One of the challenges in cancer research is determining the molecular events occurring at cell surfaces of normal and tumor cells that account for the differing cell-cell interactions, immunological properties, and altered rates of endocytosis. The purpose of this project is to develop and apply a new type of imaging technique based on the principle that cell surfaces and other components will photoemit electrons under the action of UV light. Photoelectron imaging (photoelectron microscopy or PEM) is the electron optics analogue of fluorescence microscopy and is capabe of extending the widely used immunofluorescence approach to much higher resolution. The mechanism of image formation is different from those of transmission or scanning electron microscopy, and the information content is complementary. In the past, this NCI grant has sponsored the development of the theory of photoelectron imaging, biophysical studies on the photoelectric effect, the first successful demonstration of photoelectron imaging of organic and biological samples, and the design and construction of the only photoelectron microscope in existence for biological studies. The major goal of the current renewal is to advance the status of photoelectron microscopy from that of an emerging technique to that of a research tool in cell biology. Within this context there are four aims: 1) to obtain improved photoelectron images of cell surfaces (e.g. PtK2 epithelial cells, human FS-2 fibroblasts, sperm cells) and to extend the imaging experiments to melanoma cells, correlating the results with scanning electron microscopy; 2) to examine the photoelectric behavior of cytoskeletal elements after removal of the plasma membrane in combined studies with immunofluorescence; 3) to continue the development of photoelectron markers based on the immunogold technique and to test this approach in studies of cell surface receptors involved in receptor-mediated endocytosis (e.g. human low density lipoprotein receptors), cell adhesion proteins (e.g. fibronectin), and tumor associated antigens; and 4) to carry out a parallel effort in instrument development, incorporating the new column, stage design, and UV system, and bringing the instrument up to the full resolution theoretically obtainable in photoelectron imaging using conventional electron optics.
Showing the most recent 10 out of 30 publications
