Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Abstract

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.

Scanning electron microscopy of cell surface antigens labeled with colloidal gold

“…We have recently demonstrated that gold particles of 13 nm in diameter can be resolved in the BE1 mode (JEOL JSM 840, with a LaBe cathode; Soligo and de Harven, 1985). Observations of immunogold labeling with the transmission electron microscope have already demonstrated that, by using gold particles of different sizes with a judicious choice of ligand, doubIe labeling experiments can be carried out (Bendayan, 1982).…”

“…It makes the labeling of those antigens that are excessively denatured by 0.2% glutaraldehyde (10 min) impossible. Our experience has shown that many surface antigens expressed on the surface of human leukocytes resist glutaraldehyde fixation under these conditions (Soligo and de Harven, 1985). It probably would be an oversimplification, however, to classify surface antigens in only two groups, "resistant" and "nonresistant."…”

Should we be counting immunogold marker particles on cell surfaces with the SEM?

“…Thus, BE rather than SE images must be studied for counting gold-labelled antigenic sites, and small particles must be used to minimize steric hindrance. We have recently shown that 20 nm (de Harven et al, 1984) and even 13 nm (Soligo & de Harven, 1985) gold particles can be resolved, using the solid state BE detector of a JEOL JSM 840 scanning electron microsocope equipped with a lanthanum hexaboride (LaB,) cathode. Although carbon coating does not completely prevent electrostatic charge, it permits accurate counting of gold particles in the BE1 mode of the SEM.…”

“…The choice o f accelerating voltage does not seem to be a very critical factor, although the resolution o f 13 n m gold particles (Soligo & de Harven, 1985) was achieved using 35 kV and the smallest available probe current ( 1 x 1 0 " A). The accelerating voltage and probe current must be adjusted for each sample to minimize the diffuse non-specific backscattered electron signal emitted by the irradiated cells.…”

Microfluidic device for a rapid immobilization of Zebrafish larvae in environmental scanning electron microscopy

“…Despite a large body of evidence using optical imaging for the characterization of zebrafish and Xenopus embryos on chip‐based devices, no attempts have been so far made to interface microfabricated chip‐based technologies with scanning electron microscopy (SEM). Because the SEM imaging is based on electron scattering at the surface rather than electron transmission, the analysis is fast and large amounts of samples can be analysed with a great depth of view . Such images are particularly appealing for small model organisms' morphological analysis by providing detailed 3D surface topographical information and yielding more information than the flat or reconstructed 3D images usually produced by confocal imaging.…”