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Visualization of silver-enhanced reaction products from protein-and immuno-colloidal gold probes by laser scanning confocal microscopy in reflection mode.

We have employed a laser scanning confocal microscope in reflection mode to directly and indirectly visualize sites of deposition of silver-enhanced reaction products from colloidal gold probes. A direct approach was used for the localization of alpha-fetoprotein receptors in human myoblasts by incubating primary cultures with an alpha-fetoprotein-gold conjugate. For an indirect approach, cultured CEM cells, derived from a human T-lymphoma cell line, were incubated with a mouse monoclonal antibody to mature T-cells, followed by a gold-labelled antibody to mouse immunoglobulins. Multiple optical sections of each sample were collected by reflection laser scanning confocal microscopy and combined into three-dimensional renderings. A (non-confocal) transmission image was generated of each field for comparative purposes. The increasing use of reflection laser scanning confocal microscopy combined with colloidal gold conjugates as biological markers will probably be of considerable advantage in cytochemical analysis.

Antigens, CD↗

High resolution size determination of 20 nm colloidal gold particles by SedFFF.

PURPOSE: Assessment of lower size limit of Sedimentation Field-Flow Fractionation (SedFFF), specifically to evaluate if the method is suitable to determine the size and size distribution of 20 nm colloidal gold particles with high resolution. METHODS: Sedimentation Field-Flow Fractionation was used to determine the size of the colloidal particles. Due to the high density of gold it was possible to extend the lower size limit of SedFFF well below 20 nm. The size distribution of a gold colloid was obtained from the peak broadening caused by the polydispersity of the sample. The peak broadening due to instrumental imperfections was determined. For comparison purpose the particles were also sized using SEM and PCS. RESULTS: The mean diameter of the particles was determined to be (20.87+/-0.05) nm, the standard deviation in size being 1.04 nm (about 5%). SEM could confirm that the particles are about 20 nm in diameter. A sizing with PCS was not possible. The particles have a strong tendency to aggregate and PCS yields a diameter that is much too large. CONCLUSIONS: At optimized analytical parameters Sedimentation Field-Flow Fractionation is an effective method to measure the size of gold particles as small as 15 nm with an accuracy of about 0.1 nm. The polydispersity of the sample can easily be determined.

Chemical Fractionation↗

A colloidal gold labeling technique for the direct determination of the surface area of eukaryotic cells.

We have developed a colloidal gold labeling technique for the direct quantitation of the cell surface area. The method is based on coating the cell surface with [195Au] colloidal gold-protein complexes followed by morphometric determination of the labeling density (gold particles/micron2 cell surface) and radiometric determination of the total number of gold particles bound per cell. The ratio of both values directly gives the cell surface area. The accuracy of the method was shown using Staphylococcus aureus cells as a model system, where the cell surface area determined with our assay (4.0 microns2) corresponded well to the value calculated from the radius of the cells (3.6 microns2). In a more complex model system J-774 mouse macrophages were labeled with different amounts of [195Au] gold-protein complexes to show that the assay is independent of the degree of saturation of the cell surface binding sites. Both high (135 Au/microns2) and low (65 Au/microns2) labeling densities resulted in a surface area of about 1200 microns2. The technique finally was applied to L-929 fibroblasts to determine the increase of the cell surface area when the cells change from a spherical to a flat monolayer state. We found that the cell surface area increased 3-fold during the spreading process. The results show that the colloidal gold labeling technique allows the direct determination of the surface area of complex eukaryotic cells. The technique is suitable for the quantitation of changes in the surface architecture known to occur in different functional states of eukaryotic cells.

Animals↗

Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles.

The gastrointestinal uptake of micro- and nanoparticles has been the subject of recent efforts to develop effective carriers that enhance the oral uptake of drugs and vaccines. Here, we used correlative instrumental neutron activation analysis and electron microscopy to quantitatively and qualitatively study the gastrointestinal uptake and subsequent tissue/organ distribution of 4, 10, 28, and 58 nm diameter metallic colloidal gold particles following oral administration to mice. In our quantitative studies we found that colloidal gold uptake is dependent on particle size: smaller particles cross the gastrointestinal tract more readily. Electron microscopic studies showed that particle uptake occurred in the small intestine by persorption through single, degrading enterocytes in the process of being extruded from a villus. To our knowledge this is the first report, at the ultrastructural level, of gastrointestinal uptake of particulates by persorption through holes created by extruding enterocytes.

Administration, Oral↗

Part III: Surface-enhanced Raman scattering of amino acids and their homodipeptide monolayers deposited onto colloidal gold surface.

