Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs--application to the mammalian corneal stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center‐to‐center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Collagen types I, III, and V constitute the thick collagen fibrils of the mouse decidua

A mammal's endometrium is deeply remodeled while receiving and implanting an embryo. In addition to cell proliferation and growth, endometrial remodeling also comprises synthesis and degradation of several molecular components of the extracellular matrix. All of these events are orchestrated by a precise sequence of ovarian hormones and influenced by several types of cytokines. As we have previously reported, an intriguing and rapid increase in collagen fibril diameter occurs in the decidualized areas of the endometrium, surrounding the implantation crypt, whereas collagen fibrils situated far from the embryo remain unchanged. Collagen fibrilogenesis is a complex molecular process coordinated by a number of factors, such as the types and amounts of glycosaminoglycans and proteoglycans associated with collagen molecules. Collagen genetic type, mechanical stress, aging, and other factors not yet identified also contribute to this development. A recent study suggests that thick fibrils from mouse decidua are formed, at least in part, by aggregation of thin fibrils existing in the stroma before the onset of decidualization. In the present ultrastructural study using single and double immunogold localization, we showed that both thin and thick collagen fibrils present in the mouse pregnant endometrium endometrium are heterotypic structures formed at least by type I, type III, and type V collagens. However, type V collagen predominates in the thick collagen fibrils, whereas it is almost absent of the thin collagen fibrils. The putative role of type V homotrimer in the rapid increase of the diameter of collagen fibrils of the mouse decidua is discussed.     

Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy

The orientation of fibrils within biological tissues is of primary importance. In this study, we propose a simple method based on second harmonic generation (SHG) microscopy to map, pixel by pixel, the orientation of the symmetry axis of the second‐order nonlinear susceptibility tensor of fibrils that produce SHG. The method uses only four images acquired at specific polarizations of the input laser beam, and can be easily and cheaply implemented on a confocal microscope. In addition to orientation informations, the method also provides polarization independent images and estimations of the ratio of the nonlinear susceptibility components. We demonstrate the relevance of our concept by studying the orientation fields of the collagen meshwork in a healthy rat liver that provides well separated fibrils. By correlating the mean orientation of the nonlinear susceptibility to the fibril orientation itself for many fibril segments, and using circular statistics, it is shown that both orientations are truly parallel at the fibril scale. Our polarimetric method allows to map fibril orientation fields, independently of individual fibril contrast in the SHG image.     

Structure and morphology of the Alzheimer's amyloid fibril

Amyloid fibrils are deposited in a number of diseases, including Alzheimer's disease, Type 2 diabetes, and the transmissible spongiform encephalopathies (TSE). These insoluble deposits are formed from normally soluble proteins that assemble to form fibrous aggregates that accumulate in the tissues. Electron microscopy has been used as a tool to examine the structure and morphology of these aggregates from ex vivo materials, but predominantly from synthetic amyloid fibrils assembled from proteins or peptides in vitro. Electron microscopy has shown that the fibrils are straight, unbranching, and are of a similar diameter (60-100 A) irrespective of the precursor protein. Image processing has enhanced electron micrographs to show that amyloid fibrils appear to be composed of protofilaments wound around one another. In combination with other techniques, including X-ray fiber diffraction and solid state NMR, electron microscopy has revealed that the internal structure of the amyloid fibril is a ladder of beta-sheet structure arranged in a cross-beta conformation.     

Protein casting: A new replication technique for observing fluid surface structures

Certain insoluble copolymers form characteristic liquid crystalline structures at water/copolymer interfaces. The formation of these structures, discernable with phase contrast microscopy, correlates with biological activities as immunological adjuvants. In order to study such structures at the electron microscopic level, it was necessary to develop a preparation technique which preserved the water/polymer interface. A technique was devised, here called “protein casting,” which uses highly concentrated bovine serum albumin (BSA) in water, subsequently fixed with glutaraldehyde, to form a replica of the water/polymer interface. These casts appeared highly accurate at low magnifications, and resolved to less than 1ønm. This technique seems ideal for accurate replication of surfaces formed by nonmiscible liquids and should prove helpful in studies of other materials in aqueous suspension.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.     

