SGER - Atomic Force Microscopy System with Single Molecule Fluorescence Capabilites

单分子研究可以提供关于生物分子和细胞复合体的结构和动力学的独特信息，这是使用传统结构技术无法获得的。这些研究将在接近生理环境的条件下进行，为直接观察单个生物分子及其复合物的功能开辟了道路。特别是扫描探针显微镜和原子力显微镜（AFM）已被证明是一种非常有用的技术，可以在纳米范围内进行分子的静态和动态研究。然而，样品必须与表面结合的要求极大地限制了动力学研究。最近，利用单光子敏感荧光显微镜在单分子水平上实时观察分子复合物的动力学方面取得了突破。然而，这种技术的分辨率受到光的波长的限制，并且不能揭示分子的结构细节。AFM与单分子荧光显微镜（SMF）的结合将使我们能够在分子水平上通过AFM识别生物系统的结构细节，并使用SMF显微镜在单分子水平上直接观察实验系统的动力学。重要的是，动力学可以在由原子力显微镜表征的分子系统上进行，并且可以定位在相对较大的表面积上。因此，本课题的主要目标是在现有的Bioscope AFM/荧光显微镜的基础上构建AFM/SMF系统。荧光系统的灵敏度将使用专门设计的样品进行测试。此外，将识别和分析集成AFM的潜在缺陷，以寻求解决方案。

Single molecule studies can provide unique information on the structure and dynamics ofbiological molecules and cellular complexes that is impossible to obtain using traditionalstructural techniques. These studies, to be performed at conditions close to physiologicalenvironments, open the way for looking directly at the function of individual biologicalmolecules and their complexes. Scanning probe microscopy and atomic force microscope(AFM) in particular has proven to be a very useful technique permitting static and dynamicstudies of molecules at nanometer range resolution. However, the requirement for the sample tobe bound to the surface restricts considerably the dynamical studies. A breakthrough hasrecently been made in the real-time observation of dynamics of molecular complexes at thesingle-molecule level using single-photon sensitive fluorescence microscopes. However, theresolution of this technique is limited by the wavelength of the light and does not revealstructural details of molecules. The combination of AFM and a single molecule fluorescence(SMF) microscope into one instrument would enable us to identify structural details ofbiological systems at the molecular level by AFM and to use the SMF microscope to observedirectly the dynamics of an experimental system at a the single molecule level. Importantly, thedynamics can be performed on molecular systems structurally characterized by AFM andlocalized on a comparatively large surface area. Therefore, the major objective of this proposalis to build AFM/SMF system based on the currently available Bioscope AFM/fluorescencemicroscope instrument. The sensitivity of the fluorescent system will be tested using speciallydesigned samples. In addition, potential pitfalls of integrated AFM will be identified andanalyzed for solutions.