"IDBR: TYPE A" Development of a High Resolution Fluorescence Force Microscope

埃默里大学被授予开发高分辨率荧光力显微镜(HRFFM)，以生成与当前方法相比在空间、取向、力和时间分辨率上高数量级的分子比力图。力是生命系统的核心，但在分子水平上研究它们的工具很少。开发基本的工具和仪器来表征和可视化物理力和生化信号之间的分子相互作用，对于全面了解生命系统中的信号通路至关重要。HRFFM仪器将通过绘制在空间、方向、力和时间分辨率上比当前细胞力成像方法高一个数量级的力来满足这些需求。研究、教育和推广工作将通过强调部队和光学显微镜而整合在一起。将与学生为科学组织K-12外展，这是一个将显微镜带到课堂上的项目，从而挑战学生可视化和探索“微观世界”。该计划将在埃默里大学的多个系扩大多学科教育，通过重组两门课程，将对生物科学和物理科学之间的相互作用感兴趣的学生聚集在一起。重要的是，将通过多种渠道向广大科学界传播关于HRFFM仪器的实用知识。首先，将开发一个力成像维基百科风格的网站，提供关于力成像的社区资源和教育信息。直接参与该项目的学生将在YouTube上发布和录制教学视频，主题包括：力芯片准备和偏振成像。还将发布分析程序、方案和参考资料，创建一个专注于细胞生物学中的分子力的互动社区。HRFFM仪器将在全国学术机构的研讨会和研讨会上展示，从而确保更广泛的细胞生物学和生物物理界充分认识到这种新工具的出现和实用。目前测量细胞力学的黄金标准-牵引力显微镜，是基于在拉伸下变形的聚合物表面。遗憾的是，牵引力显微镜固有的分子特异性、~1微米空间分辨率、~1 S时间分辨率和~1 NN力敏感性的缺乏掩盖了细胞机械转导的分子基础。例如，牵引力数据以nN/微米为单位报告；其灵敏度比测量分子力所需的分辨率低三个数量级。HRFFM仪器将解决这些缺点，并以PN范围的力分辨率、~ms的时间分辨率、~20 nm的空间分辨率、化学特异性和活细胞中的三维力矢量来测绘力。此外，HRFFM仪器将提供一个连接单分子FRET和超分辨率成像社区的公共平台，并在细胞生物学和单分子生物物理学领域之间建立独特的联系。该奖项由两个项目联合颁发：(1)生物基础设施部(生物科学局)的生物研究仪器开发，(2)物理部(数学和物理科学局)的生命系统物理学。

An award is made to Emory University to develop a high resolution fluorescence force microscope (HRFFM) to generate molecularly specific force maps orders of magnitude higher in spatial, orientation, force, and time resolution compared with the current methods. Forces are central to living systems, but few tools exist to investigate them at the molecular level. Developing the basic tools and instrumentation to characterize and visualize the molecular interplay between physical forces and biochemical signals is paramount in gaining a global understanding of signaling pathways in living systems. The HRFFM instrument will address these needs by mapping forces that are orders of magnitude higher in spatial, orientation, force, and time resolution compared with the current methods for cellular force imaging. The research, education, and outreach efforts will be integrated though their emphasis on forces and optical microscopy. K-12 outreach will be organized with Students for Science, a program that brings microscopes to the classroom, thus challenging students to visualize and probe the "micro- world". The program will expand multidisciplinary education across multiple departments at Emory University though restructuring of two courses that will bring together students interested in the interface between the biological and physical sciences. Importantly, practical knowledge about the HRFFM instrument will be disseminated to the broad scientific community through multiple channels. Firstly, a force imaging Wikipedia style website providing a community resource and educational information on force imaging will be developed. Students working directly on the project will post and record instructional YouTube videos on topics including: force chip preparation and polarization imaging. Analysis programs, protocols and references will also be posted, creating an interactive community focused on molecular forces in cell biology. The HRFFM instrument will be showcased at workshops and seminars across academic institutions nationally, thus ensuring that the broader cell biology and biophysics communities are fully aware of the advent and utility of this new tool.The current gold standard for measuring cellular mechanics, traction force microscopy, is based on polymer surfaces that deform under tension. Unfortunately, the lack of molecular specificity, ~1 µm spatial resolution, ~1 s time resolution, and ~1 nN force sensitivity inherent to traction force microscopy obscures the molecular underpinnings of cellular mechanotransduction. For example, traction force data is reported in units of nN/µm2; three orders of magnitude less sensitive than the resolution required for measuring molecular forces. The HRFFM instrument will address these shortcomings and map forces with pN-range force resolution, ~ms time resolution, ~20 nm spatial resolution, chemical specificity, and 3D force vectors in living cells. Moreover, the HRFFM instrument will provide a common platform bridging the single molecule FRET and super-resolution imaging communities, as well as creating a unique connection between the fields of cell biology and single molecule biophysics.This award is being made jointly by two Programs: (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Physics of Living Systems, in the Division of Physics (Math and Physical Science Directorate).