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

杜克大学细胞和生物表面工程中心申请资金购买原子力显微镜（AFM）仪器，供中心内的教师共享使用。中心内目前没有可供共享使用的此类仪器。相当多的核心研究人员在其他机构使用了原子力显微镜。要求的数字仪器多模式/BioscopeTM AFM允许同时光学显微镜和AFM成像。我们建议通过将全内反射荧光显微镜（TIRFM）纳入其光学系统，进一步扩展BioscopeTM AFM的功能。该仪器将加强细胞与生物表面工程跨学科中心六名成员（来自四个部门）在以下研究项目中的研究：(1)表征晶体，二维细菌s层蛋白质阵列和由s层纳米模板引导的纳米模式蛋白质阵列；(2)阐明肌肉骨骼细胞机械信号转导的机制；(3)为多分析物集成光波导传感器设计的光模式抗体阵列的表征；(4)内皮细胞在新型微图案细胞粘附配体基质上附着和扩散过程的定量成像；(5) DNA芯片的表征：硅衬底上多路、空间定位的寡核苷酸阵列；(6) AFM分析运动蛋白；(7)建立一个生物物理模型，将蛋白质配体识别能量与原子力显微镜测量的分子间力联系起来。获得一台原子力显微镜也将显著增强杜克大学细胞和生物表面工程中心的教育使命。该中心最近获得了惠特克基金会颁发的特殊机会奖，以提高该项目在本科和研究生阶段的教育水平。该中心致力于开发创新的实验课程，利用现代实验技术来理解、量化和可视化分子和细胞水平上对生物现象至关重要的基本力量、压力和识别过程。原子力显微镜在生物相关环境中实时研究这些现象的能力，加上其相对易于使用，使其成为正在开发的新研究生课程的许多实验室组成部分的理想实验技术。

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.