Photoelectron imaging of cytoskeletal elements

The established electron microscope techniques have richly contributed to the current understanding of cytoskeletal structure. The purpose of this paper is to demonstrate the kinds of images of cytoskeletal structures obtained by photoelectron microscopy (PEM or photoelectron imaging), which has only recently been applied utilizes the fact that specimens exposed to UV light emit electrons (the photoelectric effect). These electrons are accelerated and focused to provide a detailed image of the exposed biological surface. There are several interdependent factors involved in imaging the cytoskeletons of cells grown in culture. Since PEM is a surface technique a major consideration is exposure of the structure of interest. A second consideration is the preservation of the three-dimensional integrity of the structures during the specimen preparation, and a third is preservation of antigenicity so that specific structures can be directly identified by antibody labeling methods. The photoelectron images of Triton-extracted cells reveal a detailed filamentous network of cytoskeletal elements. Prefixation of cells with a crosslinking agent (DTSP) prior to Triton extraction has little or no effect on the final image, whereas brief prefixation with 0.025% glutaraldehyde preserves more of the surface lamina and cytoplasmic organelles such as mitochondria. A comparison of immunofluorescence and photoelectron micrographs of the same cells shows a large correspondence in the fibers observed, and demonstrates the higher resolution of PEM. Direct identification of specific cytoskeletal elements in the photoelectron micrographs is illustrated using microtubule distributions in CV-1 cells by antibody decoration or, more generally, by the use of photoemissive gold markers on antibodies directed against microtubules.