Ultrastructure and composition of the cell surfaces of infection structures formed by the fungal plant pathogen Colletotrichum lindemuthianum

A variety of microscopical techniques and molecular probes have been used to study the ultrastructure and composition of the cell surfaces of the conidia (i.e. spores) and infection structures produced by the hemibiotrophic fungal plant pathogen Colletotrichum lindemuthianum. The fungal conidium germinates to produce a germ-tube, the tip of which swells to produce a domed, melanized appressorium which adheres firmly to the plant surface. Penetration of the cuticle and cell wall is followed by the development of a biotrophic intracellular hypha, which is surrounded by an invagination of the host plasma membrane. Freeze-substitution of C. lindemuthianum germlings showed that conidia are coated with a dense layer of fibrillar material. This 'spore coat' contains irregularly shaped pores, giving it a reticular appearance. Negative staining of germlings revealed the presence of numerous long, flexuous fibres or fimbriae, protruding from the surfaces of germ-tubes and appressoria. Colloidal gold was used to visualize fungal extracellular proteins. The colloidal gold stained a fibrillar sheath around germ-tubes, whereas appressoria were surrounded by a halo, comprising an inner unstained region and a stained perimeter. The carbohydrate composition of the cell surfaces of the conidia and infection structures was studied by labelling cells with rhodamine- and fluorescein-conjugated lectins. The results showed that the extracellular matrices of germ-tubes and appressoria are very similar in composition, but differ from those of conidia and intracellular hyphae. Monoclonal antibodies have been prepared to germlings and infection structures of C. lindemuthianum and their use has provided further evidence that the extracellular matrices around germ-tubes and appressoria have several glycoproteins in common. The results also show that the cell surface of C. lindemuthianum becomes specialized during biotrophic development inside host cells.     

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray μCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses.     

Atomic force microscopy analysis of enveloped and non-enveloped viral entry into, and egress from, cultured cells

Since its invention, the atomic force microscope has been used to image a wide variety of biological samples, including viruses. Viral entry into, and egress from, cultured cells has been extensively studied using numerous scientific techniques and to a limited extent using atomic force microscopy. One of the main structural differences that can exist between viruses is the absence, or presence, of an envelope and this factor has consequences for the mode of viral entry and egress. In this study, the entry into, and egress from, cultured cells of enveloped and non-enveloped viruses were investigated using atomic force microscopy. No significant cell surface changes were observed following infection with enveloped or non-enveloped viruses. Although roughness analysis of viral entry revealed cell smoothing post-infection, no differences between the roughness values of enveloped and non-enveloped viral entry were observed. Line analysis of viral entry revealed minor differences between cells infected with an enveloped rather than a non-enveloped virus. These differences may represent a distinction between the uptake processes of enveloped and non-enveloped viruses. Studies of viral egress revealed that infected cells were undergoing cytopathic changes. Whilst topographic, height and roughness differences clearly occurred between virally- and mock-infected cells, no significant differences were elucidated between enveloped and non-enveloped viral egress.     

Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells

Cryofixation by high‐pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighboring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualization of membrane release events. African horse sickness virus (AHSV) is a nonenveloped virus that employs two different release mechanisms from mammalian cells, i.e., lytic release through a disrupted plasma membrane and a nonlytic budding‐type release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualization, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilizing other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titers in tissue culture. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Cytotoxicity assessment of a gold nanoparticle-chitosan nanocomposite as an effi cient support for cell immobilization: comparison with chitosan hydrogel and chitosan-gelatin

Cell-based biosensors have become a research hotspot in the biosensors and bioelectronics fields. The main feature of cell-based biosensors is immobilization of living cells on the surface of transducers. Different types of polymers which are used as scaffolds for cell growth should be biocompatible and should have reactive functional groups for further attachment of biomolecules. In this work, cell attachment and proliferation on chitosan hydrogel, chitosan-gelatin and gold nanoparticle-chitosan nanocomposite membranes was studied. Characterization of the membranes was performed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Cytotoxicity assessment on HEK293 cells was carried out for all membranes using the MTT assay. Cell morphology and viability were assessed to evaluate cell attachment and proliferation. Regarding cell studies, the findings revealed that none of the membranes induced cytotoxic effects. However, the data showed that gold nanoparticle nanocomposite membranes improved HEK293 attachment and adhesion more than other membranes, indicating that it provides an effective surface for immobilizing cells for sensing applications.     

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.     

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV-1 on the surface of HIV-1-infected H9-lymphocytes

Immunofluorescence staining techniques at present, when applied to follow the expression of HIV-1-specific antigens on infected cells, only give the information that the antigens detected are localized in the outer region of the membrane of the infected cell. We therefore set up a procedure using magnetic polystyrol particles coated with antibodies specific for the HIV-1 antigens under study, in combination with scanning electron microscopy. We were able to demonstrate that p24 and p18 structural antigens are clearly expressed on the surface of HIV-1-infected H9 lymphocytes. This means that there was no steric hindrance for structures of cell-like size specific for HIV-1 antigens to interact with their target antigens. Other antigens may be hidden in membrane structures and are therefore inaccessible, for example, to the beads used here, which were of a similar size to antigen-specific cells in vivo. The results of this model system must be seen with respect to the interaction of antigen-specific cell-mediated immunity with full antibody-dependent cellular cytotoxicity, or without cytotoxic T lymphocytes, the mediator function of antibodies.     

Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future. Microsc. Res. Tech. 77:153–160, 2014. © 2013 Wiley Periodicals, Inc.     

Atomic force microscopy imaging of retroviruses: Human immunodeficiency virus and murine leukemia virus

Retroviruses are membrane‐enveloped, RNA‐containing viruses that produce a wide range of threatening diseases in higher animals. Among these are human immunodeficiency virus (HIV), which produces acquired immune deficiency syndrome (AIDS) in humans, and murine leukemia virus (MuLV), which produces leukemias in rodents. We have obtained the first atomic force microscopy (AFM) images of these two retroviruses, both isolated from culture media and emerging from infected cell surfaces. The HIV virions are 127 nm diameter on average, and those of MuLV are 145 nm, although there are wide distributions about the means. The AFM images show the arrangement of the envelope protein, responsible for host cell entry, on the surfaces of both virions. Disruption of the viruses using detergents or physical means allowed us to visualize interior structures, including the outer shells of both MuLVand HIV, the cores of MuLV, and the nucleic acid of HIV complexed with core proteins. Using immunolabeling techniques borrowed from electron microscopy, we were able to demonstrate the binding of gold‐labeled antibodies directed against the envelope protein of MuLV. The AFM images are revealing, not only in terms of surface topology, but in terms of interior features as well, and they reveal the eccentricities and uniqueness of individual virus particles rather than yielding the average member of the population. Further application of AFM to viruses associated with other pathologies may ultimately have a significant impact on the diagnosis and treatment of virus‐promoted diseases.     

Force spectroscopy of hepatocytic extracellular matrix components

We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular “footprints” indicated that the cells secrete large polymers (e.g., 3.5 μm contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.     

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.     

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.     

Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy

Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the “gold standard” for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723–730, 2015. © 2015 Wiley Periodicals, Inc.     

Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16–24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium–formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.     

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.     

Proliferation study and microscopy evaluation on the effects of tannic acid in human fetal osteoblast cell line (hFOB 1.19)

Tannic acid (TA) is a phenolic compound that might act directly on osteoblast metabolism. The study was performed to investigate the effects of TA on the proliferation, mineralization, and morphology of human fetal osteoblast cells (hFOB 1.19). The cells were divided into TA‐treated, untreated, and pamidronate‐treated (control drug) groups. Half maximal effective concentration (EC₅₀) values for TA and pamidronate were measured using MTT assay. The EC₅₀ of hFOB 1.19 cells treated with TA was 2.94 M. This concentration was more effective compared to the pamidronate (15.27 M). Cell proliferation assay was performed to compare cell viability from Day 1 until Day 14. The morphology of hFOB 1.19 was observed via inverted microscope and scanning electron microscope. Calcium (Ca) and phosphate (P) were assessed using energy‐dispersive X‐ray (EDX) analysis. Furthermore, the mineralization of hFOB 1.19 was determined by von Kossa staining (P depositions) and Alizarin Red S staining (Ca depositions). The number of cells treated with TA was significantly higher than the two control groups at Day 10 and Day 14. The morphology of cells treated with TA was uniformly fusiform‐shaped with filopodia extensions. Besides, globular‐like structures of deposited minerals were observed in the TA‐treated group. In line with other findings, EDX spectrum analysis confirmed the presence of Ca and P. The cells treated with TA had significantly higher percentage of both minerals at Day 3 and Day 10 compared to the two control groups. In conclusion, TA enhances cell proliferation and causes cell morphology changes, as well as improved mineralization.     

The use of rhodamine 6G and fluorescence microscopy in the evaluation of phospholipid-based polymeric biomaterials

A technique is described that allows the staining and subsequent visualization of polymers that contain the phosphorylcholine (PC) group. These materials are useful as bulk materials or coatings for the fabrication of medical devices. The staining method employs rhodamine 6G, which can be simply and rapidly applied to the polymer coating and imaged using fluorescence microscopy. The specificity of the staining for the PC polymers makes this technique suitable for the evaluation of a wide range of substrates and provides qualitative information on coating uniformity, coverage and morphology. It can be used to examine the durability of, and defects in, the coating. Statistical analysis of the fluorescent intensity by measuring the pixel value during imaging can allow for the method to be used as a quality control tool.     

Photoelectron microscopy of erythrocyte ghosts; imaging of lectin binding sites with colloidal gold markers

Images of human red cell ghosts have been obtained by photoelectron microscopy (photoemission electron microscopy or PEM) without any staining, metal coating or shadowing. Membrane folding, when it occurs in these collapsed structures, shows up clearly even though the membrane itself is only 5–10 nm in thickness. Using red cell ghosts as a model system, it is shown that colloidal gold can act as a photoemissive marker. Specific lectin binding sites on the cell surface were labeled with a colloidal gold-wheat germ agglutinin complex. The gold particles were readily detectable against the weaker emission from the cell surface.     

Photoelectron microscopy of erythrocyte ghosts: imaging of lectin binding sites with colloidal gold markers

Images of human red cell ghosts have been obtained by photoelectron microscopy (photoemission electron microscopy or PEM) without any staining, metal coating or shadowing. Membrane folding, when it occurs in these collapsed structures, shows up clearly even though the membrane itself is only 5-10 nm in thickness. Using red cell ghosts as a model system, it is shown that colloidal gold can act as a photoemissive marker. Specific lectin binding sites on the cell surface were labeled with a colloidal gold-wheat germ agglutinin complex. The gold particles were readily detectable against the weaker emission from the cell surface.