Preparation Of Thin Sections Research Articles

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.

Ultraviolet Resonance Raman Spectroscopy of Bulk and Microscopic Human Colon Tissue

A sensitive ultraviolet resonance Raman (UVRR) system, which integrates bulk and microscopic capabilities, is described for use with human tissue. The system incorporates a quasi-continuous tunable laser excitation source in the range 200–300 nm, a liquid nitrogen-cooled charge-coupled device (CCD), a single grating spectrometer, dielectric longpass laser line rejection filters, and cooled specimen holders to minimize bio- and photo-degradation. The system was used on normal human colon tissue, and was compared to our previously used Nd:YAG-based system. Spectra with higher signal-to-noise ratio (S/N) than has previously been achieved, and with less than 5% photobleaching, are presented. The present system outperforms our previous system by improving the S/N in bulk normal colon spectra by a factor of greater than 10. We show for the first time UVRR spectra collected from the various microscopic regions of human colon in a thin section preparation, and the line shapes from the different microscopic morphological components are related to the bulk colon spectra. At 251 nm the epithelial cells and the lamina propria are the major contributors to the UVRR spectrum of bulk colon tissue. Our data suggest that combined bulk and microscopic UVRR spectroscopy of human tissue could provide a valuable tool for bioassay and histochemistry.

Preparation of polished thin sections from poorly consolidated regolith and sediment materials

Abstract Good quality polished thin sections can be prepared from poorly consolidated, wet, fragile and friable regolith and sediment materials in a standard earth science thin section laboratory, with only minor additional equipment. Materials are dried over an acetone bath, impregnated with a polyester resin mix, and cut and polished using minor modifications of standard hard rock thin sectioning equipment and techniques. The sections produced are suitable for both optical and electron microscopy, and electron probe microanalysis.

Applicability of a combined staining method with fluorochromes for the visualization of microbial cells in a soil thin section

Staining of microbial cells in a soil thin section provides useful information about the role of soil as an ecological niche by relating the in situ distribution of microorganisms to the state of pores under undisturbed conditions. For technical improvement, we developed a procedure consisting of a combined staining method with 5-sulfofluorescein diacetate (SFDA), a metabolic type fluorochrome, and magnesium salt of l-anilino-8-naphthalene sulfonic acid (Mg-ANS), an adsorption type fluorochrome, in order to detect and differentiate the living microbial cells from the dead ones in a single soil thin section. Using images under a fluorescence microscope, we also examined the applicability of SFDA for staining microbial cells embedded in soil thin sections, which had seldom been attempted. Through this study, we confirmed the following facts: i) combined staining with SFDA and Mg-ANS can be achieved without major modification of the general procedure for thin section preparation, ii) staining with SFDA can be applied to soil thin sections, and iii) detection of in situ distribution and differential identification of the living and dead microbial cells may be possible by the proposed combined staining method.

An X-ray scanning technique to determine soil macroporosity by chemical mapping

Soil micromorphological studies have used various techniques for pore analysis, including optical measurements, electron microscopy and computed tomography. This study examined a method based on X-ray photon mapping, in which a scanner obtains the sample's X-ray spectrum, selects those portions which have been specified as the elements to map and then only separates that part of the spectrum to be analysed by the software. With this new technique, the resulting chemical maps are composed of the distribution of selected elements within the sample and the only digitising is to convert the analogue X-ray signal into a digital signal that can be computer processed. The approach provided two-dimensional chemical maps of soil blocks, clearly showing macropore sizes and distributions and enabling calculation of macroporosity values. The results showed mean macroporosity values of 17–18%. The main benefits of the technique are the precision of data and image processing and the elimination of laborious thin section preparation, as hardened soil blocks can be scanned automatically.

Preparation of Thin Sections

Abstract The optical examination of a rock sample in thin section is the quickest and most economical method for classifying rock type and determining which analytical route to follow. Thin sections for transmitted light are the most common, but there are also: Polished Thin Sections • Polished sections are used for classification and identification of minerals that cannot be determined in standard thin sections. They are also essential for microprobe analysis. Minute mineral grains are analyzed by bombarding them with a focused bean of electrons, which generate x-rays, characteristic of the elements within the grains. X-rays are identified and quantified to determine the chemical composition of minerals.

