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体的功能开辟了道路。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，可以在纳米范围内对分子进行静态和动态研究。然而，样品必须结合到表面的要求在很大程度上限制了动力学研究。用单光子灵敏荧光显微镜在单分子水平上实时观察分子络合物的动力学最近取得了突破性进展。然而，这项技术的解决方案受到光的波长的限制，并且不能揭示分子结构的细节。将AFM和单分子荧光显微镜结合在一起，将使我们能够通过AFM在分子水平上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，这种动力学可以用原子力显微镜对分子体系进行结构表征，并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生物显微镜AFM/荧光显微镜仪器的基础上建立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，还将确定并分析集成AFM的潜在陷阱以寻求解决方案。

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.