) We will develop novel imaging technologies for real-time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g. DNA region, RNA region, protein), and to do so at relatively low cost. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. The general scheme is based on novel concepts for single-celled and single-molecule detection and characterization recently demonstrated in our laboratory. We are therefore in a unique position to respond to this particular RFA. Four distinct but interrelated goals are identified: (1) development of a diffusion-based single-molecule detection system which permits rapid and highly confident detection of hybridization to a DNA probe or binding to an antibody in the presence of a large excess of unhybridized probe molecules or free antibodies, respectively. This will lead to single-event homogeneous assays of molecular alterations of DNA and proteins, respectively, in biological tissues: (2) development of a microscale single-molecule electrophoresis system for the rapid detection and identification of specific targets in the presence of a large excess of similar species. This will extend single-molecule homogeneous assays to small molecules relevant to molecular profiling. It may even be possible to detect directly targeted species in biological tissues without introducing additional probes: (3) development of high-speed high-throughput cell screening protocols based on the above concepts and specialized data treatment software for rapid 'on-line"""""""" analysis of the images with an emphasis on confidence in target recognition and reduction in post-imaging data work-up; (4) demonstration of actual analysis of human cells and tissue samples. with outside collaboration, to evaluate and to validate the technology developed, and to further optimize the performance regarding speed, ruggedness and accuracy. Initially, we plan to target one model virus and one known region of a DNA.