Preparation of thin Sections of (znxcdy)s Fine Particles by an Ultramicrotome

Abstract The low voltage phosphors should be efficiently excited with an electron beam value lower than 500V. Thus the properties of low voltage phosphors are directly affected by the surface states of phosphors. Recently, to improve the properties of these phosphors, much analysis has been carried out focused on the surfaces of phosphors using by FESEM, AES or XPS etc.. Nevertheless, the microstructure of phosphors around the surface has not been made clear due to the resolution limits of the above equipments and the difficulties of sample preparation. In this study, in order to analyze the microstructure of phosphors around the surface, we have prepared the thin sections of the (ZnxCdy)S phosphors by an ultramicrotome and carried out high resolution electron microscope(HREM) study on the thin sections. To carry out ultramicrotomy, the particles of phophors were embedded with acrylic resin (methyl methacrylate: n-butyl methacrylate= 4:6, Benzoyl peroxide 1.5%) in a capsule.

Application of Confocal Laser Scanning Microscopy in Bone

The optical sectioning method of confocal laser scanning microscopy provides higher resolution than standard light microscope techniques. The use of optical rather than physical sections for detailed histological analyses of bone obviates the need for either decalcification or complex plastic embedding processes which are required as a routine for the preparation of thin microtome sections. In this study we have used confocal laser scanning microscopy for the morphological analyses of fresh unembedding human cortical bone, bone allograft and bone cement interfaces. Our results have indicated that such an approach has provided a relatively easy and rapid means for the assessment of the histology of normal and pathological bone.

Adherence ofBilophila wadsworthiato Cultured Human Embryonic Intestinal Cells

Adherence ofBilophila wadsworthiato the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76–100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0–25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence ofB. wadsworthiato Int 407 cells is mediated by an outer membrane or cell wall component.

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.

Preparation of Thin Sections of Coated Fibers for Characterization by Transmission Electron Microscopy

A method of preparing transmission electron microscopy (TEM) specimens of coated ceramic fibers has been developed, which produces large electron transparent areas due to the minimal preferential milling of the fiber, coating, or epoxy matrix. Multiple individual fibers or tows are impregnated with a high‐temperature epoxy and contained to assure a high fiber‐to‐epoxy volume ratio. The samples are then sectioned and mechanically thinned either parallel or normal to the fiber axes using a wedge polisher on diamond lapping films to achieve a thickness of less than 5 μ.m. The thinned sample is then ion‐milled to electron transparency in less than 30 min, giving representative specimens of the coating, fiber, and coating‐fiber interface. This technique is also well suited to preparing extremely flat specimens for scanning electron microscopy analysis of thin coatings. Examples of TEM sections of coated fibers prepared using this technique are presented.

Evaluation of polyester, epoxy and acrylic resins for suitability in preparation of soil thin sections for in situ biological studies

Micromorphological investigations which aim to integrate both physical and biological features of soils are particularly demanding with respect to the quality of the thin sections produced for such studies. Rapid preparation of thin sections is also often desirable. The suitability of a range of resins for the rapid preparation of high-quality thin sections appropriate for biologically oriented studies was tested. Preparative procedures were investigated using four undisturbed soils (albic, calcic, humic, and argillic horizons) chosen to exhibit contrasting properties. Roots of sown Lolium perenne, their associated AM-mycorrhizas, and indigenous fungi and actinomycetes of the soils were used as indicators of the quality of preservation of biological material in sections of 15 μm thickness. Crystic 17449 and Palatal P50-01 proved most suitable, producing durable sections in 2–3 weeks with excellent preservation of roots, microbes and inorganic features in all the soils tested.

