Quantification of gold labeled cell surface antigens by SEM: Current progress and remaining problems

1990 ◽  
Vol 48 (3) ◽  
pp. 34-35
Author(s):  
Etienne de Harven ◽  
Davide Soligo ◽  
Roy McGroarty ◽  
Hilary Christensen ◽  
Richard Leung ◽  
...  
Keyword(s):  
Cell Surface ◽  
Immunogold Labeling ◽  
Target Antigen ◽  
Facs Analysis ◽  
Becton Dickinson ◽  
Cell Surface Antigens ◽  
Human T Lymphocyte ◽  
Backscattered Electron ◽  
Imaging Mode ◽  
Electron Imaging

Taking advantage of the high elemental contrast of particles of colloidal gold observed in the backscattered electron imaging(BEI) mode of the SEM (1,2), the human T lymphocyte was chosen as a model system to study the potential value of immunogold labeling for the quantification of cell surface expressed molecules. The CD3 antigen which is expressed on all human T lymphocytes and is readily identified by the LEU-4 murine monoclonal antibody (Becton Dickinson, Mountain View, CA) followed by a gold conjugated goat anti-mouse Ig polyclonal antibody was chosen as a model target antigen. When quantified by non-EM methods, using radio-iodinated probes or FACS analysis, approximately 30,000 to 50,000 copies of this antigen per cell are enumerated.The following observations were made while attempting to quantify the same molecule by SEM after specific immunogold labeling:Imaging in the SE vs BE mode: The numbers of gold markers counted in the secondary electron (SE) imaging mode are considerably lower than those counted on the same cells in the backscattered electron (BE) imaging mode.

scholarly journals Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing

2019 ◽  
Vol 400 (3) ◽  
pp. 343-350 ◽  
Author(s):  
Steffen Dickopf ◽  
Matthias E. Lauer ◽  
Philippe Ringler ◽  
Christian Spick ◽  
Peter Kern ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Surface Binding ◽  
Cell Surface Antigens ◽  
Peripheral Blood Mononuclear ◽  
Variable Regions ◽  
High Flexibility

Abstract A novel bispecific antibody format was applied to generate T cell-engaging antibodies. The TriFab format is a trivalent IgG-shaped entity composed of two Fab arms that bind to antigens on the surface of tumor cells, which are linked via flexible peptides to a CD3 binding moiety that replaces the CH2 domains of conventional IgGs. The distinctive feature of these T cell recruiting bispecifics is that their CD3 variable regions are incorporated between domains, rather than N- or C-terminally fused to an Fc or antibody fragments. T cell recruiting TriFabs resemble in size and shape, are expressed and show biophysical properties similar to regular IgGs. Transmission electron microscopy (TEM) demonstrates high flexibility of the cell surface binding arms as well as target antigen accessibility of the interspersed CD3 binding domain. Functional co-culturing assays of peripheral blood mononuclear cells (PBMCs) and different tumor cell lines (MCF7 and A431) revealed a dose-dependent T cell-mediated cytotoxicity that was induced by the TriFabs targeting either LeY or EGFR cell surface antigens.

scholarly journals Simultaneous visualization of myeloperoxidase reactivity and immunogold labeling by backscattered electron imaging with the scanning electron microscope.

1987 ◽  
Vol 35 (2) ◽  
pp. 267-269 ◽  
Author(s):  
D Soligo ◽  
E P de harven
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Cell Surface ◽  
Cell Types ◽  
Myeloperoxidase Activity ◽  
Backscattered Electron Imaging ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Simultaneous Visualization ◽  
Scanning Electron

We describe a method to label circulating human granulocytes with an immunogold marker and then incubate them for demonstration of myeloperoxidase activity. The cells were observed in the backscattered electron imaging (BEI) mode of the scanning electron microscope. This permits simultaneous visualization of cell surface morphology, the immunological surface marker, and the cytochemical reactivity of each individual cell. Enhanced identification of the various cell types and more precise characterization of cell surface features in the different steps of leukocyte differentiation are expected to result from application of this technique.

Multiparameter Approaches to FACS Analysis of Human Leucocyte Cell Surface Antigens

1984 ◽  
pp. 621-630 ◽  
Author(s):  
N. L. Warner ◽  
L. L. Lanier ◽  
A. Jackson ◽  
G. Babcock ◽  
R. Evans
Keyword(s):  
Cell Surface ◽  
Surface Antigens ◽  
Facs Analysis ◽  
Cell Surface Antigens

Remarks on the application of backscattered electron imaging (BEI) to the scanning electron microscopy (SEM) of biological structures

1983 ◽  
Vol 41 ◽  
pp. 484-487
Author(s):  
Etienne de Harven
Keyword(s):  
High Resolution ◽  
Incident Beam ◽  
Spot Size ◽  
Primary Electron ◽  
Critical Point Drying ◽  
Cell Shapes ◽  
High Resolution Images ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Scanning Electron

Biological ultrastructures have been extensively studied with the scanning electron microscope (SEM) for the past 12 years mainly because this instrument offers accurate and reproducible high resolution images of cell shapes, provided the cells are dried in ways which will spare them the damage which would be caused by air drying. This can be achieved by several techniques among which the critical point drying technique of T. Anderson has been, by far, the most reproducibly successful. Many biologists, however, have been interpreting SEM micrographs in terms of an exclusive secondary electron imaging (SEI) process in which the resolution is primarily limited by the spot size of the primary incident beam. in fact, this is not the case since it appears that high resolution, even on uncoated samples, is probably compromised by the emission of secondary electrons of much more complex origin.When an incident primary electron beam interacts with the surface of most biological samples, a large percentage of the electrons penetrate below the surface of the exposed cells.

