Life Sciences Commons^™

Labeling With Nanogold And Undecagold: Techniques And Results, James F. Hainfeld

Scanning Microscopy

A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and allow many new probes to be made with peptides, nucleic acids, lipids, drugs, and other molecules. A new fluorescent-gold conjugate is useful for examining localization by fluorescence microscopy, then visualizing the same label at the ultrastructural level in the electron microscope.

Accessing Nuclear Structure For Field Emission, In Lens, Scanning Electron Microscopy (Feisem), T. D. Allen, G. R. Bennion, S. A. Rutherford, S. Reipert, A. Ramalho, E. Kiseleva, M. W. Goldberg

Scanning Microscopy

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not …

Use Of Colloidal Gold And Neutron Activation In Correlative Microscopic Labeling And Label Quantitation, B. J. Darien, P. A. Sims, K. T. Kruse-Elliott, T. S. Homan, R. J. Cashwell, A. J. Cooley, R. M. Albrecht

Scanning Microscopy

Albumin was conjugated to 16 nm gold particles (Alb-Au16) and infused into anesthetized pigs to determine if plasma, tissue and bronchoalveolar lavage (BAL) fluid concentrations of gold could be quantitated by neutron activation (Au198). Additionally, transmission electron microscopy (TEM) of lung and liver samples was evaluated for sites of gold distribution and morphological changes. The minimum concentration of gold detected by neutron activation ranged between 1.4 and 1.9 ppb (ng/gm of sample). No gold was detected in the plasma of pigs prior to Alb-Au16 infusion, while mean post infusion concentrations were 1.037 ± 0.69 ppm (μg/gm plasma, ±SD). The concentrations …

Gold, Electron Microscopy, And Cancer Therapy, James F. Hainfeld

Scanning Microscopy

Radioactive gold has properties suitable for radiotherapy and can provide lethal irradiation to cells. If the gold is conjugated to a targeting molecule, such as an antibody, it may be possible to specifically deliver the dose to tumor cells. Various gold particles are possible candidates and include gold colloids with adsorption of antibodies or gold clusters with covalent attachment. Different sizes of gold particles are available and some may be preferred for certain situations. Problems with intravenous injection and in vivo delivery are numerous, and a more tractable application is the direct instillation into the urinary bladder of radiogold immunoconjugates …

Prerequisites Of High Resolution Scanning Electron Microscopy, René Hermann, Martin Müller

Scanning Microscopy

Cryotechniques must be employed throughout all preparation and observation steps in order to extract high resolution scanning electron microscopical information from biological material.

Cryoimmobilization, followed by freeze-drying and metal-shadowing at low temperature, yields optimal structural information of T4 polyheads used as a test specimen. Freeze-substitution of frozen T4 polyheads and subsequent freeze-drying renders the substructures recognizable but less crisp than freeze-drying from aequous solutions. Critical point drying of ethanol dehydrated chemically fixed, or freeze-substituted test specimens results in complete loss of discrete polyhead structure.

In-lens field-emission scanning electron microscopes and highly sensitive electron detectors are instrumental prerequisites in achieving transmission …

Strategies For Improving The Cytochemical And Immunocytochemical Sensitivity Of Ultrastructurally Well-Preserved, Resin Embedded Biological Tissue For Light And Electron Microscopy, Jan A. Hobot, Geoffrey R. Newman

Scanning Microscopy

Many techniques for processing tissue into resin are available, varying from conventional room temperature to low temperature procedures. The problem is to choose an appropriate method to suit the biological specimen under study. Room temperature approaches with aldehyde and osmium fixation do not give optimal retention of immunoreactivity. Osmium can be removed from sections, but recovery of immunosensitivity is reduced. Osmium post-fixation can be omitted, but heat polymerization of resins causes tissue extraction and loss of immunoreactivity. Alternative techniques rely on the use of milder polymerization methods and avoid osmium. However, while providing an improvement, this alone is not sufficient …

Scanning Electron Microscopy Of Immuno-Gold Labeled Antigens Associated With Bladder Cancer, E. De Harven, J. G. Connolly, Y. Wang, W. Hanna, G. Bootsma

Scanning Microscopy

Scanning electron microscopy (SEM), in the backscattered electron imaging (BEI) mode, has been used to study the topographical distribution of colloidal gold labeled antigens expressed on the luminal surface of the bladder urothelium in biopsies from three categories of patients: 1) normal controls; 2) patients with a history of bladder cancer but no pathological diagnosi s at time of cystoscopy; and 3) patients with overt transitional cell carcinoma (TCC) of various histopathological stages and grades. Cold cup biopsies were processed for immuno-SEM according to a previously described method. Antigens under investigation were: 1) ABH blood group antigens; and 2) those …

Observations Of Colloidal Gold Labelled Platelet Microtubules: High Voltage Electron Microscopy And Low Voltage-High Resolution Scanning Electron Microscopy, R. M. Albrecht, J. Prudent, S. R. Simmons, J. Pawley, J. J. Choate