Rotation cryotomy: Medical and scientific value of a new serial sectioning procedure

Preparation of thin serial sections for comparative macromorphologic investigations has always represented a grave technical problem, especially in the case of regions in which bone as well as soft tissue are to be documented within their natural relations to each other in any desired sectional plane. Non-decalcified specimens up to the size of a whole cadaver are embedded in physiologic medium, precisely positioned, and deep-frozen to a specimen-ice block. A newly developed device, working on the basis of blades rotating at high speed, allows quick, successive removal of sections from the surface of the specimen block, with a thickness of each section infinitely variable between 0.1 and 5 mm. Following each cut, the new surface of the block can be documented photographically or on videotape for macromorphologic evaluation. So far more than 1,000 human, animal, and botanical specimens have been sectioned and evaluated with this method. In none of the cases were specimens damaged. Furthermore, any desired sectional plane could be adjusted; consequently a definite correlation between these sections and previous sonography, magnetic resonance (MR), or computed tomography (CT) images could be established. As serial cryosectioning becomes available to a far wider circle of medical and natural scientists, high-quality results should be obtained at lower costs. © 1996 Wiley-Liss, Inc.

Stain Characteristics of Thin Ground Sections of Hydrophobic Resin-Embedded Equine Hooves.

For a light microscopic examination from a large amount of horse hoof specimen, we usually apply a grinding technique using a hydrophobic resin to prepare a thin ground section. After preparation of the resin-embedded thin ground section (RGS), we stain it without prior deresinization or heating in any stages of the staining procedures in order to intensify the stain outcome. This technique includes an advantage to prevent RGS from modification. We do not study, however, the stain characteristics of the RGSs of the hoof tissues which are made by the above procedures. Therefore, we compared the RGSs (n=11) with the standard paraffin-embedded sections (PSs) (n=11) with H.E. stain, Ayoub-Shklar method and Dane's method. The H.E. stained RGS did not essentially differ from the one of the PS, but two special stain methods showed different stain characteristics compared with the stain outcome of the PS. From these results, H.E. stain is an applicable stain technique for the non-deresinized thin ground sections of the horse hooves.

Preparation of successive thin sections of fine oxide particles by an ultramicrotome

A large number of monodispersed fine oxide particles, which are uniform in shape, size and composition, have been developed as ideal advanced materials. Recently, by the newly developed gel-sol method, various shapes of micron-sized monodispersed hematite particles (α-Fe2O3) were produced, such as peanut-type, ellipsoidal and pseudocubic shapes. To understand the growth mechanism of these particles, it is necessary to investigate their internal structures. In this study, in order to analyze three dimensional internal structure of these particles, we have prepared their successive thin sections with an ultramicrotome and carried out HREM study on these successive thin sections.Monodispersed peanut-type and pseudocubic particles were prepared by the standard procedures of the “gel-sol method. For carrying out ultramicrotomy, these particles were embedded with acrylic resin (methyl methacrylate: n-butyl methacrylate=3:7 or 4:6, benzoyl peroxide 1.5-1.7%) in a gelatin capsule. After trimming with a glass knife, successive thin sections of the particles were obtained by a diamond knife with the optimum slicing condition. The slicing speed and thickness were set to be 0.6 mm/sec and 70 nm, respectively.

Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 μm) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 μm) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

Determination of Slag and Fly Ash Content in Hardened Concrete

Abstract Some agencies limit the slag (ground-granulated blast-furnace slag) replacement of portland cement in some instances. In other cases, minimum slag replacements are specified. Therefore, there is a need to be able to quantify the slag content to show conformance with specifications. The two methods proposed here are (1) X-ray diffraction (XRD) of ignited mixtures and (2) optical microscopy on thin sections. The XRD method involves ignition of the mortar fraction of concrete at 950 to 1050°C to devitrify the unreacted slag. The resulting crystalline melilite component is then compared to that in an ignited sample of slag from the same source. The optical method involves preparation of thin sections of the concrete and point counting to obtain the number of unhydrated slag grains. These are compared to a set of calibration standard sections. The optical technique can also be used for estimating fly ash content and similarly, grain mounts have been used to detect the presence of both fly ash and slag substitution in portland cements. The paper describes both these techniques in detail and discusses limitations. When properly calibrated, either technique can be used to estimate the slag replacement level to approximately ±10%. As well, several case histories are described where these procedures have proved to be extremely useful.