Backscattered Electron Imaging of GaAs/AlGaAs Super Lattice Structures with an Ultra-High- Resolution SEM

1988 ◽  
Vol 46 ◽  
pp. 204-205
Author(s):  
Kazumichi Ogura ◽  
Michael M. Kersker
Keyword(s):  
High Resolution ◽  
Layer Structure ◽  
High Sensitivity ◽  
Beam Current ◽  
Electron Beam Current ◽  
Lattice Structures ◽  
Super Lattice ◽  
Different Types ◽  
Backscattered Electron ◽  
Electron Imaging

Backscattered electron (BE) images of GaAs/AlGaAs super lattice structures were observed with an ultra high resolution (UHR) SEM JSM-890 with an ultra high sensitivity BE detector. Three different types of super lattice structures of GaAs/AlGaAs were examined. Each GaAs/AlGaAs wafer was cleaved by a razor after it was heated for approximately 1 minute and its crosssectional plane was observed.First, a multi-layer structure of GaAs (100nm)/AlGaAs (lOOnm) where A1 content was successively changed from 0.4 to 0.03 was observed. Figures 1 (a) and (b) are BE images taken at an accelerating voltage of 15kV with an electron beam current of 20pA. Figure 1 (c) is a sketch of this multi-layer structure corresponding to the BE images. The various layers are clearly observed. The differences in A1 content between A1 0.35 Ga 0.65 As, A1 0.4 Ga 0.6 As, and A1 0.31 Ga 0.69 As were clearly observed in the contrast of the BE image.

The distribution of antigen on the surface of RBL-2H3 rat basophilic leukemia cells observed by back-scattered electron imaging

1986 ◽  
Vol 44 ◽  
pp. 274-275
Author(s):  
R.F. Stump ◽  
J.R. Pfeiffer ◽  
JC. Seagrave ◽  
D. Huskisson ◽  
J.M. Oliver
Keyword(s):  
Cell Surface ◽  
Dynamic Properties ◽  
Leukemia Cells ◽  
Antigen Binding ◽  
Scattered Electron ◽  
Ige Receptor ◽  
Receptor Complexes ◽  
Rat Basophilic Leukemia Cells ◽  
Electron Imaging ◽  
Basophilic Leukemia

In RBL-2H3 rat basophilic leukemia cells, antigen binding to cell surface IgE-receptor complexes stimulates the release of inflammatory mediators and initiates a series of membrane and cytoskeletal events including a transformation of the cell surface from a microvillous to a lamellar topography. It is likely that dynamic properties of the IgE receptor contribute to the activation of these responses. Fewtrell and Metzger have established that limited crosslinking of IgE-receptor complexes is essential to trigger secretion. In addition, Baird and colleagues have reported that antigen binding causes a rapid immobilization of IgE-receptor complexes, and we have demonstrated an apparent increase with time in the affinity of IgE-receptor complexes for antigen.

A pre-embedding, protein a-gold immunolabelling technique for cytocentrifuged cell monolayers for lm and tem

1986 ◽  
Vol 44 ◽  
pp. 220-221
Author(s):  
K. Chien ◽  
I.P. Shintaku ◽  
A.F. Sassoon ◽  
R.L. Van de Velde ◽  
R. Heusser
Keyword(s):  
Cell Surface ◽  
Staining Method ◽  
Protein A ◽  
Surface Antigens ◽  
Cell Suspensions ◽  
Cellular Morphology ◽  
Cellular Phenotype ◽  
Cell Surface Antigens ◽  
Cell Monolayers ◽  
Diagnostic Histopathology

Identification of cellular phenotype by cell surface antigens in conjunction with ultrastructural analysis of cellular morphology can be a useful tool in the study of biologic processes as well as in diagnostic histopathology. In this abstract, we describe a simple pre-embedding, protein A-gold staining method which is designed for cell suspensions combining the handling convenience of slide-mounted cell monolayers and the ability to evaluate specimen staining specificity prior to EM embedding.

Preparation And elemental analysis of ancient fibers

1987 ◽  
Vol 45 ◽  
pp. 410-411
Author(s):  
Allen Angel ◽  
Kathryn A. Jakes
Keyword(s):  
Cross Sections ◽  
Physical Structure ◽  
Energy Dispersive ◽  
Archaeological Sites ◽  
X Ray ◽  
Long Term Storage ◽  
Backscattered Electron ◽  
Electron Imaging

Fabrics recovered from archaeological sites often are so badly degraded that fiber identification based on physical morphology is difficult. Although diagenetic changes may be viewed as destructive to factors necessary for the discernment of fiber information, changes occurring during any stage of a fiber's lifetime leave a record within the fiber's chemical and physical structure. These alterations may offer valuable clues to understanding the conditions of the fiber's growth, fiber preparation and fabric processing technology and conditions of burial or long term storage (1).Energy dispersive spectrometry has been reported to be suitable for determination of mordant treatment on historic fibers (2,3) and has been used to characterize metal wrapping of combination yarns (4,5). In this study, a technique is developed which provides fractured cross sections of fibers for x-ray analysis and elemental mapping. In addition, backscattered electron imaging (BSI) and energy dispersive x-ray microanalysis (EDS) are utilized to correlate elements to their distribution in fibers.

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