Free-floating cryostat sections for immunoelectron microscopy: Bridging the gap from light to electron microscopy

Frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone for two reasons: some skin basement membrane zone proteins are labile to routine chemical fixation, and skin is not amenable to vibratome sectioning. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slide-mounting, usually limit immunohistochemical study to the light microscopy level. In the present study, we introduce use of unfixed, free-floating cryostat sections for characterization of immunolocalizations of selected skin basement membrane proteins at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy. In this regard, the method renders itself useful when results of immunolabeling experiments need to be elucidated quickly at histological and ultrastructural levels as required for diagnostic and accelerated investigative strategies.     

Sinusoidal endothelial cells of the liver: Fine structure and function in relation to age

Liver endothelial cells form a continuous lining of the liver capillaries, or sinusoids, separating parenchymal cells and fat-storing cells from sinusoidal blood. Liver sinusoidal endothelial cells differ in fine structure from endothelial cells lining larger blood vessels and from other capillary endothelia in that they lack a distinct basement membrane and also contain open pores, or fenestrae, in the thin cytoplasmic projections which constitute the sinusoidal wall. This distinctive morphology supports the protective role played by liver endothelium, the cells forming a general barrier against pathogenic agents and serving as a selective sieve for substances passing from the blood to parenchymal and fat-storing cells, and vice versa. Sinusoidal endothelial cells, furthermore, significantly participate in the metabolic and clearance functions of the liver. They have been shown to be involved in the endocytosis and metabolism of a wide range of macromolecules, including glycoproteins, lipoproteins, extracellular matrix components, and inert colloids, establishing endothelial cells as a vital link in the complex network of cellular interactions and cooperation in the liver. Fine structural studies in combination with the development of cell isolation and culture techniques from both experimental animal and human liver have greatly contributed to the elucidation of these endothelial cell functions. Morphological and biochemical investigations have both revealed little changes with age except for an accumulation of iron ferritin and a decrease in the activities of glucose-6-phosphatase, Mg-ATPase, and in glucagon-stimulated adenylcyclase. Future studies are likely to disclose more fully the role of sinusoidal endothelial cells in the regulation of liver hemodynamics, in liver metabolism and blood clearance, in the maintenance of hepatic structure, in the pathogenesis of various liver diseases, and in the aging process in the liver.     

Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix

Angiogenesis is critical to wound repair. Newly formed blood vessels participate in provisional granulation tissue formation and provide nutrition and oxygen to growing tissues. In addition, inflammatory cells require the interaction with and transmigration through the endothelial basement membrane to enter the site of injury. Angiogenesis, in response to tissue injury, is a dynamic process that is highly regulated by signals from both serum and the surrounding extracellular matrix (ECM) environment. Vascular endothelial growth factor, angiopoietin, fibroblast growth factor, and transforming growth factor beta are among those most potent angiogenic cytokines in wound angiogenesis. The cooperative regulation of them is essential for wound repair. Migration of endothelial cells and development of new capillary vessels during wound repair is dependent on not only the cells and cytokines present but also the production and organization of ECM components both in granulation tissue and in endothelial basement membrane. The ECM regulates angiogenesis by providing scaffold support and signaling roles. They also serve as a reservoir and modulator for growth factors. Laminins are the major noncollagenous ECM of endothelial basement membrane. Two newly recognized laminins, 8 and 10, are the major laminins produced by human dermal microvascular endothelial cells. Laminin 10 is highly expressed in blood vessels around skin wounds. Laminin 8 promotes dermal endothelial cell attachment, migration, and tubule formation. Integrins with either beta 1 or alpha v subunits are the major cellular surface receptors for ECM molecules and mediate the interactions between cells and ECM during wound angiogenesis.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the structural and immunocytochemical examination of biological specimens by electron microscopy

A simple and reliable method to make resinless sections for electron microscopy was recently developed by using polyethylene glycol (PEG) as a transient embedding media. In this paper the practical procedure of this PEG method is described in detail. Normal ultrastructure of several types of in-situ cells in resinless sections is demonstrated. The cytoplasmic matrix of all in-situ cells examined is revealed to consist of the microtrabecular lattice. A result from application of this technique to immuno-electron microscopy is also illustrated. This method is shown to have potential in overcoming the problem of intracellular penetration of macromolecular antibodies. Several artifacts caused by failures in specimen preparations are displayed. The real or artifactual nature of the microtrabecula is briefly discussed.     

