Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Novel techniques of preparing TEM samples for characterization of irradiation damage

Focus ion beam preparation of transmission electron microscopy (TEM) samples has become increasingly popular due to the relative ease of extraction of TEM foils from specific locations within a larger sample. However the sputtering damage induced by Ga ion bombardment in focus ion beam means that traditional electropolishing may be a preferable method. First, we describe a special electropolishing method for the preparation of irregular TEM samples from ex‐service nuclear reactor components, spring‐shaped spacers. This method has also been used to prepare samples from a nonirradiated component for a TEM in situ heavy ion irradiation study. Because the specimen size is small (0.7 × 0.7 × 3 mm), a sandwich installation is adopted to obtain high quality polishing. Second, we describe some modifications to a conventional TEM cross‐section sample preparation method that employs Ni electroplating. There are limitations to this method when preparing cross‐section samples from either (1) metals which are difficult to activate for electroplating, or (2) a heavy ion irradiated foil with a very shallow damage layer close to the surface, which may be affected by the electroplating process. As a consequence, a novel technique for preparing cross‐section samples was developed and is described.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

Influence of nanostructure composition on its morphometric characterization by different techniques

Morphometric characterization of nanoparticles is crucial to determine their biological effects and to obtain a formulation pattern. Determining the best technique requires knowledge of the particles being analyzed, the intended application of the particles, and the limitations of the techniques being considered. The aim of this article was to present transmission (TEM) and scanning (SEM) electron microscopy protocols for the analysis of two different nanostructures, namely polymeric nanoemulsion and poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles, and to compare these results with conventional dynamic light scattering (DLS) measurements. The mean hydrodynamic diameter, the polydispersity index, and zeta potential of the nanostructures of polymeric nanoemulsion were 370.5 ± 0.8 nm, 0.133 ± 0.01, and ?36.1 ± 0.15 mV, respectively, and for PLGA nanoparticles were 246.79 ± 5.03 nm, 0.096 ± 0.025, and ?4.94 ± 0.86 mV, respectively. TEM analysis of polymeric nanoemulsion revealed a mean diameter of 374 ± 117 nm. SEM analysis showed a mean diameter of 368 ± 69 nm prior to gold coating and 448 ± 70 nm after gold coating. PLGA nanoparticles had a diameter of 131 ± 41.18 nm in TEM and 193 ± 101 nm in SEM. Morphologically, in TEM analysis, the polymeric nanoemulsions were spherical, with variable electron density, very few showing an electron‐dense core and others an electron‐dense surface. PLGA nanoparticles were round, with an electron‐lucent core and electron‐dense surface. In SEM, polymeric nanoemulsions were also spherical with a rough surface, and PLGA nanoparticles were round with a smooth surface. The results show that the “gold standards” for morphometric characterization of polymeric nanoemulsion and PLGA nanoparticles were, respectively, SEM without gold coating and TEM with negative staining. Microsc. Res. Tech. 77:691–696, 2014. © 2014 Wiley Periodicals, Inc.     

Low-voltage transmission electron microscopy reveals SV40 viral particles within secretory granules in pancreatic cells

Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast. Results indicate that following infection of the cell entry and trafficking of the viral particles are present in unique cellular compartments such as ER, dense bodies, and secretory granules.     

Effect of indirubin‐3‐monoxime against lung cancer as evaluated by histological and transmission electron microscopic studies

The aim of this study is to evaluate the antitumor effect of indirubin‐3‐monoxime and its mode of action in benzo(α)pyrene [B(α)P] induced lung cancer in A/J mice. Light microscopic examination of lung sections of [B(α)P] induced lung cancer mice revealed the presence of adenocarcinoma characterized by extensive proliferation of alveolar epithelium and loss of alveolar spaces. The control lung tissue showed a normal architecture with clear alveolar spaces. Interestingly the indirubin‐3‐monoxime treated groups showed the reduced adenocarcinoma with appearance of alveolar spaces. Transmission Electron Microscopic (TEM) studies of lung sections of [B(α)P] induced lung cancer mice showed the presence of phaemorphic cells with dense granules and increased mitochondria. The lung sections of mice treated with indirubin‐3‐monoxime showed the presence of shrunken, fragmented, and condensed nuclei implying apoptosis. The effects were dose dependent and prominent in 10 mg/kg/5 d/week groups suggesting the therapeutic role of indirubin analogue against this deadly human malignancy. Here, our results indicate that indirubin‐3‐monoxime brings antitumor effect against [B(α)P] induced lung cancer by its apoptotic action in A/J mice. Microsc. Res. Tech. 73:1053–1058, 2010. © 2010 Wiley‐Liss, Inc.     

