High Speed Atomic Force Microscopy for Real Time Imaging of Biological Processes

摘要在显微镜的所有形式中，显微镜是研究生物结构和功能的最广泛使用的工具。具有亚秒级时间分辨率和纳米级分辨率的生命和运动生物系统的研究在生物学研究中正变得越来越重要。需要对动态过程进行更快、高分辨率成像的生物学研究的一个关键例子是通过酶、化学和物理过程降解纤维素材料。改善纤维素降解是高效生产乙醇的重要一步，对于发展适合运输的可持续燃料供应来说，很可能对国家至关重要。这个项目是开发一个基于原子力显微镜(AFM)的系统，用于生物样品的纳米级成像，比目前的AFM快几个数量级。该项目将为动态生物过程的实时和高分辨率成像开发新的方法和仪器。该项目解决了原子力显微镜研究界的一个关键限制，同时建立在当前高速原子力显微镜系统的进步之上。这种快速扫描原子力显微镜的技术进步是一种主动、自驱动的微悬臂。该系统还将利用一种新的钝化方案，在生物成像中遇到的所有液体环境中提供稳健的操作。此外，有源探头的尺寸将被微型化，以保持快速的动力学和精细的操作。这一快速扫描系统的应用将是对里氏木霉纤维素酶降解纤维素的实时成像。该系统将可视化降解机理，并确定这一过程的动力学。这项研究的更广泛影响将是从根本上改进生物学和生物技术领域的科学家和工程师可用的表征方法。该项目将使全国各地显微镜实验室中运行的高速扫描系统成为可能，生物学家可以在那里利用视频速率原子力显微镜获得动态生物过程的最快和最高分辨率的图像。该显微镜还将能够对脂膜、蛋白质和分子马达、细胞和其他生物系统的表面特征进行高速分子识别。第一步将是研究纤维素和纤维素酶之间的动力学相互作用，目的是提高纤维素乙醇的产量。这些进步的首要任务是生产可转移到其他商业AFM系统的技术，直接实现在各种研究和行业实验室进行的扫描探头研究。有了该项目的贡献，动态纳米级成像和操作可以扩展到视频率，以类似于扫描电子显微镜或视频光学显微镜的方式。拟议的计划还将为学生创造教育机会，通过专注于研究生培训、本科生参与和K-12外展努力，了解物理科学、生物科学和纳米技术之间的联系。

AbstractIn all of its forms, the microscope is the most widely used tool in the investigation of biological structure and function. The study of living and moving biological systems, with sub-second time scale resolution and with nanometer length scale resolution, is becoming increasingly important in biological research. A critical example of biological research that requires faster, high resolution imaging of dynamic processes is the degradation of cellulosic materials by enzymatic, chemical, and physical processes. Improved cellulose degradation is an important step to efficient ethanol production and will likely be critical to the nation for development of a sustainable supply of fuel suitable for transportation. This project is to develop a system, based on the Atomic Force Microscope (AFM), for nanometer scale imaging of biological samples that is several orders of magnitude faster than current AFMs. The project will develop both new methodologies and instrumentation for the real-time and high-resolution imaging of dynamic biological processes. The project addresses a critical limitation for the AFM research community, while building upon the advances of current high-speed AFM systems. The technological advancement of this fast-scanning AFM is an active, self-actuating microcantilever. The system will also utilize a novel passivation scheme to provide robust operation in all liquid environments encountered in biological imaging. Moreover, the dimensions of the active probe will be miniaturized, to retain fast dynamics as well as delicate operation. The application of this fast-scanning system will be the real-time imaging of the degradation of cellulose by the cellulase enzyme from the fungus Trichonderma reesei. This system will visualize the mechanism of degradation and determine the kinetics of this process. The broader impact of this research will be to radically improve the characterization methods available to scientists and engineers working in biology and biotechnology. High speed scanning systems operating in microscopy labs across the country, where biologists could utilize video rate atomic force microscopy to obtain the fastest and highest resolution images of dynamic biological processes, would be enabled by this project. The microscope will also be capable of high speed molecular recognition for surface characterization of lipid membranes, proteins and molecular motors, cells and other biological systems. The initial step will be an investigation of the kinetic interactions between cellulose and cellulases, with the aim of improving production of cellulosic ethanol. An overarching priority for these advancements is to produce technology that is transferable to other commercial AFM systems, directly enabling the scanning probe research conducted at various research and industry labs. With the contributions of this project, dynamic, nanoscale imaging and manipulation can be extended to video rates, in a manner akin to scanning electron microscopy or video optical microscopy. The proposed program will also create educational opportunities for students to learn about the interface between physical science, biological science, and nanotechnology by focusing on graduate training, undergraduate involvement, and K-12 outreach efforts.