Comparison of polyethylene glycol and diethylene glycol distearate embedding methods for the preservation of fungal cytoskeletons

Hyphae of the fungus Basidiobolus magnus were embedded in extractable polyethylene glycol (PEG) or diethylene glycol distearate (DGD) to prepare embedment-free sections in order to seek components of the cytoskeleton that may be obscured in epoxy-embedded sections. All methods showed that hyphae possess an intricate filamentous matrix, which is apparently unoriented. However, the appearance of the cytoskeleton depended on the embedding medium, the solvent used during embedding, and whether cells were fixed by conventional fixation or freeze-substitution. PEG proved to be the best embedding medium for fungal cells because DGD caused cell shrinkage and produced a more lamellar than filamentous matrix. Also, the cytoskeletal filaments were clearer in freeze-substituted cells than in conventionally-fixed cells. Since we failed to find microtubules in the embedment-free sections, we re-embedded cells in Epon to discern whether microtubules or other cytoplasmic components had changed. Neither PEG nor DGD adequately preserved microtubules as compared to regular Epon-embedded sections, and other cellular structures were significantly altered. Therefore, alternative methods need to be employed to further characterize fungal cytoskeletons.     

Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.     

Imaging of collagen type III in fluid by atomic force microscopy

Type III collagen is a component of the basement membrane of endothelial cells, and may play a role in the interaction between hemostatic system proteins and the basement membrane of blood vessels. To begin to investigate these structural interactions, we have imaged type III collagen in solution by atomic force microscopy. A 20 microg/ml solution of type III collagen in bicarbonate buffer (pH 9.5) from calf skin was deposited onto a freshly cleaved mica substrate. Atomic force microscopy images were acquired using a fluid cell and tapping mode with oxide-sharpened silicon nitride probes 2, 3, and 4 hours after deposition of the collagen onto the mica. Two-hour preparations displayed fibrillar networks with well-defined sites of nucleation and lateral growth. At 3 and 4 hour polymerizations, more mature fibrils of increasing lengths, diameters, and complexity were observed. Fibrils appeared to be aligning and twisting (helical formation) to form a mature fibril with a higher mass per unit area. Interestingly, the mature fibrils appeared larger centrally with tapered ends displaying declining slopes. These observations compare favorably with those previously published on collagen type I assembly [Gale et al. (1995) Biophys. J. 68:2124-2128]. High resolution atomic force microscopy images of type III collagen in solution should provide a template for observation of the interactions between basement membrane components and hemostatic system proteins present in cardiovascular disease.     

Ultrastructure of endothelial cells under flow alteration

Endothelial cells are stable and quiet in normal animals. They arrange regularly and have a smooth lumen surface and thin endothelial wall. According to Thoma's principle (1893) and Kamiya and Togawa's principle (1980) on the relationship of the vascular diameter to flow alteration, blood flow is in equilibrium to the diameter and in a physiological state. That is to say, there is no fast flow or slow flow. To understand the nature of the endothelial cells, we should investigate endothelial cells under flow alteration to break the equilibrium state. Endothelial cells under increased flow were studied in arteries with an arteriovenous fistula or in the capillaries of myocardium with volume-overloaded hearts or of the skeletal muscle by electrical stimulation. Those under decreased flow were studied by the closure of the fistula or by ceasing the stimulation. Endothelial cells in the coarctation of the arteries were also observed. Endothelial cells were activated by increased flow in the arteries and capillaries, while they were inactivated by decreased flow. Endothelial activation is characterized as lumen protrusions, increase of cytoplasmic organelles, abluminal protrusions, basement membrane degradation, internal elastic lamina degradation in the arteries, and sproutings in the capillaries. These are ultrastructurally comparable to angiogenesis. Endothelial inactivation is characterized by the decrease of endothelial cell number with apoptosis, which is ultrastructurally comparable to angioregression. We assume that endothelial cells respond to increased flow by angiogenesis and to decreased flow by angioregression.     

