The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study

Summary The arrangement of cortical microtubules in tobacco protoplasts is described using the following techniques: 1. Transmission electron microscopy (TEM) of thin sections of whole protoplasts, 2. TEM of negatively stained protoplast ghosts, and 3. Indirect immunofluorescence microscopy of protoplast ghosts. Ghosts were prepared by attaching freshly isolated protoplasts to glass coverslips or formvar/carbon-coated grids with poly-L-lysine and then bursting them either osmotically or by detergent treatment in the presence of a microtubule stabilizing buffer. Osmotic bursting of protoplasts yielded large pieces of plasma membrane with attached microtubules. These preparations proved very useful for measuring the density and length of cortical microtubules. Detergent treatment dissolved the plasma membrane and altered the distribution of cortical microtubules.     

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0 degree C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37 degrees C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.3 approximately 3 micron thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 200 approximately 1,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0 degree C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37 degrees C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stereomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.     

A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.     

Detection of surface-bound ligands by freeze-fracture autoradiography

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.     

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and preembedding immunogold labelling

Summary A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.     

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.     

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Summary Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0° C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37° C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.33 m thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 2001,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0° C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37° C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stercomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.     

Topology of penicillin-binding protein 1b of Escherichia coli and topography of four antigenic determinants studied by immunocolabeling electron microscopy.

A method has been developed to study the orientation of proteins in the cytoplasmic membrane of Escherichia coli. Vesicles from sonicated cells were incubated in droplets on electron microscope support grids in sequence with a monoclonal antibody (MAb) against a protein with an unknown orientation (PBP 1b) followed by a MAb against a periplasmic component (peptidoglycan). The different MAbs were made visible with 5- and 10-nm gold-conjugated secondary antibodies, respectively. PBP 1b appeared to colabel with peptidoglycan. The labeling of PBP 1b in membrane vesicles with MAbs against four different epitopes was further used to estimate the number of PBP 1b molecules per cell. Approximately 1,400 PBP 1b molecules per cell grown in broth were labeled. The spatial distribution of the epitopes of the MAbs was studied by immunocolabeling of pairs of MAbs and by competitive antibody-binding inhibition. It could be tentatively concluded that the four epitopes form a cluster of antigenic determinants which occupy less than half of the surface of PBP 1b.     

Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopy: receptor distributions revealed by silver enhancement, backscattered electron imaging, and digital image analysis

Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

Neutral endopeptidase, a major brush border protein of the kidney proximal nephron, is directly targeted to the apical domain when expressed in Madin-Darby canine kidney cells

We have used a retroviral vector containing both the cDNA for rabbit neutral endopeptidase (EC 3.4.24.11; NEP) and the neomycin resistance gene to promote the expression of NEP in a polarized Madin-Darby canine kidney (MDCK) cell line. Cells resistant to G418 (a neomycin synthetic analog) were analyzed with a fluorescence-activated cell sorter to isolate a homogeneous population of cells which stably expressed NEP at their surface. When cells grown in Petri dishes were labeled with an antibody to NEP coupled to colloidal gold and examined under the electron microscope, a strong labeling of microvilli was observed, whereas very few particles were present on the basolateral domain, suggesting that the polarized distribution of this enzyme typical of proximal tubule cells is maintained in this MDCK cell population. To study more accurately the mechanism by which MDCK cells target NEP to the apical surface, cultures were grown to confluence on Costar Transwell chambers and used for pulse-chase experiments with [35S]methionine. Immunoprecipitation of recombinant NEP was then performed by adding an anti-NEP polyclonal antibody to the apical or basolateral surface of intact monolayers and by analyzing immunoprecipitates by gel electrophoresis and fluorography. Our results suggest that NEP is delivered directly to the apical domain and does not transit through the basolateral domain of the plasma membrane. This NEP-expressing MDCK cell line therefore constitutes a new model for investigating the molecular basis of apical membrane targeting in polarized epithelial cells.     

Three-dimensional cytomorphology in fine needle aspiration biopsy of medullary thyroid carcinoma.

OBJECTIVE: To elucidate three-dimensional (3-D) cytomorphology in fine needle aspiration biopsy (FNAB) of medullary thyroid carcinoma (MTC). STUDY DESIGN: ENAB was performed on tumors from five patients with MTC. The aspirate was stained and observed under a light microscope (LM). The aspirate was also fixed, dehydrated, critical point dried, spattered with gold ions and observed with a scanning electron microscope (SEM). For transmission electron microscopy (TEM), the specimen was fixed, dehydrated, embedded in an Epon mixture, cut with an ultramicrotome, mounted on copper grids, electron doubly stained with uranium acetate and lead citrate, and observed with TEM. Findings under SEM were correlated with those under LM and TEM. RESULTS: Under SEM, 3-D cytomorphology of MTC displayed a disorganized cellular arrangement with indistinct cell borders in three cases. The cell surface was uneven and had granular protrusions that corresponded to secretory granules observed under TEM. In one case with multiple endocrine neoplasia type IIB, there were abundant granules on the cell surface. In one case of sporadic MTC with multinucleated tumor giant cells and small cells, granular protrusions also were noted on the cell surface. CONCLUSION: Granular protrusion was a characteristic finding in FNAB of MTC tinder SEM and might be helpful in the differential diagnosis.     

