High Performance Terahertz Spectroscopy Systems for Advanced Biological Studies

近场显微镜系统，如扫描隧道显微镜、原子力显微镜、扫描光学显微镜和电子显微镜，可以获得生物样品非常详细的结构信息。然而，它们的光谱和操作限制了它们在研究生物过程动力学方面的应用。替代的单分子方法使用不同的标记技术来表征生物分子的结构动力学。标记活性部位附近的生物分子可能是一项重大挑战，可能会影响许多生物过程中分子的自然行为。为了解决这些局限性，本项目提出了一种创新的扫描太赫兹纳米显微镜系统，作为一种强大的无标记生物学研究工具，以促进生物物理学的研究。建议的无标记探测系统将允许研究生物分子在自然条件下的复杂行为，同时避免对标记反应进行详尽的遗传和生化表征。该系统通过全封装和光纤耦合的平台，为实际环境中的生物学研究提供了极大的灵活性(大面积扫描、同时光激发和添加外部化学刺激的灵活性)。因此，建议的研究将有益于开展单分子生物物理、细胞结构、纳米医学、蛋白质折叠等研究的生物研究社区。作为我们传播的一部分，我们与生物研究社区成员密切合作，利用和评估开发的太赫兹光谱系统用于各种生物学研究。为了吸收从事这种跨学科研究的研究生，建立了一个专门的培训计划，为这些研究活动招募本科生和暑期实习生，并特别优先从代表性不足的群体中招聘有才华的本科生和研究生。太赫兹波为生物分子的无标记表征和研究生物系统的结构、动力学和操作提供了前所未有的功能。这是因为太赫兹光子能量可以与重生物分子中分子的低结合能相媲美，为区分蛋白质和通过太赫兹光谱提供关于其构象状态的信息提供了一个平台。此外，由于生物分子的不同太赫兹特征依赖于它们的分子间和分子内的振动和旋转，太赫兹光谱可以研究活细胞及其在各种生物系统中的相互作用，包括细胞的新陈代谢和繁殖，以及通过细胞膜从环境到细胞的化学转移和可能的构象变化。此外，由于太赫兹光谱可以捕捉飞秒尺度的动态变化，它非常适合于研究蛋白质重排、折叠和与其他生物分子结合过程中的分子运动动力学。尽管太赫兹技术前景看好，但由于现有太赫兹光谱分析系统的低灵敏度和有限的空间分辨率，太赫兹技术在生物学研究中的范围和潜在用途仍然受到限制。拟议的纳米显微镜系统解决了这两个限制，同时与最先进的系统相比，提供了显著更高的灵敏度和带宽。该系统由等离子体光导太赫兹源和电光晶体组成，分别用于宽带太赫兹波的产生和探测。等离子体太赫兹源和电光晶体与一种新型的太赫兹探头集成在一起，该探头包括一个锥形波导，用于将产生的太赫兹光束聚焦到具有纳米焦距的生物样品上，并将从样品反射的太赫兹光束耦合到用于检测的电光晶体上。太赫兹探测器旨在允许在纳米级进行太赫兹光谱分析，而不会对光谱带宽产生重大影响。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Near-field microscopy systems such as Scanning Tunneling Microscopy, Atomic Force Microscopy, Scanning Optical Microscopy, and Electron Microscopy can yield highly detailed structural information of biological samples. However, their spectral and operational constraints have limited their use for studying dynamics of biological processes. Alternative single-molecule approaches use various labeling techniques for characterizing structural dynamics of biomolecules. Labeling biomolecules near their active sites can be a major challenge and can affect the natural behavior of molecules in many biological processes. To address these limitations, this project proposes an innovative Scanning Terahertz Nanoscopy system as a powerful label-free biological study tool to advance the research in biophysics. The proposed label-free probing system would allow studying the complex behavior of biomolecules under native conditions, while avoiding exhaustive genetic and biochemical characterization of labeling reactions. The proposed system offers significant flexibility for biological studies in practical settings (flexibility in large area scanning, simultaneous optical excitation, and adding external chemical stimuli) through a fully packaged and fiber-coupled platform. Therefore, the proposed research would benefit the biological research communities conducting research on single-molecule biophysics, cellular structure, nanomedicine, protein folding, etc. As a part of our dissemination, we work closely with the members of the biological research community to utilize and evaluate the developed terahertz spectroscopy system for various biological studies. A special training program is constructed to assimilate graduate students working on this type of interdisciplinary research, undergraduate students and summer interns are recruited for these research activities and special priority is given to recruitment of talented undergraduate and graduate candidates from underrepresented groups. Terahertz waves offer unprecedented functionalities for label-free characterization of biomolecules and studying the structure, dynamics and operation of biological systems. This is because terahertz photon energies are comparable with the low binding energies of molecules inside heavy biomolecules, offering a platform for differentiating proteins and providing information about their conformation states through terahertz spectroscopy. Additionally, since distinct terahertz signatures of biomolecules are dependent on their intermolecular and intramolecular vibrations and rotations, terahertz spectroscopy enables investigating living cells and their interaction inside various biological systems including cell metabolism and reproduction as well as chemical transfer from the environment to cell through cell membrane and possible conformational changes. Moreover, since terahertz spectroscopy can capture femtosecond-scale dynamic variations, it is very well suited for investigating kinetics of molecular motions during protein rearrangement, folding, and binding to other biomolecules. Despite its great promises, the scope and potential use of terahertz technology for biological studies is still limited by low sensitivity and limited spatial resolution of existing terahertz spectroscopy systems. The proposed nanoscopy system solves both limitations, while offering significantly higher sensitivities and bandwidths compared to the state-of-the-art. The proposed time-domain terahertz spectroscopy system consists of a plasmonic photoconductive terahertz source and an electro-optic crystal for broadband terahertz wave generation and detection, respectively. The plasmonic terahertz source and the electro-optic crystal are integrated with a novel terahertz probe, which consists of a tapered waveguide used for focusing the generated terahertz beam onto the biological sample with nanoscale focus dimensions and coupling the reflected terahertz beam from the sample to the electro-optic crystal for detection. The terahertz probe is designed to allow terahertz spectroscopy at the nanoscale without a considerable impact on the spectral bandwidth.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.