Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Abstract

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 speciesabout 20 A-is determined by the known maximum distance for molecular excitation by fast electrons. Direct extrapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. 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.The fluorescence of proteins, nucleic acids, and fluoresceincoupled antibody are excited not only by ultraviolet light, but also by fast electrons. The electron effect is localizedindividual fluorescing groups (or fluorophors), such as the tryptophan and tyrosine residues of protein (1) MATERIALS AND METHODS Scanning Electron Microscope. The microscope used in this work is the AMR 1000 (Advanced Metals Research Corporation, Waltham, Mass.). The electron source is thermionic, and high vacuum is provided by an oil diffusion pump with liquid nitrogen trap. Light from the microscope filament normally gives a serious background for the photon detector used to detect fluorescence; the background is reduced by misaligning the filament mechanically and restoring a portion of the lost beam current by adjustment of the electromagnetic alignment currents. The measurements reported below were made at 20 kV accelerating voltage. Beam currents on target ranged from 5 X 10-13 amp (= 3 X 106 electrons/sec) to 1 X 10-9 amp ( = 6 X 109 electrons/ sec). The specimen-support stub was maintained at room temperature (220).Photon Detector. An aluminum mirror in the shape of a half-paraboloid (6) with axis horizontal covers the specimen. The electron beam of the microscope traverses a vertical hole in the mirror and intersects the specimen at the focus of the paraboloid. Fluorescent light collected by the mirror is passed to a Pyrex tube, aluminized on the inside, which conducts it to a quartz window at the wall of the vacuum chamber. Outside, the light is dispersed by a Jarrell-Ash 0.25 m grating monochromator and a selected wavelength band is detected by an EMI 9789QB bi-alkali photomultiplier tube placed at the exit slit. The complete light collection and detector system has an overall theoretical efficiency of 2%, bu...

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

“…la and lb.) At 30 kV, using the formula UD = A/IT for the destruction cross-section (see Hough et al, 1976) we get UD = 3 x 10-5 nm2, in good agreement with our earlier estimates. The destruction cross-section is expected to vary inversely with the microscope voltage, and such a variation is at least approximately shown by Fig.…”

“…A convenient and comprehensive bibliography of cathodoluminescence has been published by Brocker & Pfefferkorn (1978). Hough et al (1976) set up a mirror system similar to that of Horl, but with the figure of a full ellipsoid of resolution and with possibly a superior optical surface (McKinney & Hough, 1977). Intrinsic fluorescences from biochemically purified, bulk samples of aromatic amino acids, protein and DNA were studied in some detail with particular attention to the destruction rate of the fluorescence and the evolution of the fluorescence spectra under the beam .…”

Carboxylic monolayer formation for observation of intracellular structures in HeLa cells with direct electron beam excitation-assisted fluorescence microscopy

“…We observed the auto-fluorescence of the intracellular structures with the D-EXA microscope. As an important next research topic, we need to measure the spectrum of autofluorescence of cells, and the biomolecules excited by the D-EXA microscope can be identified with comparison of the cathodoluminescence spectra of biological molecules [9,10]. The damages of carboxylic monolayer film with the irradiation of electron beam have not been observed during our experiments.…”

Immuno-Fluorescence Scanning Electron Microscopy of Biological Cells

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