Blue molybdenum oxides: A stain for light and electron microscopy

Blue molybdenum oxides (molybdenum blues) have been prepared from aqueous phosphomolybdic acid solutions and applied to thin and semi-thin sections of glutaraldehyde-fixed, epoxy-embedded tissues. A light blue colour and high electron opacity were found in mast cell granules, the secretion content of goblet cells, and cytoplasmic granules in Drosophila salivary glands. The possibility that binding of blue molybdenum oxides to polyhydroxylic components accounts for the staining and contrasting reactions in some cell structures is briefly discussed.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM)

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland‐related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d ‐glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2‐ to 3‐µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in‐solution ASEM for histology and to study vesicle secretion systems. Further, the high‐throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine‐related diseases.     

Antennal multiporous sensilla: their gustatory features for host recognition in female parasitic wasps (insecta, Hymenoptera: Platygastroidea).

In parasitic wasps, various kinds of antennal plates have been interpreted as olfactory organs due to the presence of numerous pores. However, on the basis of ultrastructural investigations, some of these multiporous plates were revealed as being release sites of exocrine glands while others were postulated to have a gustatory function. Such sensilla, present only on female antennae, show unique features, found exclusively in Platygastroidea, with some differences between Scelionidae and Platygastridae. The cuticular apparatus consists of a short basiconic shaft with an apical multiporous area, the pores of which are covered by movable structures. The cellular components are made up of a remarkable number of sensory neurons, from 200 to 220, and an undefined number of sheath cells. These multiporous sensilla have tubular accessory glands that release their secretion through the socket sensillum only in scelionids. These morphological characters, combined with behavioral observations, strongly suggest a gustatory function, although electrophysiological studies are still needed to confirm this. A new terminology for antennal multiporous plates is discussed in relation to their different functions.     

Secretory profile of metapleural gland cells of the leaf-cutting ant Acromyrmex coronatus (Formicidae: Attini)

Ants present a pair of metapleural glands located at the posterolateral end of the thorax. Because of its importance in the social organization of ants, the present study was aimed at describing the morphophysiology of this gland in three worker castes of Acromyrmex coronatus, focused on secretory activity using histological and histochemical techniques. Our findings revealed that the secretory and the storage portions of this gland are connected by extracytoplasmic portion of canaliculi that drain the secretion from each secretory cell to the collecting chamber. This secretion contains glycoproteins. In minor workers, the secretion contains higher levels of polysaccharides when compared to that of major workers, supporting the role of the metapleural gland in the maintenance of the fungus garden. The nucleus as well as cytoplasm of secretory cells were strongly positive for RNA indicating that these cells are active in the synthesis of proteins and lipids, compounds found in the final secretion. The variant of the CEC revealed that the secretory activity of the entire gland is synchronous, as all cells exhibit the result.     

Intramandibular glands in different castes of leaf‐cutting Ant,Atta laevigata (Fr. Smith, 1858) (Formicidae: Attini)

Intramandibular glands have been poorly studied in polymorphic ants, where the differences between castes were unsufficiently scrutinized. Leaf‐cutting ants possess one of the most complex systems of communication and labor division, which is polymorphic well as age polyethism, and makes them an ideal model for the study of intramandibular glands. This study has investigated the occurrence of intramandibular glands in female castes and subcastes of Atta laevigata. The mandibles of the queen, medium, and minor workers, and soldiers were submitted to histological, histochemical, ultrastructural, and morphometric analyses. The class‐3 gland cells and the epidermal gland with a reservoir were found in all the castes. The queens and soldiers showed a higher number of class‐3 gland cells, distributed within the mandible as well as a greater gland size in comparison to the workers. The histochemical tests, periodic acid‐Schiff (PAS), mercury‐bromophenol, and Nile blue, were similar for the class‐3 gland cells and epidermal glands with a reservoir. However, the tests evidenced differences between the castes, with carbohydrates strongly positive in all of them, whereas neutral lipids were found in the queen and soldiers. The protein was weakly positive in the queen, whereas in the soldier, medium, and minor workers these reactions were strongly positive in the intramandibular glands. Our findings in A. laevigata suggest that intramandibular glands are directly involved in labor division and consequently in chemical communication between the castes. Microsc. Res. Tech. 78:603–612, 2015. © 2015 Wiley Periodicals, Inc.     

Light and transmission electron microscopic structure of skin glands and dermal scales of a caecilian amphibian Gegeneophis ramaswamii,with a note on antimicrobial property of skin gland secretion

Amphibian skin secretions contain a variety of bioactive compounds that are involved in diverse roles such as communication, homeostasis, defence against predators, pathogens, and so on. Especially, the caecilian amphibians possess numerous cutaneous glands that produce the secretory material, which facilitate survival in their harsh subterranean environment. Inspite of the fact that India has a fairly abundant distribution of caecilian amphibians, there has hardly been any study on their skin and its secretion. Herein, we describe, using light microscopy and electron microscopy, two types of dermal glands, mucous and granular, in Gegeneophis ramaswamii. The mucous glands are filled with mucous materials. The mucous‐producing cells are located near the periphery. The granular glands are surrounded by myoepithelial cells. A large number of granules of different sizes are present in the lumen of the granular gland. The granule‐producing cells are present near the myoepithelial lining of the gland. There are small flat disk‐like dermal scales in pockets in the transverse ridges of the posterior region of the body. Each pocket contains 1–4 scales of various sizes. Scanning electron microscopic (SEM) study of the skin surface showed numerous funnel‐shaped glandular openings. The antibacterial activity of the skin secretions was revealed in the test against Escherichia coli, Klebsiella pneumoniae, and Aeromonas hydrophila, all gram‐negative bacteria. SEM analyses confirm the membrane damage in bacterial cells on exposure to skin secretions of G. ramaswamii.     

Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.     

Light scattering microscopy with angular resolution and its possible application to apoptosis

An inverted microscope has been modified for light scattering experiments with high angular resolution in combination with transmission, wide‐field fluorescence or laser scanning microscopy. Supported by simulations of Mie scattering, this method permits detection of morphological changes of 3T3 fibroblasts on apoptosis and formation of spherically shaped cells of about 20 μm diameter, in agreement with visual observation. Smaller sub‐structures (e.g. cell nuclei) as well as cell clusters may possibly contribute to the scattering behaviour. Results of 2‐dimensional cell cultures are confirmed by 3‐dimensional multicellular spheroids of 3T3 fibroblasts and HeLa 2E8 cervix carcinoma cells, where in most cases no morphological changes are discernable. This offers some advantage of light scattering microscopy for label‐free detection of apoptosis and may represent a first step towards label‐free in vivo diagnostics.     

Three‐dimensional morphometric comparison of normal and apoptotic endothelial cells based on laser scanning confocal microscopy observation

Three‐dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H₂O₂ to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H₂O₂ can induce apoptosis effectively by regulating the activity of apoptosis‐related biomolecules, including pro‐apoptotic factors p53 and Bax, and anti‐apoptotic factor Bcl‐2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus‐to‐cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction. Microsc. Res. Tech. 76:1154–1162, 2013. © 2013 Wiley Periodicals, Inc.     

Direct attachment of cell suspensions to high-pressure freezing specimen planchettes

We describe a procedure for high‐pressure freezing (HPF) of cultured cells using the HPF aluminium planchettes as a substrate. Cells are either grown directly on planchettes covered with Matrigel or allowed to attach to poly‐l ‐lysine‐coated planchettes. This method allows for rapid transfer of the cells into the HPF and minimizes physical and physiological trauma to the cells. Furthermore, the yield of well‐frozen cells approaches 100% for every cell type we have tried so far. In this report, we show well‐preserved ultrastructure in mitotic and interphase HeLa cells, isolated gastric parietal cells and isolated gastric glands. Immunogold labelling of H⁺/K⁺‐ATPase is shown in parietal cells of isolated gastric glands embedded in LR White resin. The aluminium planchettes appear to have little effect on cell physiology, as demonstrated by the fact that parietal cells cultured for 24–28 h on the planchettes retain their responsiveness to stimulation with histamine.     

The distribution of T lymphocytes,B lymphocytes,and dendritic‐like cells of the anal tonsil in the laboratory shrew,Suncus murinus

We investigated the distribution of T lymphocytes, B lymphocytes, and S‐100 protein‐immunoreactive dendritic‐like in the anal tonsil of the laboratory shrew, Suncus murinus. In adult animals, T lymphocytes were located mainly at the periphery of the anal tonsil, especially around small blood vessels. B lymphocytes were located in the central and subepithelial region of the anal tonsil, which includes primary lymphoid follicles, and in which there are small numbers of scattered T lymphocytes. B and T lymphocytes were distributed over 72.7 and 27.3% of the tonsillar area, respectively. However, their areas of distribution were not clearly distinguished. The areas containing B lymphocytes were enriched in S‐100 protein antibody‐immunoreactive cells, which exhibited a dendritic shape. These S‐100‐positive cells appeared to be identical to the follicular dendritic cells (FDC) seen in the follicles of lymphoid organs. These results suggest that the anal tonsils constitute one of the gut‐associated lymphoid tissues (GALT), and that a function of the anal tonsil includes the capture of intruding antigens that would generate protective antibody responses. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Embossed‐carving processing of cytoskeletons of cultured cells by using focused ion beam technology

The focused ion beam (FIB) technology has drawn considerable attention in diverse research fields. FIB can be used to mill samples at the nanometer scale by using an ion beam derived from electrically charged liquid gallium (Ga). This powerful technology with accuracy at the nanometer scale is now being applied to life science research. In this study, we show the potential of FIB as a new tool to investigate the internal structures of cells. We sputtered Ga⁺ onto the surface or the cross section of animal cells to emboss the internal structures of the cell. Ga⁺ sputtering can erode the cell surface or the cross section and thus emboss the cytoskeletons quasi‐3 dimensionally. We also identified the embossed structures by comparing them with fluorescent images obtained via confocal laser microscopy because the secondary ion micrographs did not directly provide qualitative information directly. Furthermore, we considered artifacts during the FIB cross sectioning of cells and propose a way to prevent undesirable artifacts. We demonstrate the usefulness of FIB to observe the internal structures of cells. Microsc. Res. Tech. 76:290–295, 2013. © 2013 Wiley Periodicals, Inc.     

