Immuno-EM using colloidal metal nanoparticles and electron spectroscopic imaging for co-localization at high spatial resolution

Multiple‐labelling immuno‐EM is a powerful tool for localizing and co‐localizing different antigens simultaneously in cells and tissues at high spatial resolution. Commonly used labels for this purpose are differently sized gold spheres. A comparison of results obtained with differently sized markers is often difficult, because the diameters of markers influence labelling efficiency. In the current study, we investigate a method for high‐resolution multiple‐labelling immuno‐EM, using equally sized colloidal markers made of different metals. Energy filtering transmission electron microscopy is used to differentiate particles based on elemental composition. The labels consist of colloidal gold, palladium and platinum‐core gold‐shell particles of approximately 6 nm in diameter, which are conjugated to different primary antibodies. Applicability of the electron spectroscopic imaging, methodology is demonstrated by labelling of actin, α‐actinin and myosin on ultra‐thin cryosections of skeletal muscle tissue.     

Determination of gold-labelled surface receptors on single cells by X-ray microanalysis

A standardless X-ray microanalytical procedure has been developed to determine the number of gold-labelled surface receptors on whole single cells. The effect of the injection of K₂PtCl₄ into mice on gold-labelled concanavalin A (Con A) receptors on peritoneal macrophages was examined with an energy dispersive X-ray detector in an SEM. The numbers of gold particles seen in electron micrographs and estimated by fluorescence photometric measurements of fluorescein isothiocyanate-labelled Con A receptors were correlated with the X-ray microanalytical results.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

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.     

Post-embedding cytochemistry with gold-labelled reagents: a review

The detection of antigens and glycoconjugates with the protein A–gold and the lectin–gold techniques, respectively, is reviewed. Special attention is directed to the necessary conditions for fixation and embedding as well as to the staining procedures of tissue sections for light and electron microscopy.     

Immunogold labeling of insulin growth factor-I receptors in elderly human skeletal muscle

Age-associated muscle wasting, or sarcopenia, can be delayed or reversed with interventions, including exercise and pharmaceutical agents. Mapping morphometric changes in the skeletal muscle insulin growth factor 1 receptor can provide valuable information regarding mechanisms controlling muscle protein metabolism. Immunocolloidal gold labeling is a powerful immunocytochemistry procedure for detecting antigens at the ultrastructural level, providing plausible biological markers of cell and tissue adaptations to stimuli. The intent here was to employ immunogold labeling to identify, localize, and quantify the insulin growth factor receptor-I (IGF-IR) in elderly human skeletal muscle. Needle biopsy specimens of the leg vastus lateralis muscle were fixed with 1% glutaraldehyde and 4% paraformaldehyde, dehydrated, and embedded in LR white resin. Pilot experiments were carried out to establish optimal dilutions of primary and secondary antibodies and to employ controls to establish staining specificity. The 6 nm gold particles were first evident when viewed at transmission electron microscopy (TEM) magnifications at 54,000x and clearest at 71,000x. Consistencies were noted in the staining patterns, with the majority of particles lying in proximity to the myofilaments. Gold particles were also found randomly along the outer membrane of the sarcolemma and the mitochondrial membranes. National Institutes of Health (NIH) Image 1.55 version software was used to measure receptor density (NIH, Bethesda, Md., USA). It appears that immunogold labeling of postembedded tissue samples is a sensitive method for detecting IGF-I receptors at the ultrastructural level.     

Imaging of immunolabeled membrane receptors in uncoated sem specimens

Epidermal growth factor receptors (EGFR) were labeled with 10 nm immunogold and examined on uncoated specimens of A431 human epidermoid carcinoma cells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (V_o). At V_o=2kV, the SE and BSE signals were too weak to identify all markers, while at V_o=3–7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increasing the V_o to above 7 kV sometimes provokes instability of the specimens. A V_o of ? 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.     