Scanning Microscopy

18 nm colloidal gold-antitubulin and 4 nm colloidal gold-antitubulin were used to label microtubules in adherent, fully spread platelets. Both sizes of marker effectively labelled microtubules in the partially extracted platelets. However only the 4 nm gold penetrated the dense microfilament matrix of the inner filamentous zone so that portions of microtubules within this cytoskeletal zone could be tracked. The gold marker could be visualized well with 1 MeV high voltage transmission EM and with 5 kV or greater secondary imaging or 20 kV backscattered imaging of carbon only coated samples. 1 kV secondary imaging permitted high resolution imaging of …

Attachment Of Plasma Membranes Of Cultured Cells To Silicon Chips For High Magnification Imaging In Scanning Electron Microscopy, Klaus-Ruediger Peters, William W. Carley

Scanning Microscopy

In the membrane preparation method described in this paper, a polylysine-coated silicon chip is adsorbed to the exposed apical surface of a cell monolayer. Upon removal, the adsorbed chip separates the plasmalemma from the residual bodies of the cultured cells. This sandwich-membrane separation approach simplifies access to the cytoplasmic aspects of both the apical and the basal plasmalemma which remains on the culture substrate and is covered to a varied extent by cytoplasmic infrastructures. To stabilize the attached membranes, small crosslinking agents are used in a controlled osmium impregnation. Large crosslinkers are avoided since they induce thickening of fine structures. …

Double-Axis Rotary Shadowing For High-Resolution Scanning Electron Microscopy, R. Hermann, J. Pawley, T. Nagatani, M. Müller

Scanning Microscopy

Thin continuous metal coatings and a scanning electron microscope-generated spot size in the range of the visualized particles, are necessary prerequisites if one hopes to extract high-resolution topographic information in the scanning electron microscope. Chemical fixation and dehydration in organic solvents at room temperature lead to severe ultrastructural artifacts which can be avoided by cryofixation and freeze-drying of the specimen. 0.9 to 2.7 nm thick homogeneous layers of chromium and germanium can be deposited onto the surface of cryofixed and freeze-dried specimens at high sub-zero temperatures by electron beam evaporation using "double-axis rotary shadowing". Theoretical calculations of the layer geometry …

Bioapplication Of Colloidal Gold In Microbiological Immunocytochemistry, Julian E. Beesley

Scanning Microscopy

Microbiological organisms are an ubiquitous group of animals encompassing bacteria, viruses, protozoa, algae and fungi. They are adapted for survival in many diverse habitats and exert a profound effect on man and his environment. Colloidal gold electron immunocytochemistry is a useful technique for studying these organisms and may be applied in several ways. The post-embedding technique is used to detect internal antigens, whilst the pre-embedding technique is employed for the detection of external antigens. In contrast the immuno-negative stain technique is applied to detect antigens on structures such as viruses or bacterial pili which may be dried down onto an …

Trifluoperazine Inhibition Of Fibrinogen Receptor Redistribution In Surface Activated Platelets: Correlative Video-Enhanced Differential Interference Contrast Light Microscopic, High Voltage Electron Microscopic And Scanning Electron Microscopic Studies, O. E. Olorundare, S. L. Goodman, R. M. Albrecht

Scanning Microscopy

Video-enhanced differential interference contrast (VDIC) light microscopy in conjunction with fibrinogen labelled colloidal gold was employed as a probe to follow the mobility of the fibrinogen receptor on platelets. Correlative studies by both high voltage and scanning electron microscopy confirms localization of labels relative to platelet ultrastructural and surface characteristics, respectively. Treatment of platelets with trifluoperazine prior to and after incubation with fibrinogen-gold labels results in a concentration dependent inhibition of receptor movement. The results obtained from this study suggest that phosphorylation of myosin by the Ca⁺⁺ -calmodulin dependent enzyme, myosin-light chain kinase, is important in the fibrinogen redistribution …

Assembly And Alignment Of Fibronectin-Coated Gold Beads Into Fibrils By Human Skin Fibroblasts, Donna M. Pesciotta Peters, Deane F. Mosher

Scanning Microscopy

The assembly of fibronectin into fibrils was monitored by high voltage electron microscopy using 18 nm colloidal gold beads bound to fibronectin (Au₁₈-fibronectin) or an amino terminal 70 kd fragment of fibronectin (Au₁₈-70 kd) that blocks the incorporation of fibronectin into disulfide bonded fibrils. Subconfluent cultures of human skin fibroblasts were incubated with the colloidal gold complexes for 0.25, 0.5, 1.5 and 5 h. In fibroblast cultures incubated with Au₁₈-fibronectin and Au₁₈-70 kd fragments for 0.25 and 0.5 h, the complexes of Au₁₈-fibronectin and Au₁₈-70 kd fragment were …

