Tunable Label Contrast on the Cell Surface: Photoelectron Imaging with Multiple Wavelength Excitation

Abstract

Photoelectron imaging is a novel way of imaging the distribution of specific cell surface components. The basic principle is the photoelectric effect, in which electrons are ejected from the specimen by UV light. There are two primary forms of image contrast in photoelectron microscopy: material contrast and topographical contrast. Material contrast, provided by differences in photoelectron quantum yields, makes it possible to select labels that appear bright against the darker background of the cell surface. Topographical contrast ensures that label distribution can be interpreted in the context of cellular structures. By varying the wavelength of the exciting light, the contrast between label and cell surface can be controlled (tuned) to produce images ranging from a primarily topographical view of the surface to those showing only the label distribution. Being an emission-based technique, photoelectron imaging shares with fluorescence microscopy the ability to image label distributions at low magnification. However, unlike fluorescence microscopy the photoelectron image is formed by electrons, which generates a greater level of attainable resolution. Label contrast and wavelength-dependent tunability of contrast is illustrated with images of silver-enhanced immunogold-labeled cell surfaces, including selective labeling of cells in mixed-cell co-cultures, the cell surface CD44 adhesion protein on human glioma cells, fibronectin patterns on human fibroblasts, and the human transferrin receptor on MCF-7 human breast carcinoma cells.