Distribution of GABA and glycine receptors on bipolar and ganglion cells in the mammalian retina

The amino acids GABA and glycine mediate synaptic transmission via specific neurotransmitter receptors. Molecular cloning studies have shown that there is a great diversity of GABA and glycine receptors. In the present article, the distribution of GABA and glycine receptors on identified bipolar and ganglion cell types in the mammalian retina is reviewed. Immunofluorescence obtained with antibodies against GABA and glycine receptors is punctate. Electron microscopy shows that the puncta represent a cluster of receptors at synaptic sites. Bipolar cell types were identified with immunohistochemical markers. Double immunofluorescence with subunit-specific antibodies was used to analyze the distribution of receptor clusters on bipolar axon terminals. The OFF cone bipolar cells seem to be dominated by glycinergic input, whereas the ON cone bipolar and rod bipolar cells are dominated by GABAergic input. Ganglion cells were intracellularly injected with Neurobiotin, visualized with Streptavidin coupled to FITC, and subsequently stained with subunit specific antibodies. The distribution and density of receptor clusters containing the alpha1 subunit of the GABA(A) receptor and the alpha1 subunit of the glycine receptor, respectively, were analyzed on midget and parasol cells in the marmoset (a New World monkey). Both GABA(A) and glycine receptors are distributed uniformly along the dendrites of ON and OFF types of parasol and midget ganglion cells, indicating that functional differences between these subtypes of ganglion cells are not determined by GABA or glycinergic input.     

Method for localization of sialic acid on cell surface and cell interior in peripheral blood: a pilot study

This article presents a method for identification and localization of cell surface and intracellular sialoglycoconjugates of peripheral blood cells. To reveal cell surface conjugates, a sample of peripheral blood was incubated with lectin after centrifugation and rinsing. For intracellular localization in leukocytes, RBCs were lysed and the membranes were permeabilized prior to cytochemical reaction. Fluorescein isothiocyanate conjugated lectins were used for visualization in fluorescence microscope. All lectins bound specifically to the surface of erythrocytes. Confocal microscopy showed surface and intracellular labeling of permeabilized leukocytes. A part of the signal in eosinophils originated from binding of anionic fluorophore to cationic granular proteins.     

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.     

QD as a bifunctional cell-surface marker for both fluorescence and atomic force microscopy

Fluorescent quantum dots (QDs) are a new class of fluorescent label and have been extensively used in cell imaging. Streptavidin-conjugated QDs have a diameter of ca. 10–15 nm; therefore when used as probes to label cell-surface biomolecules, they can provide contrast enhancement under atomic force microscopy (AFM) and allow specific proteins to be distinguished from the background. In addition, the size and fluorescent properties potentially make them as probes in correlative fluorescence microscopy (FM) and AFM. In this study, we tested the feasibility of using QD-streptavidin conjugates as probes to label wheat germ agglutinin (WGA) receptors on the membrane of human red blood cells (RBCs) and simultaneously obtain fluorescence and AFM images. The results show that the distribution of QDs labeled on human RBCs was non-uniform and that the number of labeled QDs on different erythrocytes varied significantly, which perhaps indicates different ages of the erythrocytes. Thus, QDs may be employed as bifunctional cell-surface markers for both FM and AFM to quantitatively investigate the distribution and expression of membrane proteins or receptors on cell surface.     

A novel method of Z-contrast imaging in STEM applied to double-labelling

A novel method of Z-contrast imaging in the scanning transmission electron microscope (STEM) is presented. The technique relies on the element dependence of the angular distribution of the scattered electrons, and is realized with a detector consisting of a set of concentric rings. It is possible to discriminate 9-nm colloidal gold and silver specifically distributed on thin sections. In addition to this practical work, numerical evaluations are used to assess the method. With two smaller markers, this approach will be useful in discriminating closely spaced antigenic sites when steric hindrance occurs with double-labelling using probes of different sizes.     

Direct visualization of colloidal gold-bound molecules and a cell-surface receptor by ultrahigh-resolution scanning electron microscopy

The structure of protein A-coated colloidal gold particles, and of macrophage cell-surface receptors conjugated with immunogold particles, was studied using an ultrahigh-resolution scanning electron microscope. Protein A, when conjugated with 15-nm gold, formed a coat completely surrounding the particle. Particles conjugated with both protein A and immunoglobulin G (IgG) were similar, but with additional protrusions formed by the IgG. IgG molecules directly bound to gold were resolved sometimes as complexes of three units, sometimes as more filamentous, V-shaped structures. On the cell surface of a macrophage reacted with a monoclonal antibody to Mac-1 antigen (the murine C3bi receptor) followed by protein A-gold, gold particles were seen to be linked via the IgG to the receptor, visualized as a round granule.     