Expression Of Major Histocompatibility Complex Antigens On Macrophages: Correlative Study Using Flow Cytometry, Radioimmunoassay, And Colloidal Gold Immunolabeling, Lynne E. Guagliardi, Donna M. Paulnock, Ralph M. Albrecht

Scanning Microscopy

Correlative scanning electron microscopy (SEM), radioimmunoassay (RIA), and flow cytometric analysis were used to characterize levels of class I and class II major histocompatibility complex-encoded (MHC) antigen expression on peritoneal exudate cells of mice chronically infected with Chlamydia psittaci. Analysis of peritoneal macrophages by all three techniques revealed a marked induction of H-2 K,D (class I) and I-A, l-E (class II) antigens on cells from infected C3H mice when compared to uninfected controls.

Scanning electron micrographs further document that the increases in class I and II MHC antigens are due to an increase in la/H-2 bearing cells as well …

Colloidal Gold Labelling Of Fibrinogen Receptors In Epinephrine – And Adp–Activated Platelet Suspensions, J. A. Oliver, R. M. Albrecht

Scanning Microscopy

It has been generally accepted for over twenty years that epinephrine stimulates platelet aggregation without inducing shape change. However, it has been recently reported that discoid platelets are not recruited into ADP-or epinephrine-stimulated aggregates. Previous work in our laboratory has suggested that platelet shape change is necessary for the binding of fibrinogen to its surface receptor, which is a prerequisite for platelet aggregation. These studies seem to indicate that epinephrine-induced platelet aggregation does involve shape change. To investigate this possibility, the extent of shape change and fibrinogen binding in suspensions of epinephrine-and ADP-activated and control platelets was assessed by scanning …

Colloidal Gold - A Powerful Tool In Scanning Electron Microscope Immunocytochemistry: An Overview Of Bioapplications, G. M. Hodges, J. Southgate, E. C. Toulson

Scanning Microscopy

Colloidal gold may be conjugated to a wide variety of macromolecules, provides a versatile system for immunocytochemical studies by various types of microscopy (light and fluorescent microscopy, scanning (SEM) and transmission (TEM) electron microscopy), and is significantly contributing to the development of SEM immunocytochemistry as a routine analytical procedure.

A comprehensive overview has been compiled of the literature on SEM bioapplications of colloidal gold. This is illustrated through a selected series of studies focussing on a) cell surface receptor-ligand interactions; b) expression of cell surface lectin-binding sites; c) surface distribution of extracellular matrix components; and d) visualization of gold-labelled cytoskeletal …

Surface Characterization Of Biomaterials By Immunogold Staining - Quantitative Analysis, Kinam Park, Scott R. Simmons, Ralph M. Albrecht

Scanning Microscopy

The labeling of target proteins by immunogold particles has been analyzed based on Einstein's law of Brownian motion. The theory was confirmed from the experiments which employed antifibrinogen gold markers to label fibrinogen molecules adsorbed on the polyethylene surface. The theory predicts that the degree of labeling depends on the concentration of gold markers, temperature, medium viscosity, size of gold markers, and staining time. Of these factors most important is the concentration of immunogold particles. Small change in the marker concentration results in a significant variation in the staining efficiency when other variables are kept constant. The effect of temperature …

Labeling Of Sweet Taste Binding Sites Using A Colloidal Gold-Labeled Sweet Protein, Thaumatin, Albert I. Farbman, Carolyn K. Ogden-Ogle, Göran Hellekant, Scott R. Simmons, Ralph M. Albrecht, H. Van Der Wel

Scanning Microscopy

Thaumatin, an intensely sweet tasting protein, was bound to colloidal gold and applied to the taste bud-bearing foliate papillae of Rhesus monkeys. Examination of thin sections of taste pores showed that gold particles were bound to merocrine secretions of Type I taste bud cells, to some cell remnants of lysed cells, and, most importantly, to small, membrane bounded blebs of cytoplasm. These blebs are thought to be shed into the pore from the tips of taste bud cell microvilli, particularly those arising from Type II cells. The binding of gold particles to microvillus tips and to the blebs suggest that …

Receptor-Mediated Binding, Endocytosis And Cellular Processing Of Macromolecules Conjugated With Colloidal Gold, Dean A. Handley

Scanning Microscopy

Receptor-mediated expression of cellular functions assures biological specificity, regulation, and control of nutritive and metabolic requirements. The study of receptor-ligand interactions is a central theme in many published reports employing colloidal gold labeling. With the ligand directly conjugated with colloidal gold, the investigator is afforded the opportunity to observe all phases of cellular binding, endocytosis, lysosomal delivery and catobolism. Reviewed are published studies employing direct ligand conjugation with colloidal gold, the relative merits and disadvantages of this type of procedure and data from recent studies investigating endothelial receptor binding of proteins in the coagulation and fibrinolysis cascades.