Identification of Biological Molecules in situ at High Resolution via the Fluorescence Excited by a Scanning Electron Beam

Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifiable in situ via the fluorescence excited by the focused electron beam of a scanning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular species-about 20 angstrom -is determined by the known maximum distance for molecular excitation by fast electrons. Direct extrapolation from an observed resolution of 900 angstrom in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 angstrom. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations.