Excitation under the scanning electron microscope of DNA‐associated fluorescence from chicken erythrocyte nuclei, polytene chromosomes and adenovirus 2 virions

Abstract

Biological structures not seen by conventional light microscopy, such as longitudinal striations in polytene chromosomes, and, at the limit of sensitivity, virions of adenovirus 2, have been detected via DNA-associated fluorescence excited under the scanning electron microscope. The maximum sensitivity realized, about 1 detected photon per 700 base pairs, falls short by about an order of magnitude of that required to achieve, in unreplicated specimens, the 2 nm intrinsic resolution of the method. A combination of DzO-HzO substitution with freezedrying provides the best unquenching procedure found for in situ DNA.DNA-associated fluorescence for light microscopy can be created by moderate exposure of the specimen in the electron microscope. I N T R O D U C T I O NSince the demonstration of electron-induced fluorescence (or cathodoluminescence, CL) in thioflavin T-stained spinach leaf by Pease & Hayes (1966), a small number of investigators in North America and Europe have attempted to develop and use the fluorescence of stained or unstained biological material as a contrast mechanism in scanning electron microscopy. Manger & Bessis (1970) reported CL from cells and proteins treated with paraformaldehyde (although, in the absence of spectra, alternative interpretations of their data are possible). Muir et al. (1971) presented CL images of fungal spores and CL spectra from unfixed and glutaraldehydefixed mammalian cells. DeMets and co-workers (1971, 1974) examined the CL and CL-spectra of selected organic compounds while Falk (1972) and Ong et al. (1973) used CL to study organic herbicide dispersal patterns on plant leaves. Horl (1972) developed a new detector system (incorporating a half-ellipsoidal mirror) and applied it in the study of adrenal and lung tissues in thick section. He obtained space distributions of intrinsic (auto-) fluorescence which varied with the wavelength band selected.Bond et al. Haggis et al. (1976) introduced improved light collection by somewhat different techniques and also attacked a significant source of background, namely light from the electron source. These authors studied intrinsic fluorescence in thick sections of Tradescantia and Eucalyptus, and also the fluorescence of 'Calcofluor White' (a laundry brightener) used as a stain for whole-mount preparations of yeast and thin sections of wheat seed. Soni et al. (1975) used CL to detect capping in mouse B lymphocytes induced by fluorescein-labelled antibody made in goat against mouse IgG. In the last two publications cited, resolutions approaching the limit of the light microscope were demonstrated. These authors, as well as Muir and coworkers and Hayes and co-workers, emphasize the need to guard against apparent CL images which are really produced by electron scattering with production of light elsewhere. Barnett et al. (1975a, b) made brief observations of intrinsic fluorescence and the fluorescence