Combined Atomic Force and Total Internal Reflection Fluorescence Microscopy for Biological Research

点击翻译按钮获取中文摘要

The Center for Cellular and Biosurface Engineering at Duke University requests funding to purchase atomic force microscopy (AFM) instrumentation for the shared use of faculty within the Center. No such instrumentation currently exists within the Center for shared use. A significant number of the core investigators have used atomic force microscopes at other institutions. The requested Digital Instruments MultiMode/BioscopeTM AFM allows simultaneous optical microscopy and AFM imaging. We propose to further extend the capabilities of the BioscopeTM AFM by incorporating total internal reflection fluorescence microscopy (TIRFM) into its optics. This instrument will enhance the research of six members in the interdisciplinary Center for Cellular and Biosurface Engineering (from four departments) in the following research projects: (1) characterization of crystalline, two-dimensional bacterial s-layer protein arrays and nanopatterned protein arrays directed by the s-layer nanotemplates; (2) elucidation of the mechanism of mechanical signal transduction in musculoskeletal cells; (3) characterization of photopatterned antibody arrays designed for a multianalyte integrated optical waveguide sensor; (4) quantitative imaging of endothelial cell attachment and spreading processes on novel, micropatterned cell-adhesive ligand substrates; (5) characterization of a DNA chip: multiplexed, spatially-localized oligonucleotide arrays on silicon substrates; (6) analysis of motor proteins by AFM; and (7) development of a biophysical model to relate protein-ligand recognition energetics to AFM-measured intermolecular forces. The acquisition of an AFM will also significantly enhance the educational mission of the Center for Cellular and Biosurface Engineering at Duke University. The Center was the recent recipient of a special opportunity award from the Whitaker Foundation for educational enhancements to the program at both the undergraduate and graduate levels. The Center is committed to developing innovative laboratory classes that utilize contemporary experimental techniques to understand, quantify, and visualize the fundamental forces, stresses, and recognition processes at the molecular and cellular levels that are central to biological phenomena. The ability of the atomic force microscope to investigate these phenomena in a biologically-relevant milieu in real time combined with its relative ease of use makes it an ideal experimental technique for a number of the laboratory components of a new graduate course under development.