This proposal from an interdisciplinary (and interschool) Institute for Medicine and Engineering (IME) at the University of Pennsylvania is to purchase an atomic force microscope to become an integral part of microforce measurement and imaging laboratory. The PI and major users, who are members of 5 departments in 2 schools, represent an experienced group of investigators in molecular biomechanics and membrane biophysics. By placement in the IME, the instrumentation is formally committed to interdisciplinary research. No AFM suitable for biological research is available in Philadelphia area universities. This facility will provide: 1. Precise measurements of surface topography and force-related events in biological systems ranging from single molecules to intact cells, simultaneous with direct observation and quantitation of molecular dynamics through fluorophore reporter molecules. 2. Interdisciplinary research training opportunities for graduate students and postdoctoral fellows. The requested instrument is a Digital Instruments Nanoscope IIIa atomic force microscope system with Bioscope and Multimode tapping (in fluid) capabilities and fluorescence optical pathways. A recent modification devised by Yanagida for subpiconewton intermolecular force microscopy will be build into the system. House in a dedicated microscope room in the IME, the system will be used for: a. Topographic imaging of biological surfaces ranging from intact cell surfaces to isolated membranes at angstrom-nanometer resolution. b. The investigation of force interactions with cell surfaces and transmission to cell cytoskeleton. c. Intermolecular force measurements at the subpiconewton level in molecular motors; actin-myosin and kinesins. d. Cell-adhesion protein interactions. e. Investigation of cell surface deformation coincident with mechanotransduction (eg transmembrane ion flux). Optical pathways will permit simultaneous collection of quantitative fluorescence measurements during intact cell and single molecule manipulations, and the Yanagida refinement will permit subpiconewton force sensitivity.
