Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Synchrotron microcomputer tomography for the non‐destructive visualization of the fish skeleton

The objective of this study was to elucidate the potential of synchrotron radiation‐based microcomputer tomography as a non‐destructive method to visualize hard tissue in fish with high resolution. The high X‐ray contrast between mineralized tissue (bone and teeth) and soft tissue permits an easy differentiation. The nature of this method avoids preparation artefacts which may occur during the preparation for histology or scanning electron microscopy. The spatial resolution is of the order of a few μm, depending on the sample. Results obtained for the species medaka (Oryzias latipes) and zebrafish (Danio rerio) are presented.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

Microstructure and growth of the lenses of schizochroal trilobite eyes

Lenses within the schizochroal eyes of phacopine trilobites are made principally of calcite, and characterization of them using light microscopy and high‐resolution electron imaging and diffraction has revealed an array of microstructural arrangements that suggest a common original pattern across the suborder. The low convexity lenses of Odontochile hausmanni and Dalmanites sp. contain calcite fibres termed trabeculae. The c axis of trabecular calcite lies parallel to the lens axis, and adjacent trabeculae are distinguished by small differences in their a axis orientations. Despite the common alignment, the boundaries between trabeculae cross‐cut the c axis as they fan out towards the lens base. Trabeculae are absent from the lens immediately beneath the visual surface, and instead, a radial fringe is present and is composed of micrometre‐thick sheets of calcite whose c axes are oriented at a low angle to the visual surface. High convexity lenses are more common than those of lower convexity among the species studied, and they have a much thicker radial fringe. Beneath this fringe, all of the lens calcite is oriented with its c axis parallel to the lens axis and it lacks trabeculae. We propose that both the high and low convexity lenses formed by rapid growth of calcite from a surface that migrated inwards from the cornea, and they may have had an amorphous calcium carbonate precursor. The trabeculae and radial fringes are unlikely to have had any beneficial effect on the transmission or focusing of light, but rather are the outcomes of an elegant solution to the problem of how to construct a biconvex lens from a crystalline solid.     

Mid-IR microspectroscopic imaging of breast tumor tissue sections

IR microspectroscopic imaging is a relatively new approach for the examination of tissue sections. In contrast to standard light microscopy based procedures, the IR approach requires neither sample staining nor fixation. The IR spectra of breast tumor tissue sections are obtained via a microscope equipped with a focal plane array detector. This enabled the simultaneous collection of individual mid-IR spectra from thousands of different sample positions with a spatial resolution near the diffraction limit. The analysis of the IR data reveals a high sensitivity of the IR approach toward changes in tissue biochemistry and variations in breast tissue architecture. Moreover, the data demonstrate the need for collecting spectra with high spatial resolution at the level of individual cells. This minimizes problems associated with tissue microheterogeneity and is an essential prerequisite for future studies aimed at developing IR microspectroscopic imaging as a complement to present diagnostic tools for breast cancer.     

Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

Non-isotopic in situ hybridization to detect chick Sox gene mRNA in plastic-embedded tissue

Summary In situ hybridization techniques have rapidly become widely used by the molecular biologist for the localization of specific nucleic acid sequences in individual cells or tissues. We describe the demonstration of Sox gene mRNA in chick tissue that has been embedded in the plastic methyl methacrylate to permit the preparation of sections for high-resolution light microscopy. Polymerization of the plastic was induced by using either N, N-dimethylaniline or N, N-3,5-tetramethylaniline. The in situ hybridization technique used was non-isotopic and used a digoxigenin-labelled probe detected with an antibody bound to alkaline phosphatase, which was then localized using X-phosphate-Nitro BT as a substrate-chromogen mix. Various pretreatments of the tissue sections were investigated, including the use of proteinase K, and heat-mediated techniques using a microwave oven and a pressure cooker. The best results were produced using pressure cooking on tissue in which the plastic had been chemically polymerized with N, N-3,5-tetramethylaniline. For the demonstration of Sox 11, this combination had a critical influence on the staining results, but for Sox 21 all protocols used produced good staining. This revised version was published online in November 2006 with corrections to the Cover Date.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

A Simple Method Using Alizarin Red S For the Detection of Calcium in Epoxy Resin Embedded Tissue

This report presents a simple procedure for staining 1-2 μm epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue 0 solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralition. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets¹, monitor dynamics in living cells^2-4, and visualize the three dimensional evolution of entire organisms^5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo⁷ and are currently being applied to disease imaging and diagnosis in clinical settings^8,9.Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole¹, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.Download video file.^{(90M, mov)}     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Elderberry bark lectin-gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations

Summary The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate.N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites ofSambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (2,6) galactose/N-acetylgalactosamine.     