“Microscopic evidences of heavy metals distribution and anatomic alterations in breaching‐leaves of Cupressus lindleyi growing around mining wastes”

In this article a study of the distribution of heavy metals in Cupressus lindleyi breaching‐leaves was done in Taxco, Guerrero. At the same, heavy metals micro‐localization was conducted in the breaching‐leaves to understand the structural changes provoked by mining waste on plants. The most abundant contaminants in soils, tailings and different plant organs (roots, stems, and leaves) were Zn, Mn, and Pb. Nevertheless, As was more accumulated in the stem and breaching‐leaves. The translocation factor and the bio‐concentration factor were less than 1. The structural changes observed were the great accumulation of starch grains and phenolic compounds in the palisade parenchyma, changes in the hypodermis cell wall and necrotic zones in the palisade parenchyma. The distribution of heavy metals in breaching‐leaves tissues was homogeneous in most of the elements. These results showed that C. lindleyi is a species that can be employed in phytostabilization of contaminated zones with mining waste because it is a native plant that does not require a lot of conditions for its development. Microsc. Res. Tech. 77:714–726, 2014. © 2014 Wiley Periodicals, Inc.     

Molecular ultrastructure of the urothelial surface: Insights from a combination of various microscopic techniques

The urothelium forms the blood–urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno‐TEM), and freeze‐fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno‐FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 77:896–901, 2014. © 2014 Wiley Periodicals, Inc.     

Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

Direct observation of liquid crystals using cryo‐TEM: Specimen preparation and low‐dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo‐sectioning approaches, as a comprehensive toolset enabling high‐resolution direct cryo‐TEM observation of a broad range of LCs. We also present comparative analysis using cryo‐TEM and replica freeze‐fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high‐pressure freezing and cryo‐sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo‐TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent‐core thermotropic LCs, comprehensive understanding of the twist‐bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. © 2014 Wiley Periodicals, Inc.     

Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.     

Advantages of embedment‐free section transmission electron microscopy

The usefulness of embedment‐free section transmission electron microscopy (TEM) is stressed for present and future morphological analyses, and several examples are demonstrated which are revealed in sections for the first time by this method: en‐face views of slit diaphragm of renal glomerulus and fenestrated diaphragm of capillary endothelium, transparency of neural myelin, attenuated endothelium and some basement laminae, labyrinth architecture of vacuoles within lipid droplets, and enhanced 3D effect of ultrastructures, the latter of which is the case in electron tomography. In addition, the biological significance of structured appearance (microtrabecular lattices) of the cytoplasmic matrix, which is disclosed by this method, are briefly reviewed in relation to the sol–gel transition of cytoplasmic heterogenous proteins. Since the ultrastructures of various cells and tissues in this method are confirmed to be well correspondent to those in conventional epoxy section TEM except for isotropic dimensional changes, and because there is no necessity for any special expensive equipments other than those for the conventional TEM, the embedment‐free section TEM method with these advantages, deserves much more wide application to the morphological research including electron tomography. Microsc. Res. Tech. 76:1257–1265, 2013. © 2013 Wiley Periodicals, Inc.     