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.     

Co-localization of the cellulose framework and cell-wall matrix in helicoidal constructions

Affinity methods (enzymes, lectins and antibodies used as specific probes) were applied in order to target cellulose and the major matrix components in cell walls of dicotyledons built up as helicoids. The probes were the enzyme cellobiohydrolase, CBH1, for cellulose, polyclonal antibodies and the lectin RCA (Ricinus communis agglutinin) for xyloglucans, and monoclonal antibodies JIM5 or JIM7 for homogalac-turonan sequences (according to their degree of esterification). Observations were performed: (i) in muro and/or on heteromolecular fractions following controlled cell-wall dissociation experiments; and (ii) at the light microscope level and/or at the electron miscroscope level by means of various visualization markers. Affinity labelling was complemented by subtractive cytochemistry and by the labelling of available anionic groups by means of cationic gold particles. Data confirm the importance of using a variety of probes, the combination of which allows the acquisition of convergent and complementary results. Concerning the particular case of helicoidal walls of elongating cells, it was shown that cellulose was always co-localized with xyloglucans and homogalacturonan polymers in zones where the cholesteric order was well defined. Cellulose was always associated with compatible hemicellulose polymers capable of binding tightly. Moreover, residual charges were always present along the microfibrils, forming an anionic coat able to repel the adjacent cellulose microfibrils. A possible role of the heteromolecular association of xyloglucan and pectate as a surfactant allowing the cholesteric assembly is hypothesized.     

Quantitative energy-filtered image analysis in cytochemistry. I. Morphometric analysis of contrast-related images

A combination of energy-filtered electron microscopy (EFEM) and an image-analyzing system (IBAS/2000) is used for morphometric analyses of cells and (reaction) products. Image contrast is objectively established and segmentation is based upon intrinsic contrasts, in ultrathin sections. Cross-sectioned platinum-stained erythrocytes are used as a model to determine optimal conditions for constant measuring results for contrast, area and perimeter. Results are related to changes in: (1) the objective-lens diaphragm diameter, (2) three most frequently used contrast modes obtainable by electron spectroscopical imaging (ESI) in a Zeiss EM 902 transmission electron microscope (e.g., global, zero loss (or deltaE - 0 eV) and deltaE = 250 eV), and (3) the number of image integrations (1-250X) acquired by real-time video. A thresholding procedure is proposed for objective segmentation of such contrast-related images and applied to measure the area fraction of nuclear chromatin and the diameter of nominal 1 nm colloidal gold particles.     

Scanning electron microscopy of Streptomyces without use of any chemical fixatives

A new, short, and quick method was developed for preparation of specimen for observing Actinomycetes of genus Streptomyces by scanning electron microscopy. The cultures were directly grown on stubs and coated with a film of gold without using any fixative and dehydrating procedures. Using this simple preparation procedure, surface of intact sporing structures of Streptomyces was observed over a range of magnifications. As the preparation procedure is so simple and rapid, this procedure could be most useful for the routine examination and identification of Streptomyces.     

Protein-A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces

Due to their high atomic number contrast in transmission electron microscopy, gold particles are ideal markers in surface replicas of cultured cells. The suitability of protein-A-coated gold particles in replica immunocytochemistry for labelling surface antigens is demonstrated using measles virus-infected cells as a model system. Labelled areas can easily be distinguished from unlabelled areas, and even markers positioned in the evaporation shadow of large structures can be accurately identified, which is a prerequisite for an exact quantification and mapping of antigen. In addition, the ultrastructure of labelled areas can still be visualized because of the small size of the marker.     

Estimation of gold-labelled macromolecules attached to cell surfaces

A method for obtaining a semi-quantitative estimation of the amount of colloidal gold label attached to a cell surface is described. The X-ray emission, in a scanning electron microscope, from an even metal coating applied by diode sputter coating is used as an internal standard. The emission from the standard is used to correct for errors which would have arisen due to factors such as variable specimen surface topography. Examples of the semiquantitative estimation of 10-nm gold-labelled wheat-germ agglutinin to L929 murine fibroblast cells are given.