Surface-enhanced Raman scattering (SERS) spectra were measured for monolayers of various amino acids: L-methionine (Met), L-cysteine (Cys), L-glycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, Leu-Leu, Phe-Phe, and Pro-Pro) deposited onto a colloidal gold surface. Orientation of amino acids and their homodipeptides, as well as specific-competitive interactions of their functional groups with the gold surface, were predicted by detailed spectral analysis of the obtained SERS spectra. The analysis performed allowed us to propose a particular surface geometry for each amino acid and homodipeptide on the gold surface. In addition, we compared the structures of these molecules adsorbed on colloidal gold and silver surfaces.

Amino Acids↗

Colloidal gold--low density lipoprotein conjugates as membrane receptor probes.

We have developed a method for conjugating low density lipoproteins (LDL) with colloidal gold. Conjugation, complete after 1 min, occurs by electrostatic adsorption of the LDL to the negatively charged gold particle. Each conjugate consists of approximately eight biologically active LDL molecules clustered around a central 19-nm gold granule. Acidic (pH 4), alkaline (pH 9), or high ionic (600 milliosmolar NaCl) environments do not dissociate the conjugate. Colloidal gold is an electron-dense, nondegradable marker that is easily identified within the cell and serves as a valuable probe for studying receptor binding and endocytosis. By using a modified method of ruthenium red staining, the LDL molecules of the conjugate can be directly visualized when they are bound to the cell surface receptor. Receptor binding (4 degrees C) of the conjugate by cultured human fibroblasts reveals that the gold granule is positioned 18-21 nm from the coated pit region of the membrane. This distance, similar to the diameter of LDL, suggests concomitant internalization of the receptor during vesicular endocytosis and early lysosomal incorporation (10 min at 37 degrees C). Continued internalization (30-60 min at 37 degrees C) results in the formation of free pools of gold within the lysosome.

Cell Membrane↗

A characterisation of dark-field imaging of colloidal gold labels in a scanning transmission X-ray microscope.

While X-ray microscopes provide images of biological specimens for which the contrast is mainly due to the difference in the absorption of carbon and oxygen when X-rays transmitted through the specimen are detected, signals other than absorption can also be used to form images. Using the Stony Brook scanning transmission X-ray microscope at the National Synchrotron Light Source, high-angle dark-field images have been formed of cells labelled with colloidal gold, with and without silver enhancement. The high density of the colloidal gold particles, or the silver particles seeded by the gold, leads to a large scattering signal, and the fact that the particle diameters are comparable to the width of the microscope point spread function results in good localisation of the label with high contrast. The dark-field images can have a greater signal to noise ratio than bright-field images acquired with the same incident X-ray dose. The theory of dark-and bright-field imaging is reviewed. Theoretical calculations of scattering from gold and silver particles are presented and good agreement is found between these and experimental dark-field images of 30 nm diameter gold particles. The signal to noise ratios of experimental bright-and dark-field images are measured and found to be in agreement with theory. Images are presented of cells labelled by immunolabelling and in situ hybridisation.

Fibroblasts↗

Processing of colloidal gold particles in the glomerular mesangium and macula densa of the duck kidney.

Bovine-serum-albumin coated colloidal gold particles were injected intravenously into healthy, adult domestic ducks. The fate of these tracer particles was followed in a time-sequence up to 4.5 days in the renal glomerular mesangium--distal tubule macula densa junction with transmission electron microscopy. The bulk of particle aggregates is 'trapped' in the mesangial channel system, phagocyted by mesangial cells, exocyted back into the mesangial channels, transported extracellularily towards the vascular hilus, rephagocyted by macula densa cells and expelled into the tubular lumen. This process requires about nineteen hours in the experimental model chosen, although it continues to take place to a much lower extent even 4.5 days after administration of the tracer. This mode of macromolecular excretion may be of interest in connection with the pathogenesis of several forms of glomerulonephritis.

Animals↗

The role of liver endothelium in the binding and uptake of ceruloplasmin: studies with colloidal gold probe.

To determine the mode of uptake of ceruloplasmin (CP) by liver, the protein was labeled with colloidal gold and infused into the portal vein. In cold almost all probes bound to the sinusoidal endothelium, and at 37 degrees C internalization via a system of coated pits and vesicles occurred. Only rarely did the probe appear to bypass the endothelium, moving to the abluminal side through the gaps between endothelial cells. In the endothelial cytoplasm, the probe was seen in coated vesicles, endosomes, tubules, and large vesicles which may have formed by fusion of endosomes and tubules. Moreover, externalization of the probe to the abluminal side was noted, and this also occurred via a system of coated vesicles. The findings suggest that the uptake of CP in the liver may be primarily a transendothelial phenomenon (transcytosis).

