 70% ethanol, but 10% buffered formalin is permissible. Depending upon one's preference, these specimens can be left unstained or be prestained before plastic embedding. Osteoid seams are stained green to jade green, or light to dark purple. Mineralized bone matrix is unstained or green. Osteoblast and osteoclast nuclei are light to dark purple, cytoplasm varies from slightly gray to pink. The identification of osteoid seams by this method agrees closely with identification by in vivo tetracycline uptake using the same section from the same biopsy. The method demonstrates halo volumes, an abnormal, lacunar, low density bone around viable osteocytes in purple. This phenomenon is commonly seen in vitamin D-resistant rickets, fluorosis, renal osteodystrophy, hyperparathyroidism, and is sometimes seen in fluoride treated osteoporotic patients. In osteomalacic bone, most osteoid seams are irregularly stained as indicated by the presence of unmineralized osteoid between mineralized lamellae. The method has been used effectively in staining new bone formation in hydroxyapatite implants and bone grafts. Old, unstained, plastic embedded undecalcified sections are stained as well as fresh sections after removal of the coverslip. This stain also promises to be valuable in the study of different metabolic bone diseases from the point of view of remodeling, histomorphometry, and pathology.

Animals↗
PubMed1984

Anatomy of layer IV in cat primary auditory cortex (AI).

The structure of neurons and axons in layer IV was studied as part of a larger inquiry into the organization of primary auditory cortex (AI) in the cat. Tissue from the convexity of the middle ectosylvian gyrus between the anterior and posterior ectosylvian sulci was studied in Golgi, Nissl, Bodian, plastic-embedded, and other preparations from adult animals. Layer IV is defined as a strip about 200-250 micron thick consisting predominantly of small non-pyramidal neurons intercalated between the pyramidal somata of layers III and V, and in which few commissurally projecting cells occur. Lying some 800-900 micron beneath the pia, layer IV has six types of neurons, as seen in Golgi-stained material from anatomically and physiologically defined AI. These include three varieties (small, medium-sized, and large) of tufted neurons with intracortically branching axons and vertically polarized, cylindrical dendritic fields. Besides the tufted cells, which are the most numerous neurons in layer IV, large multipolar, double bouquet, and spiny stellate cells are scattered through layer IV. Each has a characteristic neuronal architecture and intracortical axonal branches. Smaller tufted cell somata dominate the upper half of layer IV (IVa), larger tufted cells are more common deep in layer IV (IVb). The average somatic area in midnuclear , plastic-embedded sections is 158 micron2. Layer IV (and layer IIIb) receive thick, probably ascending fibers, forming narrow, vertical terminal fields. These axons may be of thalamic origin and overlap with alternating, 50-75-micron-wide columns of somata and neuropil in layer IV. Layer III pyramidal cell axons often project to layer IV and ramify vertically and horizontally. The average height-width ratio of the dendritic domains of layer IV cells is about 3.6:1. The vertically disposed dendrites of layer IV cells, the columnar arrangement of their local axonal branches, and the polarized form of intrinsic and extrinsic axons collectively reinforces the columnar pattern in layer IV. Many layer IV cells structurally resemble neurons in layer IV in the primary visual and somatic sensory cortex. However, most AI cells have a pronounced columnar arrangement of their somata and an elongated, narrow form. The axons of many layer IV cells preserve this vertical arrangement and often branch in layer III and, to a lesser degree, in layer V.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Congenital blood cysts of the heart valves.

Congenital blood cysts of the heart valves are found most commonly on the tricuspid and mitral valves of fetuses and infants. Hearts available following 38 random autopsies of fetuses and infants 2 years of age or younger were examined. Blood cysts were found in 18 cases (47 per cent) in which ages ranged from 26 weeks of gestation to 11 months. The cysts varied in diameter, from microscopic to 3 mm. Affected valves had from one to 20 cysts. Light microscopic examination of serially sectioned paraffin-embedded tissue and plastic-embedded tissue and scanning electron microscopic examination revealed connections between the cyst lumens and ventricles via small endothelium-lined channels. The cyst structure suggested formation from ventricular endothelial infoldings in the valve leaflet base, which bulged into the atrium because of the pressure gradient present during valve closure. Blood cysts are a common finding in neonates dying of various causes and probably have no clinical significance. There is no association with asphyxia as previously described. Blood cysts may persist and enlarge to form giant cysts of the heart valves.

Aortic Valve↗
PubMed1986

Acellular scanning electron microscopy of spicular renal amyloidosis.

Two human renal biopsies containing glomerular amyloid deposits organized into spicular formations (spicular amyloid) were studied by scanning electron microscopy following removal of the cellular components (acellular SEM). Following SEM studies, portions of the same acellular tissue were embedded in paraffin and plastic for light microscopy and transmission electron microscopy, respectively. Spicular deposits by acellular SEM appear as tapering conical formations interconnected by a delicate branching network of fibrils, which imparts a higher degree of organization than previously appreciated by two-dimensional LM and TEM. Silver stains of paraffin- and plastic-embedded acellular tissue showed persistence of argyrophilia in spicular deposits, while acellular TEM showed that the spicules appeared comprised "purely" of amyloid fibrils without visible contaminating material. We conclude that the argyrophilia of spicular amyloid is an inherent feature of the parallel organization of fibrils rather than a result of incorporation of glomerular basement membrane or cell components and that spicular amyloid deposits have a higher degree of organization than is apparent by two-dimensional studies.

Amyloid↗