Electron Microscopy Markers Research Articles

FM Dye Photo-Oxidation as a Tool for Monitoring Membrane Recycling in Inner Hair Cells

Styryl (FM) dyes have been used for more than two decades to investigate exo- and endocytosis in conventional synapses. However, they are difficult to use in the inner hair cells of the auditory pathway (IHCs), as FM dyes appear to penetrate through mechanotransducer channels into the cytosol of IHCs, masking endocytotic uptake. To solve this problem we applied to IHCs the FM dye photo-oxidation technique, which renders the dyes into electron microscopy markers. Photo-oxidation allowed the unambiguous identification of labeled organelles, despite the presence of FM dye in the cytosol. This enabled us to describe the morphologies of several organelles that take up membrane in IHCs, both at rest and during stimulation. At rest, endosome-like organelles were detected in the region of the cuticular plate. Larger tubulo-cisternal organelles dominated the top and nuclear regions. Finally, the basal region, where the IHC active zones are located, contained few labeled organelles. Stimulation increased significantly membrane trafficking in the basal region, inducing the appearance of labeled vesicles and cistern-like organelles. The latter were replaced by small, synaptic-like vesicles during recovery after stimulation. In contrast, no changes in membrane trafficking were induced by stimulation in the cuticular plate region or in the top and nuclear regions. We conclude that synaptic vesicle recycling takes place mostly in the basal region of the IHCs. Other organelles participate in abundant constitutive membrane trafficking throughout the rest of the IHC volume.

FM Dye Photoconversion for Visualizing Synaptic Vesicles by Electron Microscopy: Figure 1.

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. The FM dyes have a unique set of properties that allows them to selectively label recycling vesicles: They reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. FM dyes can also be used as endocytic markers in electron microscopy (EM) through a procedure termed photoconversion (or photooxidation), as described here. Fluorescent dye molecules generate free radicals (reactive oxygen species) when subjected to strong illumination. These short-lived radicals readily oxidize any molecules found in the immediate vicinity of the fluorophore. When photoconversion of FM dyes is performed while the preparation is bathing in diaminobenzidine (DAB), a dark brown precipitate forms after the DAB is oxidized. Thus, illumination turns FM-labeled organelles into dark electron-dense ones. The technique results in a substantial increase in the resolution of FM dye labeling studies (with the obvious caveat that it is restricted to fixed preparations).

Immunoelectron microscopy of human spermatozoa: Immunelektronenmikroskopisch Untersuchung menschlicher Spermien

A number of immunocytochemical methods using various markers for electron microscopy have been developed in recent years. The immunogold technique has been especially effective in histotopochemical studies. The value of this technique for demonstrating sperm antigens results from the high electron density of gold, which makes it easily detectable under the electron microscope. The high resolution of the electron microscope permits precise localization of immunologic reactions in the sperm cell. Light microscopy findings can thus be elucidated. We tested a number of monoclonal antibodies that react with sperm antigens. Of three techniques for preparing the spermatozoa, the pre-embedding method and marking of cryoultra-microtome sections proved best.

Horseradish peroxidase cDNA as a marker for electron microscopy in neurons

A reliable and convenient marker of gene transfer in neurons is lacking for electron microscopy. To facilitate the use of molecular genetic approaches in such studies, we introduce the use of horseradish peroxidase (HRP) cDNA as a marker that identifies transfected neurons for correlated light and electron microscopy (EM). By flanking the cDNA with an endoplasmic reticulum (ER)-retention signal, expressed HRP was localized and retained inside the ER of neurons. The construct was placed under the control of the synapsin promoter resulting in neuron-specific expression of HRP. After transfection of hippocampal neurons in culture and visualization of HRP with diaminobenzidine (DAB) after fixation, both cell bodies and proximal dendrites of HRP-positive neurons were readily identifiable by light microscopy. After processing for EM, the heavily DAB-labeled ER was obvious in both cell bodies and dendrites of transfected neurons. In addition, the labeling of distal dendrites, axons, and synapses was apparent. The structural preservation of transfected neurons was optimal as is typical for glutaraldehyde-fixed tissue. Due to the ER-retention signal, HRP was contained inside the ER and the ultrastructure of all other components was not occluded and was quantitatively accessible. These characteristics make HRP co-transfection a potentially powerful tool for modern electron microscopy in both cell biology and neuroscience.