Morphology and histology of P. argentinus (Crustacea, Decapoda, Caridea) digestive tract.

This work describes the morphology and histology of the P. argentinus digestive tract. The foregut comprises the mouth, oesophagus, and stomach and is lined by a simple cylindrical epithelium overlain by cuticle. There are tegumental glands in the oral region and in the first portion of the oesophagus and of the hindgut. The cardiac stomach is an oval dorsal sac in the cephalothorax and has no calcified structures. The pyloric stomach comprises an upper chamber and a lower gland filter. The filter consists of an outer row of elongated setae and an inner row of dorsally curved setae forming longitudinal channels 16-18 microm wide. The midgut runs from the dorsal chamber of the pyloric stomach to the sixth abdominal somite without caeca. The hindgut runs from the sixth abdominal somite to the ventral anus. The mid-gut epithelium comprises dominant cylindrical cells and small undifferentiated cells in the first portion. The hindgut wall presents longitudinal folds, conspicuous muscular bundles, and a folded cuticle. The digestive tract of P. argentinus is basically similar to that of most of decapods. The absence of calcified structures in the stomach and the width of the longitudinal channels in the filter are related to the predominantly detritivorous diet.     

Ultrastructure of the external sensory apparatus of Gyrodactylus gasterostei Gläser, 1974

The aim of this study, therefore, was to investigate the ultrastructure of gyrodactylid sensilla and to ascertain how these may be employed in the colonisation of new hosts using the Gyrodactylus gasterostei Gläser, 1974—Gasterosteus aculeatus L. model. As Gyrodactylus has no specific transmission stage in its life‐cycle, movement between hosts must be achieved by strategies employed by the adult. This study suggests that certain sensilla, presented for the first time in Gyrodactylus gasterostei, may serve as chemoreceptors and mechanoreceptors and possible photoreceptors. The results of this study provide photographic data using scanning electron microscope (SEM) and transmission electron microscopy (TEM) concerning the sensory structures that are found on the tegument and in the sub‐tegumental zone of G. gasterostei that infect 3‐spine sticklebacks (Gasterosteus aculeatus L.). For this reason, it is vital to understand the factors underlying transmission to a new host, and a detailed, ultrastructural examination of the sensory structures that are used may improve current understanding of the receptors that Gyrodactylus species employ to interpret both their host and ambient environments. Such information may assist in the interpretation of transmission behaviors, particularly their responses to chemical or physical cues which gyrodactylids employ in host location during the transmission process. Microsc. Res. Tech. 77:740–747, 2014. © 2014 Wiley Periodicals, Inc.     

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 m² cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.     

Oogenesis and programmed cell death of nurse cells in the endoparasitoid,Pteromalus puparum

In this study, we describe the features of oogenesis in the endoparasitoid, Pteromalus puparum, as well as the distinct type of programmed cell death of the nurse cells through conventional light and fluorescent markers for apoptosis and immunofluorescent analysis. Oogenesis in this endoparasitoid is divided into five stages, of which stages 1–2, 3–4, and 5 are corresponding to previtellogenic growth, yolk uptake, and the formation of egg envelopes, respectively. From these studies, we demonstrate two critical events, which are vitellogenin absorption and rapid transfer of nurse cell content, resulting in remarkable increase in the volume of oocytes during oogenesis in this endoparasitoid. Vitellogenin absorption initiates in the oocyte of early stage 3, and bulk transfer of nurse cell content into the oocyte occurs at stage 4 of oogenesis in P. puparum, which is mainly characterized with the programmed cell death in the nurse cell complex. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Morphology and histochemistry of the intramandibular glands in Attini and Ponerini (Hymenoptera, Formicidae) species

An understanding of the internal morphology of the ant mandible is important in explaining the relationship between the exocrine system and the behavioral and phylogenetic characteristics of different subfamilies of Formicidae. In this study, we investigated the occurrence of intramandibular glands in ants from the Ponerini (Ponerinae) and Attini (Myrmicinae). These ants possess glands from classes I and III, and secretory epithelial cells with a reservoir. The intramandibular glands show a distinct histology in the studied species, varying in their location, degree of development, and chemical content. Using this information, it is possible to hypothesize that the glands from different tribes produce different substances, which may indicate a variety of functions, depending on the chemical nature of the cellular constituents. A cladistic analysis using the characters of the intramandibular glands separated both tribes, suggesting that structural differences in the intramandibular glands may contribute to future phylogenetic studies of the Formicidae.