Animals↗

[Light and electron microscopic demonstration of acidic glycoconjugates by means of postembedding staining procedures using cationic colloidal gold].

The cytochemical detection of acidic glycoconjugates located in rat gastrointestinal epithelia was performed using tissue sections embedded in Lowicryl K4M and cationic colloidal gold (CCG). CCG was prepared from poly-L-lysine and colloidal gold solution (10 nm). The staining of CCG was amplified after a photochemical silver reaction using silver acetate as an ion donor at the light microscopic level. The staining solutions adjusted to pH 2.5, CCG (2.5) and pH 1.0, CCG (1.0) were used for the characterization of carboxylated and sulfated glycoconjugates. At light microscopic level, CCG (2.5) stained several cell types of mucous cells in the rat gastrointestinal tract, while CCG (1.0) stained selectively mucous cells in gastric pits, glandular pylorus, upper crypts of the proximal colon and entire crypts of the distal colon. At the electron microscopic level, both CCG (2.5) and CCG (1.0) labeled the trans side of the Golgi apparatus and mucous granules in some mucous cells. These results are consistent with previous glycoconjugates histochemistry of the rat gastrointestinal tract at both the light and electron microscopic levels. CCG (2.5) and CCG (1.0) staining methods are useful and reliable postembedding staining procedures for the light and electron microscopic demonstration of intra- and extracellular acidic glycoconjugates.

Animals↗

Comparative ultrastructural morphology of aurosomes produced by colloidal gold and soluble gold salts.

Intra-articular administration of colloidal gold produced aurosomes which contained spherical electron-dense granules and also often some very fine electron-dense particles. Intra-articular administration of aurothioglucose produced aurosomes of a morphology similar to that reported to occur after administration of sodium aurothiomalate. These aurosomes produced by soluble gold salts are characterised by particle and granule-studded membranous formations which present in profile as rod-like and curled filamentous structures. On the basis of morphological differences two types of aurosomes should be recognised, the "Type I or granular aurosome" and the "Type II or membranous aurosome".

Animals↗

Immuno-scanning electron microscopy of normal and leukemic leukocytes labeled with colloidal gold.

The immunogold method, utilizing 40 nm colloidal gold particles which can be selectively visualized with the scanning electron microscope (SEM) in the backscattered electron imaging mode was used for the study of blood cells incubated with various monoclonal antibodies. Numerous anti-leukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types and even to recognize their different maturation stages. Clearcut differences in surface morphology exist among peripheral blood normal leukocytes and even among the principal lymphocyte subclasses. Marked heterogeneity in surface morphology is, on the other hand, evident when studying precursors or leukemic cells. Immature cells show, nevertheless, relatively smooth surfaces while some distinct surface features appear on cells already committed toward a specific differentiation lineage. Hairy cells can also be precisely identified, especially when in small number in heterogeneous populations, combining their typical surface morphology with their positivity for B1 and Leu M5 monoclonal antibodies.

B-Lymphocytes↗

Application of colloidal gold to ultrastructural studies.

Progress in recognition of structure and function in a variety of cells has always been linked to development of new research techniques. Beyond doubt, immunocytochemical techniques belong to such new methods. However, the techniques had not been widely applied to electron microscopy until colloidal gold was introduced as a label. This paper presents the pre-embedding and the post-embedding techniques and the applied markers, which are used in immunocytochemical techniques at the ultrastructural level. The potential of colloidal gold techniques is discussed. Particular attention is paid to methods of reaction amplification. Application of gold is illustrated with our own results.

Animals↗

In vivo demonstration of subcellular localization of anti-transferrin receptor monoclonal antibody-colloidal gold conjugate in brain capillary endothelium.

To study transcytosis at the blood-brain barrier (BBB) in vivo, we used the internal carotid artery perfusion technique in rats. Brain uptake of the OX26 anti-transferrin receptor antibody (IgG2a) coupled to 5-nm colloidal gold (OX26-Au) was morphologically examined after infusion or perfusion into the carotid artery for 10 min. The brain tissue was subsequently perfusion-fixed with 2% glutaraldehyde. By light microscopy, silver enhancement of vibratome sections revealed staining of the vascular tree. Electron microscopy showed binding of gold particles at the luminal plasma membrane of brain capillary endothelia, endocytosis in vesicles (50-100 nm), and particles beyond the abluminal plasma membrane. Studies were performed with an IgG2a isotype control, UPC10. Internal carotid artery infusion of [125I]-UPC10 showed no evidence of brain uptake or binding to endothelium. However, microvessel structures were identified after silver enhancement of vibratome sections of brain following internal carotid artery infusion of 5-nm colloidal gold conjugates of UPC10 (UPC10-Au). The morphologically observed binding of UPC10-Au to brain microvessels may be induced by gold conjugation. These studies describe the use of gold conjugates of antibodies to delineate the subcellular pathway involved in transcytosis through the BBB.