The interrelation of the size and substantivity of dyes; the role of van der Waals attractions and hydrophobic bonding in biological staining

Summary Using a pH signature criterion, it was found that whereas electrostatic attractions and repulsions were paramount in the binding of low molecular weight acid and basic dyes to tissue sections, high molecular weight dyes were also bound non-electrostatically.By studying the effects on staining of adding to aqueous dyebaths agents destroying the iceberg structure of water, the importance of hydrophobic bonding was established. It was noticed that the hydrophobic elastic fibres were stained by large dyes from dyebaths inhibitory both to electrostatic attractions and hydrophobic bonding (i.e. using acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths). This indicated that strong van der Waals attractions occurred, at least with some substrates. Supporting this idea was the observation that in tissue sections benzoylated before staining (i.e. made less acidophilic but more hydrophobic) additional structures were stained when using large acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths.Applications of the size-substantivity relationship were suggested, e.g. commenting on a standard stain for basic proteins; explaining the modes of action of traditional stains for elastic fibres and amyloid; rationalising the varied substantivities of tetrazolium salts; and finally suggesting guide lines for use in the design of new staining methods.     

Factors influencing extract of Hibiscus sabdariffa staining of rat testes

Abstract

Some plant extracts can be used in biology and medicine to reveal or identify cellular components and tissues. We investigated the effects of time and concentration on staining of histological sections of rat testes by an acidified extract of Hibiscus sabdariffa. An ethanolic extract of H. sabdariffa was diluted using 1% acetic acid in 70% ethanol to stain histological sections of testes at concentrations of 0.2, 0.1 and 0.05 g/ml for 5, 10, 15, 30, 45 and 60 min. The sections of testes were stained deep red. The staining efficiency of H. sabdariffa was greater at a high concentration and required less time to achieve optimal staining. H. sabdariffa is a strongly basic dye that can be used for various diagnostic purposes. Staining time and concentration must be considered to achieve optimal results.     

Nuclear magnetic resonance microscopy ofAncistrocladus heyneanus

Summary The tropical lianaAncistrocladus heyneanus, which is known for its biologically active naphthylisoquinoline alkaloids, has been studied by nuclear magnetic resonance (NMR) microscopy for the first time. The spatial resolution of the cross-sectional NMR images was of the order of 20 m. Quantitative NMR relaxation time images of the root and the shoot show great contrast between different tissue regions. In addition, we observed the regional distribution of chemical compounds inAncistrocladus heyneanus by chemical-shift NMR microscopy. The NMR imaging results were compared with light and fluorescence microscopic images and reveal the excellent tissue characterization using NMR technology.Abbreviations NMR nuclear magnetic resonance - CSI chemical-shift magnetic resonance imaging - FOV field of view - TE echo time - TR repetition time     

Polychromatic staining of epoxy semithin sections: a new and simple method

A simple, rapid method is described for the polychromatic coloration of semithin sections, which is applicable to material routinely processed for transmission electron microscopy. Material fixed with a glutaraldehyde-paraformaldehyde mixture and postfixed in osmium tetroxide with or without potassium ferrocyanide and embedded in different types of resin (Durkupan-ACM, Spurr resin, Taab resin) can be used. Constant and homogenous results are obtained with this technique, the staining procedure being achieved at room temperature in no more than 10 min. Sections of 0.5–1 m in thickness are oxidised and bleached. After washing, sections are stained in two steps with carbol methylene blue/carbol gentian violet solution and pararosaniline solution. Using the method described in this paper, a polychromatic coloration of the different cells and tissues was obtained (epithelial cells in various shades of blue-violet, connective tissue and elastic laminae of blood vessels in pink or red, etc.). This procedure provides greater contrast between cytoplasm and nuclei, and among the different types of cells and tissues than is seen with toluidine blue, which is very useful for observation and photography of semithin sections. Polychromatic methods found in the literature are normally complex and require a lengthy staining time or cannot be applied on material routinely processed for transmission electron microscopy. Our method is simple, rapid and can be used on any type of material routinely processed for transmission electron microscopy and embedded in epoxy resins.