Gold finder: a computer method for fast automatic double gold labeling detection,counting, and color overlay in electron microscopic images

This work presents a computerized method to identify, detect, evaluate, and, by colored overlay, present gold particle pairs in electron microscopy (EM), even in wide-field views. Double gold immunolabeled specimens were analyzed in a LEO 912 electron microscope equipped with a 2k x 2k-pixel slow-scan cooled CCD camera connected to a computer with analySIS 3.1 PRO image processing software. The acquisition of a high-resolution and high-dynamic-range image by the camera allowed correct segmentation of the gold particles, separating them from other cell structures and from the substrate. Particle identification was performed by a classification module designed by us. Based on shape and size, the computer recognized the group of small particles and classified them as either singular or clustered and differentiated these from the single bigger type. The final image shows the particle types separated and colored, and indicates the total number of objects encountered in the specific region of interest. Moreover, a montage tool allowed us to obtain final representative images of large microscopic fields, which on analysis by the Gold Finder module provided information on the distribution and localization of antigens comparable to that provided by the wide-field light microscope images.     

Distribution of insulin binding sites on Leydig cells of rat testes using insulin-coated gold particles.

The distribution of insulin binding sites in Leydig cells dispersed with collagenase from rat testes was studied using insulin-coated gold particles as an electron opaque ligand. Using electron microscope is convenient to distinguish Leydig cells among a variety of cells in crude preparations by their ultrastructural characteristics. Leydig cells were shown to possess insulin-binding sites on their plasma membranes. Initial binding sites of insulin were located to the microvillous surfaces. Following binding, receptor-ligand complexes seemed to move to the intermicrovillous plasma membrane, then to be internalized. Two modes of the internalization were confirmed. Most of the receptor-ligand complexes on Leydig cells appeared to be internalized via large, uncoated plasma membrane invaginations, while the remainder became internalized via small pits into vesicles. The receptor-ligand complexes were subsequently transferred to large subsurface vacuoles with electron-lucent lumens believed to correspond to endosomes. The reason why IGCs on the postendosomal pathway moving toward lysosomes was also discussed.     

Distribution of insulin binding sites on Leydig cells of rat testes using insulin-coated gold particles

Summary The distribution of insulin binding sites in Leydig cells dispersed with collagenase from rat testes was studied using insulin-coated gold particles as an electron opaque ligand. Using electron microscope is convenient to distinguish Leydig cells among a variety of cells in crude preparations by their ultrastructural characteristics. Leydig cells were shown to possess insulin-binding sites on their plasma membranes. Initial binding sites of insulin were located to the microvillous surfaces. Following binding, receptor-ligand complexes seemed to move to the intermicrovillous plasma membrane, then to be internalized. Two modes of the internalization were confirmed. Most of the receptor-ligand complexes on Leydig cells appeared to be internalized via large, uncoated plasma membrane invaginations, while the remainder became internalized via small pits into vesicles. The receptor-ligand complexes were subsequently transferred to large subsurface vacuoles with electron-lucent lumens believed to correspond to endosomes. The reason why IGCs on the postendosomal pathway moving toward lysosomes was also discussed.     

Scanning electron microscopy of cell surface antigens labeled with colloidal gold

A method is described and discussed that permits the specific labeling of the surface of prefixed cells with the colloidal gold marker viewed with the scanning electron microscope. Its value depends exclusively on the use of backscattered electron imaging. Its advantages include the possibility of preserving the surface features of the labeled cells, the ease with which specificity can be established, the possibility of making total counts of the labeled surface antigenic sites, and the possibility of achieving distinct labeling for two different antigens expressed on the surface of the same cell.     

Colloidal gold probes for the identification of virus particles: An appraisal

Electron microscope examination of negatively stained preparations continues to be the method of choice for the diagnosis of virus particles although in some instances an immunological test is necessary. Colloidal gold immunocytochemical probes are becoming increasingly popular for electron microscopy and their suitability for the identification of virus particles is assessed.Virus particles were immunolabelled in situ on plastic/carbon coated electron microscope grids with specific antibody and colloidal gold probes. The labelling obtained was specific, definite and with very little background. The technique is very sensitive, very quick, and since a minimum of preparation is needed it appears to possess considerable potential for virus diagnosis.     

Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography

Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.     

Silver-enhanced colloidal gold as a cell surface marker for photoelectron microscopy

Colloidal gold labeling in conjunction with silver enhancement was investigated as a labeling technique for photoelectron microscopy (PEM). PEM uses UV-stimulated electron emission to image uncoated cell surfaces, and markers for cell surfaces need to be sufficiently photoemissive to be clearly visible against this background. Label contrast provided by 6 nm or 20 nm colloidal gold markers alone was compared to that provided by 6 nm markers after silver enhancement, using both direct and indirect labeling methods for fibronectin on human fibroblast cell surfaces. In all cases, details of the fibrillar fibronectin labeling distribution which were barely discernible before silver enhancement became highly visible against the cellular surface features. Two factors evidently contribute to the pronounced increase in label contrast with silver enhancement: (1) Increased particle size, which was documented by transmission electron microscopy, and (2) increased photoemission resulting from a silver coating on the enhanced gold markers, compared with the protein coating on the unenhanced gold markers. These data demonstrate that silver enhancement of colloidal gold labeling patterns in PEM images is a highly effective method for localization of specific sites on cell surfaces.