Animals↗

Detection of mucus glycoconjugates in human conjunctiva by using the lectin colloidal gold technique in TEM. I. A quantitative study in normal subjects.

We applied a specific cytochemical reaction to characterize the glycoconjugates produced by goblet and non-goblet epithelial cells of normal human conjunctiva. For this purpose we utilized the lectins, proteins of vegetal origin, which are extremely sensitive in binding glycosidic residues. In particular, we used WGA, PNA, SBA and ConA conjugated with colloidal gold as ultrastructural marker for Transmission Electron Microscopy. This technique allowed us also to perform a quantitative analysis, by counting colloidal gold particles present on mucus granules. In this way we analyzed the content both of goblet and non-goblet epithelial cells. In the former, WGA, PNA, SBA and ConA receptors, here reported in decreasing density, were present. In the latter WGA was always positive, SBA and PNA sometimes were negative, ConA was always negative. We speculate the different contribution to mucus production by these two sources may be important in evaluating tear film stability alterations occurring in those diseases in which non-goblet epithelial cell vesicles increase.

Carbohydrate Metabolism↗

Simultaneous electron microscopic detection of colloidal gold labeled ligand and adenylate cyclase. Influence of concanavalin-A on adenylate cyclase activity of fibroblasts.

Concanavalin-A-colloidal gold (Con-A-G) complex and adenylate cyclase activity were detected simultaneously in electron microscopic preparations of human fibroblast cultures, by a combined histochemical technique. The colloidal gold particles appeared as round bodies which could be readily differentiated from the amorphous product of the adenylate cyclase enzyme reaction. The combined technique makes possible the simultaneous visualization of the bound ligand (i.e. of its binding site), and of the enzyme activated by the ligand. Treatment of the cells with Con-A accounted for a considerable increase in intracellular adenylate cyclase activity. The activity increase was disproportionally greater than the amount of bound ligand, and it also appeared in localizations showing no indication of ligand binding. Treatment of the fibroblasts with Con-A was followed by internalization of the ligand and the enzyme inside at least seemingly segregated vesicles.

Adenylyl Cyclases↗

[Preparation of monoclonal antibodies against morphine and preliminary application of its colloidal gold conjugates].

AIM: To prepare monoclonal antibodies (mAbs) against morphine. METHODS: Morphine was conjugated to rabbit serum albumin or bovine serum albumin, respectively and the BALB/c mice were immunized with the conjugated. The mAbs against morphine were obtained by hybridoma technique. The urine samples were detected with the paper chromatography based on crosslinked of the purified mAb with colloidal gold. RESULTS: Three mAbs against morphine were obtained and the sensitivity detected by paper chromatography reached to 200 microg/L. CONCLUSION: The successful preparation of the mAb and its colloid gold conjugates may be useful for field check.

Animals↗

Demonstration and cytochemical analysis of anionic sites on ejaculated boar spermatozoa: a scanning electron microscopy study using cationised colloidal gold.

The plasmalemma of spermatozoa bears negative charges as is the case for most mammalian cells. This has been concluded from the sperm cell's electrophoretic behaviour and from labelling experiments with various cationic probes followed by transmission electron microscopy of ultrathin sections. An overall view of the cell surface, however, is necessary in order to assess the distribution and density of the anionic sites adequately. We, therefore, used scanning electron microscopy in combination with cationised colloidal gold labelling to analyse the presence of anionic sites on ejaculated boar spermatozoa. Incubations were performed at pH 3.5, 2.5 and 1.0. Labelling was specific and bound gold particles were unequivocally identified using the backscattered electron signal. The chemical nature of the anionic sites involved was investigated by treating spermatozoa with pronase, phosphatase and neuraminidase as well as by methylation, acid hydrolysis and beta-elimination prior to cationised gold labelling. Our results suggest that besides phosphates, carboxyl groups are predominantly accountable for the binding of cationised colloidal gold. Presumptive macromolecules bearing these anionic sites are phospholipids and sialic acid residues. The combination of methods presented herewith should be of value in order to elucidate charge interactions which have been shown to play a role in cellular recognition events and adhesion.

Acid Phosphatase↗