Tracking Individual Biomolecules via Fluorescence Modulation and Feedback

该奖项的研究目标是利用先进的控制工程方法，为反馈跟踪显微镜提供新的性能机制。 反馈跟踪显微镜是研究单个大分子动力学的一种强大的新技术，它已经开始为我们理解DNA等生物大分子做出重要的新贡献。在此领域的先前实验已被限制为相对大的分子的研究，并需要使用许多荧光染料来标记所研究的分子。 我们将在这个NSF奖项下追求的技术进步将使反馈跟踪显微镜应用于广泛的中等大小的分子成为可能，仅使用少数荧光染料进行标记。 我们将通过应用现代控制理论方法来设计改进的反馈跟踪算法，并通过开发先进的信号处理电子设备和运动控制硬件来实现这些进步。 这些成果将包括关于改进算法和仪器的出版物，以及以电子格式免费提供的更详细的技术文档。如果成功，这项研究的结果将使单分子生物物理学和生物化学的新实验成为可能，从而有可能将反馈跟踪显微镜应用于例如单个酶和核酶。 反馈跟踪可以大大提高荧光相关光谱（FCS）和荧光共振能量转移（FRET）等传统光学测量技术的灵敏度。 这是合理的，希望跟踪FCS和跟踪FRET测量单酶/核酶将加深我们的理解，随机动力学和持久的异质性，在这些重要类别的生物分子。 PI将把这些工作的结果纳入他的应用控制理论的入门研究生课程中，由NSF资助的研究生将受益于现代工程和生物物理学/生物化学界面的高度跨学科培训。

The research objective of this award is to utilize advanced methods of control engineering to enable a new performance regime for feedback tracking microscopy. Feedback tracking microscopy is a powerful new technique for studying the dynamics of individual macromolecules, which has already begun to make significant new contributions to our understanding of biological macromolecules such as DNA. Prior experiments in this area have been limited to the study of relatively large molecules and have required the use of many fluorescent dyes to label the molecule under study. The technical advances we will pursue under this NSF award will make it possible to apply feedback tracking microscopy to a wide range of molecules of moderate size using only a few fluorescent dyes for labeling. We will make these advances by applying modern methods of control theory to design improved feedback tracking algorithms, and through the development of advanced signal-processing electronics and motion-control hardware. Deliverables will include publications on the improved algorithms and apparatus, as well as more detailed technical documentation to be made freely available in electronic format.If successful, the results of this research will enable new experiments in single-molecule biophysics and biochemistry, making it possible to apply feedback tracking microscopy for example to individual enzymes and ribozymes. Feedback tracking can be used greatly to enhance the sensitivity of traditional optical measurement techniques such as fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET). It is reasonable to hope that tracking-FCS and tracking-FRET measurements on single enzymes/ribozymes will deepen our understanding of stochastic kinetics and persistent heterogeneity in these important classes of biomolecules. The PI will incorporate the results of such work in his introductory-graduate level course on applied control theory, and the graduate student funded by this NSF award will benefit from a highly interdisciplinary training at the interface of modern engineering and biophysics/biochemistry.