Immunomagnetic separation reagents as markers in electron microscopy

Antibodies coupled to magnetic particles have been employed for immunomagnetic cell isolation, but their consequent use for electron microscopy (EM) has not been evaluated. We used commercial antibodies coupled to iron-dextran to isolate T cells and monocytes/macrophages by immunomagnetic adsorption from normal human peripheral blood mononuclear cells. Subsequently, we studied the association of electron-dense immunomagnetic reagents with cell membranes. CD14-positive monocytes/macrophages isolated from fixed peripheral blood mononuclear cells retained electron-dense beads on the plasma membrane, while live cells internalized them. Flow cytometry and electron microscopy measurements of the percentage of cells that bound a CD4-specific immunomagnetic reagent in pan-T cell isolates (containing numerous T cell subtypes) were indistinguishable. The immunomagnetic reagent associated with cells could be secondarily labeled by secondary antibody coupled to colloidal gold. This study shows that these reagents used for cell isolation or just labeling, remain associated with their targets at the cell membrane. Immunomagnetic reagents allow “capturing” of rare cells from complex mixtures, purifying and concentrating them in a single step for subsequent electron microscopy. The large number of commercially available immunomagnetic reagents specific for different human, mouse and rat antigens provides additional resources for visualization of cellular ultrastructure.

Colloidal Gold and Its Application in Cell Biology

Publisher Summary This chapter discusses the scope and variety of applications of the colloidal gold method. The advantages of colloidal gold as a marker in electron microscopy (EM) immunocytochemistry include the particulate nature of colloidal gold, which allows a fine localization of marked sites, and the size range, which guarantees high flexibility in lateral resolution. Gold particles are negatively charged and can be complexed by noncovalent electrostatic adsorption with various macromolecules forming stable and bioactive gold-ligand complexes termed “gold probes,” and the characteristic X-ray signals emitted by gold could be used to image and quantify cell-bound gold markers by the application of X-ray microanalytical techniques and appropriate computer programs. The introduction of ultrasmall gold particles with increased penetration capabilities has enlarged the potential of colloidal gold reagents, particularly in the field of light microscopy. The addition of polymers is a well-established method for controlling the stability of colloidal dispersions. Nonionic macromolecules—such as polyethylene glycol and polysaccharides—as well as charged macromolecules can be adsorbed onto a metal surface. The adsorption of macromolecules onto colloidal gold depends on a number of factors, such as (1) the stability of the colloid itself, (2) the concentration, shape, configuration, and isoelectric point of macromolecules, and (3) the ionic strength, pH, and temperature of a suspending medium.

Problems in the production and use of 5 nm avidin-gold colloids.

Over the past 5 years we have encountered several problems in the production and use of 5 nm avidin-gold colloids for markers in electron microscopy. These problems include flocculation of colloids during reduction of chloroauric acid, insoluble gold pellets following ultracentrifugation, and non-specific binding of avidin-gold colloids to biological membranes. We are able to avoid these problems by: avoiding the use of crystalline chloroauric acid; succinoylating egg white avidin prior to adsorption on the gold sols; resuspending the pellets following ultracentrifugation in 5 mM phosphate buffer, pH 7.5; and using the avidin-gold colloids within 4 weeks of production.

Histofluorescence and ultrastructural observations of small intensely fluorescent (SIF) cells in the superior sympathetic ganglion of the guinea pig.