Postembedding colloidal-gold immunocytochemistry of noncollagenous extracellular matrix proteins in mineralized tissues.

Immunocytochemistry is a powerful tool for investigating protein secretion, extracellular matrix assembly, and cell-matrix and matrix-matrix/mineral relationships. When applied to the tissues of bones (bone and calcified cartilage) and teeth (dentin, cementum, and enamel), where calcium phosphate-containing extracellular matrices are the predominant structural component related to their weight-bearing and masticatory roles, respectively, data from immunocytochemical studies have been prominent in advancing our understanding of mineralized tissue modeling and remodeling. The present review on the application of postembedding, colloidal-gold immunocytochemistry to mineralized tissues focuses on the advantages of this approach and relates them to conceptual, theoretical, and experimental data currently available discussing matrix-mineral interactions and extracellular matrix formation and turnover in these tissues. More specifically, data are summarized regarding the distribution and role of noncollagenous proteins in different mineralized tissues, particularly in the context of how they interface with mineral, and how this relationship might be affected by the various tissue-processing steps and immunocytochemical strategies commonly implemented to examine the distribution and function of tissue proteins. Furthermore, a technical discussion is presented that outlines several different possibilities for epitope exposure in mineralized tissues during preparation of thin sections for transmission electron microscopy. Cell biological concepts of protein secretion by cells of the mineralized tissues, and subsequent extracellular matrix assembly and organization, are illustrated by examples of high-resolution, colloidal-gold immunolabeling for osteopontin, bone sialoprotein, and osteocalcin in the collagen-based mineralized tissues and for enamel protein (amelogenin) in enamel.

The herpes simplex virus gene UL26 proteinase in the presence of the UL26.5 gene product promotes the formation of scaffold-like structures.

The herpes simplex virus type 1 (HSV-1) polypeptides encoded by genes UL26 and UL26.5 are thought to form a scaffold around which the capsid shell assembles. The UL26 gene specifies a proteinase that cleaves both itself and the UL26.5 gene product. To study the structure and function of the UL26 and UL26.5 gene products, the proteins were expressed in cells infected with recombinant baculoviruses containing the genes under the control of the polyhedrin promoter. Both polypeptides were made in large amounts, approaching the levels of polyhedrin protein expressed in wild-type baculovirus. The UL26 polypeptide behaved in a similar manner to the protein made in HSV-1-infected cells, cleaving itself rapidly into the capsid proteins VP21 and VP24 and converting the UL26.5 product more slowly into the capsid protein VP22a. The results of immunoblot analysis using antisera specific for the amino-terminal region of the UL26 polypeptide suggested that both the first and second ATGs in the UL26 open reading frame were recognized as translational start signals but the first ATG was the preferred initiation codon as is the case in HSV-1-infected cells. Electron microscopic examination of thin section preparations of cells infected with both the UL26.5- and UL26-recombinant baculoviruses revealed the presence of large numbers of small spherical particles, often arranged in a semi-crystalline array. These clusters of scaffold-like particles were not present in cells infected with UL26-recombinant baculovirus but were observed occasionally in UL26.5-recombinant baculovirus-infected cells. The results suggest that the proteinase, in the absence of other HSV capsid proteins, stimulates the formation of large numbers of scaffold-like particles present either as semi-crystalline arrays or as dispersed structures.

Preparation of Thin Ground Section of Resin-Embedded Hooves for Histologic Analysis.

A method for grinding thin sections o f resin-embedded hooves for histological analysis is described. Ten percent neutral formalin fixed material is dehydrated in a graded ethanol series, then embedded in resin. After polymerization, sections are cut to a width of 0.5-1.0 mm with diamond blade saw, and then ground to a thickness of 30-40 μm. The ground sections thus obtained are considered to be suitable for histological study of hooves.