Photoelectron Microscopy: A New Approach to Mapping Organic and Biological Surfaces

Abstract

A general method of imaging organic and biological surfaces based on the photoelectric effect is reported. For the experiments, a photoelectron emission microscope was constructed. It is an ultrahigh vacuum instrument using electrostatic electron lenses, microchannel plate image intensifier, cold stage, hydrogen excitation source9 and magnesium fluoride optics. The organic surfaces examined were grid patterns of acridine orange, fluorescein, and benzo(a)pyrene on a Butvar surface. A biological sample, sectioned rat epididymis, was also imaged by the new photoelectron microscope. Good contrast was obtained in these initial low magnification experiments. These data demonstrate the feasibility of mapping biological surfaces according to differences in ionization potentials of exposed molecules. A number of technical difficulties, such as the intensity of the excitation source, must be solved before high resolution experiments are practical. However, it is probable that this approach can be useful, even at low magnifications, in determination of the properties of organic and biological surfaces.Spectroscopic labeling techniques are becoming increasingly useful in studies of membranes and other biological surfaces. Labeling or tagging with organic dye molecules has long been recognized as a useful approach (1). The techniques are, of course, becoming more refined and the useful region of the electromagnetic spectrum has been greatly extended. The common techniques now include fluorescence (2, 3), optical absorption (3), electron spin resonance (3, 4), and nuclear magnetic resonance spectroscopy (3, 5). All of these techniques can yield information regarding molecular motion and orientation of molecules, and the polarity of specific binding sites. However, these spectroscopic methods do not determine the positions of the labels or distinguish between surface and bulk properties of the specimen. This is especially troublesome when dealing with biological surfaces (e.g., cell surfaces, nerve endings, and membranes of organelles). Understanding mechanisms of drug action, cell adhesion, membrane structure, immunological responses, and loss of contact inhibition in malignant cells require a knowledge of the relative positions, environments, and population densities of binding sites on the surface. It is clear that new microscopic techniques are needed that can be combined with existing spectroscopic methods in studies of biological surfaces. It was to develop new microscopic techniques that we began several years ago to examine the photoelectric effect of organic and biological surfaces.A typical experiment is depicted in Fig. 1. The specimen is placed in a vacuum chamber and is then subjected to ultraviolet light. If the energy of the light source (ha) is sufficiently high, the sample surface can emit electrons (photoionize) as well as fluoresce. This intrinsic photoionization depends on the ionization potentials of various functional groups on or very near the surface. The process for individual molecules is desc...

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