Amine-containing small intensely fluorescent (SIF) cells are ubiquitous in vertebrate sympathetic ganglia and, in some species, SIF cells have been identified as interneurons. The hypothesis proposed in this study is that SIF cells in superior sympathetic ganglia of the guinea pig function as interneurons, with efferent connections characteristic for the species. Fluorescence (catecholamine) microscopy and 5-hydroxydopamine marker for electron microscopy were used to study SIF cells, their processes and connections in this ganglion. Brightly fluorescent fibers were seen attached to virtually all SIF cells, and were of two types. The first type, single or arranged in cords, interconnected elements of the SIF-cell system; these apparent linkages joined individual SIF cells as well as adjacent clusters. The electron-microscopic evidence for synaptic contacts between SIF cells warrants the claim that integrated action is a presumed function of these elements. The second type of SIF-cell process was generally of greater length. These individual, branching fibers made presumed connections with dendrites of most principal ganglionic neurons. This arrangement suggested by histofluorescence preparations was confirmed by electron microscopy to involve synaptic connections, and the postsynaptic element was shown to be continuous with the perikaryon of the principal ganglionic neuron. Ultrastructural evidence that collections of dense-cored vesicles occur within processes of both principal ganglionic neurons and SIF cells, in proximity to unsheathed portions of plasma membrane, leads to the conclusion that interstitial diffusion of catecholamine from both may occur; the finding of SIF cell processes adjacent to fenestrated blood vessels suggests that catecholamine may also be transported through capillaries.

Immunocytochemistry Markers in Electron Microscopy: Peroxidase and Colloidal Gold

Immunocytochemistry Markers in Electron Microscopy: Peroxidase and Colloidal Gold

Ultrastructural immunogold labelling of human Langerhans cells enriched epidermal cell suspension.

Colloidal gold particles are well suited as markers in electron microscopy. Indirect immunogold staining was used to identify cell membrane antigens defined by monoclonal antibodies OKT6 and BL6 on human Langerhans cells (LC) in suspensions. Isolated epidermal cells were obtained by skin trypsinization and enriched or depleted in OKT6 positive on BL6 positive LC using the panning method: incubation of OKT6 or BL6 preincubated cells on immunoglobulin coated dishes. Indirect immunogold staining was then performed after prefixation in 2% paraformaldehyde. In LC enriched suspensions, only LC exhibited a specific membrane labelling with OKT6 or BL6 recognized by the presence of small evently distributed gold granules. Neither Birbeck granules, nor other cytoplasmic organelles, were labelled. No other epidermal cells were found positive. In LC depleted suspensions, no labelling was observed. Immunogold labelling on LC enriched suspensions after panning is now in progress for the qualitative evaluation and the quantitative analysis of cell surface constituents and antigens expressed by human dendritic epidermal cells.

Colloidal gold as a cytochemical marker in electron microscopy

Colloidal gold is orange to red, displays electron-opaque properties and is capable of strong emission of secondary electrons. As gold particles can be produced in different sizes and can be labelled with macromolecules which keep their specific properties, gold markers have found uses in light and fluorescent microscopy, and many applications in scanning and transmission electron microscopy.

Distribution of intraventricular horseradish peroxidase in normal and hydrocephalic cat brains.

The distribution of intraventricularly injected horseradish peroxidase (HRP) was examined in the ependymal and subependymal tissue of both normal and experimental hydrocephalic cats by light and electron microscopy. At different time intervals after the intracisternal injection of kaolin, the hydrocephalic ventricles were perfused at various intraventricular pressures with mock cerebrospinal fluid (CSF) containing HRP. The uptake and localization of HRP by periventricular brain tissue was studied with respect to transventricular absorption of cerebrospinal fluid. The results of both light and electron microscopic studies have shown that in normal, acute or chronic hydrocephalic cats perfused at either high or low pressure the distribution of HRP is similar. The enzyme was localized mainly to the intercellular spaces, between ependymal cells, glial cells, and in perivascular spaces (in and across the basement membrane of subependymal blood vessels). HRP did not penetrate endothelial tight junctions even in chronic hydrocephalic cats undergoing significant rates of transventricular absorption of CSF. It is concluded that since the distribution of HRP in brain is determined by diffusion, its use as a tracer for the bulk movement of CSF through nervous tissue is limited. It is, however, an excellent extracellular space marker in electron microscopy.