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。 反馈跟踪显微镜是一种用于研究单个大分子动力学的强大新技术，它已经开始为我们对 DNA 等生物大分子的理解做出重大的新贡献。该领域之前的实验仅限于研究相对较大的分子，并且需要使用许多荧光染料来标记所研究的分子。 我们将在该 NSF 奖项下追求的技术进步将使仅使用少量荧光染料进行标记即可将反馈跟踪显微镜应用于各种中等大小的分子。 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取得这些进步。 可交付成果将包括有关改进算法和设备的出版物，以及以电子格式免费提供的更详细的技术文档。如果成功，这项研究的结果将促成单分子生物物理学和生物化学的新实验，使得将反馈跟踪显微镜应用于单个酶和核酶等成为可能。 反馈跟踪可极大地提高传统光学测量技术的灵敏度，例如荧光相关光谱（FCS）和荧光共振能量转移（FRET）。 我们有理由希望对单一酶/核酶的追踪 FCS 和追踪 FRET 测量将加深我们对这些重要生物分子类别的随机动力学和持久异质性的理解。 PI 将把这些工作的成果纳入他的应用控制理论研究生入门课程中，由 NSF 奖项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.