Letter | Published:

Ultrastructure of Human Leucocytes synthesizing DNA

Naturevolume 204page894 (1964) | Download Citation



THE application of autoradiographic techniques in the localization of radioactively labelled compounds in cells by electron microscopy has been developed by a number of workers1. The direct coating of ultra-thin sections with photographic emulsion has the disadvantage that long exposure times are required to obtain positive autoradiographs. Furthermore, it is difficult to locate labelled cells in the electron microscope by this method when their frequency is low. In the experiments recorded here the characteristics of the ultrastructure of cells in DNA synthesis were examined by using a ‘thick–thin’ technique2. Two examples of proliferation in atypical lymphocytes were investigated by this method: normal human lymphocytes cultured with phytohæmagglutinin3–5, and freshly isolated leucocytes from patients suffering from infectious mononucleosis (glandular fever). The cells were incubated with thymidine (3HT) (10 mc./ml., specific activity 1.9 c./mmole) for 30 min and then fixed with osmic acid and embedded in methacrylate. A thick section (about 0.25µ) was cut from a suitable part of the block and mounted on a glass slide. The contiguous ultra-thin section was then cut and mounted on an electron microscope grid. The methacrylate was removed from the thick section with chloroform and the section coated with Ilford L4 emulsion and stored at 4° C. Three weeks later it was developed in Kodak D19B and stained with toluidine blue. A map of the whole section was made from a mosaic of light photomicrographs and the cells with positive autoradiographs noted. This map was used to locate the position of the DNA-synthesizing cells in the ultra-thin section, which were then examined and photographed in the electron microscope.

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  1. Medical Unit, St. Mary's Hospital, W.2

    • E. H. COOPER
    •  & D. R. INMAN
  2. Electron Microscope Unit, Imperial Cancer Research Fund, London, W.C.2

    • E. H. COOPER
    •  & D. R. INMAN


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