Dancing to a somewhat different rhythm: Cell migration along the natural basement membrane

Much of our understanding of the events which underlie cell migration has been derived from studies of cells in tissue culture. One of the components that mediates this process is the dynamic actin-based microfilament system that can reorganize itself into so-called stress fibers that are considered essential components for cell motility. In contrast, relatively few studies have investigated cell movement along an extracellular matrix (ECM) which is known to influence both cellular organization and behavior. This opinion/viewpoint article briefly reviews cell migration during corneal endothelial wound repair along the tissue’s natural basement membrane, Descemet’s membrane. Because the tissue exists as a cell monolayer it affords one an opportunity to readily explore the effect of cell/matrix influences on cell motility. As such, cell movement along this substrate differs somewhat from that found in vitro and migrating endothelial cells also demonstrate an ability to move along the ECM without the benefit of having an organized actin cytoskeleton.     

The histochemical basis of quantitative histology*

The interaction between histochemistry and microscopy for quantifying the morphology and function of cells in sections of tissue is reviewed. In principle, the morphological parameters of cells can be measured in suitably-stained sections using image analysers. In practice, however, the individual cells often cannot be distinguished from each other in such systems. This problem might be overcome, at least in muscle, by visualizing types III and IV collagen in the connective tissue and basement membranes surrounding the cells. The functional capacities of cells within tissues can be inferred by measuring the activities of their key enzymes with quantitative histochemical techniques. At present, the wrong enzymes are frequently chosen for this purpose. Flux-generating and physiologically significant enzymes are preferable, but this requires new histochemical techniques to be devised and validated quantitatively for use with the computer-assisted analytical microscopes that are now becoming available commercially.     

Atmospheric scanning electron microscopy and its applications for biological specimens

Scanning electron microscopy in ambient conditions (Air‐SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air‐SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)‐SEM. Sharpness was lower than that of FE‐SEM, but foot process of podocytes was observed at high magnification. Air‐SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air‐SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air‐SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.     

Laminins and their roles in mammals

Laminins are alpha-beta-gamma heterotrimeric components of all basement membranes. Laminins are now known to play the central role in organizing and establishing the basement membrane. The diversity of laminins allows them to impart special structural and signaling properties to the basement membrane. Of the 12 known laminin chain genes, 10 have been either found to be mutated in humans or experimentally mutated in mice. This has led to great progress over the last several years towards understanding both the functions of laminins and the reasons for their great diversity. In this review, I will summarize the in vivo studies in mice and humans that have contributed to this new knowledge.     

Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope

A new one-sided tandem scanning microscope (OTSM) has been utilized to optically section a transparent cornea and ocular lens with submicron depth and transverse resolution. Its high resolution, image quality, and contrast, together with color capability, real-time operation, and easy alignment, have advantages over previous confocal systems. Planes within the nuclei of surface epithelial cells, the epithelial basement membrane, nerve fibers, the interior of endothelial cells, and the transverse structure of the lens fibers are readily observed. The utility of real-time microscopic observation of live biological structures provides a new paradigm for cell biology and diagnostic ophthalmology.     

Fabrication,characterization and evaluation of the effect of PLGA and PLGA–PEG biomaterials on the proliferation and neurogenesis potential of human neural SH-SY5Y cells

In recent years with regard to the development of nanotechnology and neural stem cell discovery, the combinatorial therapeutic strategies of neural progenitor cells and appropriate biomaterials have raised the hope for brain regeneration following neurological disorders. This study aimed to explore the proliferation and neurogenic effect of PLGA and PLGA–PEG nanofibers on human SH-SY5Y cells in in vitro condition. Nanofibers of PLGA and PLGA–PEG biomaterials were synthesized and fabricated using electrospinning method. Physicochemical features were examined using HNMR, FT-IR, and water contact angle assays. Ultrastructural morphology, the orientation of nanofibers, cell distribution and attachment were visualized by SEM imaging. Cell survival and proliferation rate were measured. Differentiation capacity was monitored by immunofluorescence staining of Map-2. HNMR, FT-IR assays confirmed the integration of PEG to PLGA backbone. Water contact angel assay showed increasing surface hydrophilicity in PLGA–PEG biomaterial compared to the PLGA substrate. SEM analysis revealed the reduction of PLGA–PEG nanofibers' diameter compared to the PLGA group. Cell attachment was observed in both groups while PLGA–PEG had a superior effect in the promotion of survival rate compared to other groups (p < .05). Compared to the PLGA group, PLGA–PEG increased the number of Ki67⁺ cells (p < .01). PLGA–PEG biomaterial induced neural maturation by increasing protein Map-2 compared to the PLGA scaffold in a three-dimensional culture system. According to our data, structural modification of PLGA with PEG could enhance orientated differentiation and the dynamic growth of neural cells.     