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

Adhesion,unfolding forces,and molecular elasticity of fibronectin coatings: An atomic force microscopy study

Fibronectin is an extracellular matrix protein that is involved in cell adhesion, growth, migration, differentiation, and wound healing. Fibronectin coatings are currently used in many laboratories for biomedical and biotechnology purposes. In this study we have investigated the adhesion and mechanical properties of fibronectin coatings. The coatings were also used to study the role of the residence time and the influence of the loading rate in nonspecific interactions. The results showed that the adhesion force between silica and fibronectin increased with loading rate delivering similar values for residence times of 1 and 2 s. Further analysis indicated that the distance to the transition state was about 0.5 nm. Moreover, the adhesion force did not vary with the loading rate for contact time of 0 s. The unfolding of fibronectin domains also depended of the Dwell time (no unfolding events were observed for zero residence time). Applied loads of 2 nN were able to stretch the fibronectin layer up to 200 nm and to unfold the three fibronectin domains, which were similar for a Dwell time of 1 and 2 s. However, the unfolding length increased with loading rate: below 2.5 µm s^?1 the obtained lengths matched the value of FN I (13.5 nm), while for higher speeds the measured values corresponded to the lengths of FN II (18 nm) and FN III (27 nm). This investigation has answered and opened new questions about the mechanical stability and function of fibronectin coatings. The results have also raised theoretical questions about the difference between specific and nonspecific interactions to be addressed in future work.     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

Sperm ultrastructure of Mytella (Bivalvia) populations from distinct habitats along the northern coast of São Paulo State,Brazil

Ultrastructural analyses of bivalve spermatozoa are relevant in studies that aim to identify taxonomic traits for the purposes of discriminating species and conducting phylogenetic studies. In the present work, spermatozoa of mussel specimens of the genus Mytella, collected from two populations living in distinct habitats, were examined by electron microscopy. The objective was to identify sperm ultrastructural taxonomic traits that could be used to differentiate Mytella species. The specimens were from populations that live in intertidal zones on the southeast coast of Brazil, either buried in muddy-sand sediment or anchored to rocky substrates. The acrosomal vesicle was conical and long, the axial rod extended from the nucleus to the acrosome, the nucleus was an oblate spheroid with a condensed chromatin, the intermediate portion contained mitochondria encircling a pair of centrioles, and there was a single flagellum. The sperm was of a primitive type. The spermatozoon ultrastructure did not distinguish the specimens buried in muddy-sand sediment from those anchored to rocky substrates. The data suggest that the specimens analyzed, despite living in distinct habitats, belong to the same species, which conchological analyses identified as M. charruana. The presence of an axial rod in their sperm cells supports the inclusion of M. charruana in the subfamily Mytilinae.     

Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.     

Effects of substrate texture and curvature on the morphology of cultured cells

The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb-c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading. Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3-c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree. This study demonstrates the necessity of being aware of substrate-induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

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.     

Image analysis of mineralized and non-mineralized type I collagen fibrils

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer-generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non-mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.     

Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described.     

Arrangement and fine structure of collagen fibrils in the decidualized mouse endometrium

The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues.     

Structure and morphology of the in situ composites of a liquid crystalline polymer and polycarbonate

In situ composites were prepared via melt blending of a liquid crystalline polymer (LCP) and polycarbonate using a twin screw extruder. The structure and morphology of these composites were analysed using both transmission electron microscopy (TEM) and scanning electron microscopy. The LCP phases were able to orientate and form in situ submicrometre fibres during the extrusion and post-extrusion drawing. TEM images as well as selected-area diffraction patterns were obtained from the materials. The effects of both composition, i.e. LCP content, and post-extrusion draw-down ratio on the development of the in situ formed LCP fibres were studied in detail. A skin–core morphological differentiation is observed in these materials where well-defined LCP fibres of higher aspect ratios were formed in the skin region. However, a significant amount of unelongated LCP particles were found coexisting with the less well-defined fibres in the core region of the extrudates. This skin–core differentiation was found to be dependent on the composition and the processing conditions, e.g. draw ratio. In this instance, electron microscopy is proven to be a powerful technique not only for direct observation of the formation, dimensions and morphology of the in situ LCP fibres, but also for the qualitative and quantitative characterization of the molecular orientation and crystalline structures in these fibres using selected-area electron diffraction. It is observed that the skin–core differentiation becomes more distinct in the in situ composites containing a higher percentage of LCP but diminishes when the material is processed at higher post-extrusion draw ratio.