Morphostructural and immunohistochemical study on the role of metallothionein in the detoxification of heavy metals in Apis mellifera L., 1758

Honey is a valuable food produced by bees from sugary substances that they gather in nature. The transformation the nectar into honey, by bees, is long and complex. Except for honey, where heavy metals are absent or are found only in traces, the bees and their products have always been considered excellent biomarkers of such contaminants. We have assumed that the absence of heavy metals in honey is due to the presence of a detoxification system in the digestive system of bees, which involves metallothioneins, proteins that have a role in the homeostatic control of essential and non‐essential metals. We have placed the beehives in three different zones: industrial, urban and rural. Investigations were carried out with ICP‐MS method for the detection of heavy metals in the guts of honey bees and honey. The metallothioneins have been identified by Immunohistochemical and Western‐blotting analisys. The investigations have shown the presence of heavy metals only in bees guts but not in honey, while the presence of metallothionein has been highlighted only in epithelium of the honey sac, demonstrating the existence of an efficient system of detoxification of heavy metals.     

Electron microscopic visualization of complementary labeled DNA with platinum‐containing guanine derivative

The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast‐enhanced heavy element. The stretched single‐chain DNA was obtained by modifying double‐stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single‐stranded DNA pieces on the support film with platinum containing guanine derivative to form base‐specific hydrogen bond; and (iii) producing a magnified image of the base‐specific labeled DNA. Stretched single‐stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum‐containing guanine derivative serves as a high‐dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom‐by‐atom analysis and it is promising way toward future DNA‐sequencing or molecular diagnostics of nucleic acids by electron microscopic observation. Microsc. Res. Tech. 79:280–284, 2016. © 2016 Wiley Periodicals, Inc.     

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde‐based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol‐ or zinc salts‐based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation‐ and temperature‐sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

A novel scale‐down cell culture and imaging design for the mechanistic insight of cell colonisation within porous substrate

At the core of translational challenges in tissue engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale‐down strategy to deconstruct complex biomatrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonisation within porous substrate in this proof‐of‐concept study, TEM specimen supporters (10–30 μm thick) with fine controlled open pores (100～600 μm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Noninvasive imaging of human dermal fibroblasts cultured on these free‐standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high‐quality micrographs of cell–cell and cell–scaffold interactions at microscale, depicted cytoskeletal structures in stretched and relaxed areas at nanoscale. Thus this novel scaled‐down design was able to improve our mechanistic understanding of tissue formation not only at single‐ and multiple‐cell levels, but also at micro‐ and nanoscales, which could be difficult to obtain using other methods.     

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.     

Electron spectroscopic imaging (ESI) of viruses using thin-section and immunolabelling preparations

Electron spectroscopic imaging (ESI) and conventional bright-field transmission electron microscopy (TEM) were applied comparatively for the analysis of the fine structure and the antigenic make-up of human immunodeficiency virus and two herpes viruses. In addition to the information obtained in conventional bright-field TEM, ESI leads to high-contrast imaging of ultrathin sections and improves the resolution of thin and thick sections, and allows a better detectability of the immunolabelling markers.     

Comparative structural morphometry and elemental composition of three marine sponges from Western Coast of India

Three marine sponges Halichondria glabrata, Cliono lobata, and Spirastrella pachyspira from the western coastal region of India were compared for their morphometry, biochemical, and elemental composition. One‐way analysis of variance was applied for spicule morphometry results. Length, width, and length:width ratio were calculated independently. The ratio of length:width varied from 35 to 42 among the grown samples, which remained in the range of 10–22 in young sample at the beginning of studies. However, no significant change was observed in spicule width compared to length. Elemental compositions of marine sponges were determined by field emission gun‐scanning electron microscope. Scanning electron microscopy data revealed that the spicules of all the three sponges were mostly composed of O (47–56%) and Si (30–40%), whereas Al (14.33%) was only detected in the spicules of C. lobata. Apart from these, K, Ni, Ca, Fe, Mg, Na, and S were additionally detected in all the three samples. Presence of heavy metals in the sponges was analyzed by inductively coupled plasma‐atomic emission spectroscopy. Results showed that iron was present in a large amount in samples, followed by zinc, lead, and copper. Microsc. Res. Tech. 77:296–304, 2014. © 2014 Wiley Periodicals, Inc.