Immunogold Labeling of Human Leukocytes for Scanning Electron Microscopy and Light Microscopy: Quantitative Aspects of the Methodology

When cell surface antigens are labeled with the colloidal gold marker, backscattered electron images (BEI) reveal all the gold particles and, therefore, permit total counts. Secondary electron images (SEI) show only a small percentage of the gold particles and are inadequate for quantitative evaluation. For determination of the cellular labelinq index, a time-consuming method implies the screening of 100 cells by scanninq electron microscopy, at a magnification of approximately 12,000 to 15,000x, with continuous SE/BE shifts. A much more efficient method is to transfer the SEM sample or its equivalent under the light microscope and to count the total number of gold labeled cells in the epi-polarization mode. The total cell count can be evaluated under UV light, taking advantage of the autofluorescence of the glutaraldehyde fixed cells

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 antileukocyte 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

Immuno-Cytochemistry with Backscattered Electrons

Some cytochemical reaction products are visible inside the cytoplasm of cells observed with the scanning electron microscope (SEM) using the backscattered electron imaging (BEI) mode. Methods can be utilized whenever they result in the deposition of heavy metal, like silver, lead or osmium at the sites of the enzymatic reaction. More recently the BEI mode of the SEM has been demonstrated to improve the detection of immunogold labeled cell surface antigens. Colloidal gold particles, 40 to 15 nm in diameter can be efficiently used for immuno-specific labeling. Moreover, cytochemical reactions can be applied to previously immunogold labeled cells, therefore combining the results of enzyme cytochemistry and of surface labeling at the level of each individual cell. The choice of fixative, incubation media, dehydration and drying methods should be guided by considerations on the sample characteristics for optimal electron scattering. Cytochemical as well as immuno-labeling reactions are not used per se but in combination with the study of cell surface morphology which needs, therefore, to be sufficiently well preserved. Coating should provide good conductivity and secondary electron emission, while emitting a minimal number of backscattered electrons. The application of these methods considerably enhances our capacity to characterize with the SEM the surface morphology of precisely identified subpopulations of many cell types

Human Chromosomes: Evaluation of Processing Techniques for Scanning Electron Microscopy

Methods for scanning electron microscopy (SEM) of chromosomes have been developed in the last two decades. Technical limitations in the study of human chromosomes, however, have hindered the routine use of SEM in clinical and experimental human cytogenetics. We compared different methodologies, including metal impregnation, air drying and specimen coating. SEM preparation of human chromosomes in which osmium impregnation is mediated by tannic acid, yielded more reproducible results when compared with osmium impregnation protocols previously described. The level of osmium impregnation was systematically evaluated by imaging chromosomes in the backscattering mode. Critical point drying and a light gold-palladium coating were essential for appropriate secondary electron imaging of chromosomes. With this method, and in a preliminary quantitative analysis, we show that our SEM technique is mere sensitive than light microscopy for the detection of aphidicolin-induced fragile sites. This technical approach is useful for chromosomal studies requiring resolution higher than that obtained by light microscopy. Also, it allows the use of clinical and archival chromosomal samples prepared by routine cytogenetic techniques

Scanning Electron Microscopy of Immuno-Gold Labeled Antigens Associated with Bladder Cancer

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 identified by the following murine monoclonal antibodies (mAbs): LEU-Ml, T16, 19A211, G4 and E7. In most cases labeling patterns were correlated with the surface features of the superficial urothelial cells as revealed in the secondary electron imaging (SEI) mode of the SEM. Results, to date, indicate that the immuno-gold labeling method is more sensitive than immuno-peroxidase, and that phenotypic heterogeneity of antigenic expression (or deletion) is a frequent observation of potential diagnostic or prognostic value

Cell Surface Changes of Hemopoietic Cells During Normal and Leukemic Differentiation: An Immuno-Scanning Electron Microscopy Study

Hemopoietic cells display a wide range of cell surface antigens which are either lineage specific or acquired during differentiation. Monoclonal antibodies can be used, in conjunction with colloidal gold markers, to identify under the scanning electron microscopy (SEM) at the single cell level, specific lineage or maturation stages in the hemopoietic bone marrow. Normal bone marrow cells, either gradient separated or purified by immuno-magnetic methods and leukemic cell samples, which can be considered as frozen stages of hemopoietic differentiation, have been studied with this method. Typical cell surface morphologies, which characterize immature progenitor cells and cells committed or differentiated towards the lymphoid, myeloid, erythroid and megakaryocytic lineage have been identified. Correlations between cell surface features and some hemopoietic cells functions have been attempted on the basis of these findings

Scanning Electron Microscope Cytochemistry of Blood Cells

The backscattered electron imaging (BEI) mode of scanning electron microscopy (SEM) has been applied to study various histo-cytochemical reactions in biological specimens since the early seventies. Due to numerous, recent technical improvements the BEI mode of SEM now belongs to the routine of many SEM laboratories. For cytochemistry, BEI has been mainly used to: visualize intracellular structures and organelles; recognize the different cell types in heterogeneous populations or tissues; study the correlations between enzymatic activities and cell surface features. We have evaluated the most relevant results obtained in the study of blood cells and the possible future applications of these techniques