Immunogold-silver staining of lymphocyte surface antigens on cells in suspension and in lymph node cryostat sections

An immunogold–silver staining (IGSS) technique for the light microscopical detection of leucocyte cell surface antigens in cell suspensions and cryostat sections is described. The specimens were first incubated with monoclonal mouse antibodies and then with colloidal gold-labelled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained and mounted. In light microscopy, the tissue architecture and the cellular morphology were well preserved. Positive cells showed dark granules on their surface membranes. Optimal labelling conditions were determined. This method proved to be a reliable tool for the enumeration of T-cells and their subsets in peripheral blood. The dense labelling permitted the use of panoptic counterstains like May-Grünwald-Giemsa or Wright's stain. This IGSS technique was used to determine the distribution of the T- and B-cell subsets in cryostat sections of reactive lymph nodes. The sensitivity of the method was comparable with that of immunofluorescence microscopy for cell suspensions and that of the biotin–avidin–peroxidase technique for tissue sections. Immunogold–silver staining was combined with enzyme cytochemistry. In dark-field or epipolarization microscopy the labelling appeared as bright granules on a dark background. With its dense granular membrane labelling and its good morphology IGSS is an ideal method for the study of particular cell types in mixed cell suspensions. In addition, it could be a general method for the detection of cell surface antigens in all kinds of cells and tissues.     

Microvasculature in gingivitis and chronic periodontitis: disruption of vascular networks with protracted inflammation.

Gingivitis occurring when bacterial plaque accumulates in the gingival crevice provides a convenient and interesting model for chronic inflammation in humans. In some patients, gingivitis progresses to the destructive lesion of periodontitis, involving the formation of periodontal pockets. The basis for pocket formation and progression is not as yet clear, although neutrophilic polymorphonuclear leukocytes (PMN) appear to play a protective role. Vascular changes appear to either facilitate or inhibit PMN function with the effect of either protecting from, or stimulating, periodontitis. Contrary to most circumstances, high endothelial cells in periodontitis are involved with PMN rather than lymphocyte emigration. Expansion of the microvasculature through increased vascular diameter and tortuosity as well as the development of high endothelial cells appears to protect from periodontitis by increasing the supply of both plasma defense factors and PMN to the tissues. Vascular changes that may oppose this and promote periodontitis are the formation of perivascular hyaline material and accumulation of basement membrane rests. The inadequate tissue turnover that accumulation of these vascular products represents can be argued as a vascular response to a chronic inflammation that has failed to eliminate the irritant. It is suggested that these vascular changes may account for the highly localized and burst-like pattern of pocket formation in periodontitis. Finally, it is possible that the recent observation that periodontitis is an independent risk factor for systemic vascular disease may reflect stimulation of acute phase protein synthesis by cytokines released by periodontal high endothelial cells.     

Nidogens-Extracellular matrix linker molecules

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.     

分散剂对超细硫酸钡粉体制备的影响

采用直接沉淀法,在反应体系中添加不同的分散剂,制得了超细硫酸钡,用TEM表征了硫酸钡粉体的形貌、大小。结果表明：不同分子量匹配的聚乙二醇(PEG)作为分散剂有较好的分散效果,当PEG加入量为4%,PEG200和PEG4000的配比为5：1,反应时间为10min时,制得的粉体分散性较好,粒径分布窄,团聚得到了有效抑制,平均粒径在300nm左右。