CAREER: Unlocking Deep-Tissue Dynamics with Ultrabroadband Multiplex Label-Free Microscopy

自几个世纪前问世以来，显微镜已经彻底改变了我们对生物世界的理解。无标记显微镜因其通过利用细胞固有的光学特征来研究生命系统的能力而脱颖而出。这消除了对染色剂的需要，从而保持生物样本的自然状态，并能够真实地表示动态细胞过程。然而，目前的无标记技术的范围受到分子灵敏度，特异性和成像深度之间的根本妥协的阻碍。本CAREER提案的目标是通过开发创新光源、激发方法和算法重建技术来克服这些障碍。该项目将开发新的光纤光源和成像平台，用于从活组织深处提取内在对比度，减轻扰动并增强分子对比度。这些进步有望实现物理学、生物学和生物医学工程的突破，直接影响到在自然环境中实时探索和诊断疾病。此外，该项目将利用麻省理工学院现有的推广计划，通过开发基于乐高的显微镜模块，为K-12学生和教师提供进入微观世界和了解成像工作原理的机会。教育和推广计划旨在激发好奇心，加深来自不同背景的学生的STEM素养，特别强调传统上在科学领域代表性不足的群体。通过将研究与教育相结合，该项目旨在扩大无标记成像的前沿领域，并培养一个更具包容性和全面性的未来科学社区。该CAREER提案的目标是建立一个专注于先进的无标记成像技术的研究计划，以提高疾病诊断和治疗评估的潜力。目前用于评估细胞代谢和组织变化的金标准方法，包括组织化学、荧光抗体标签和遗传编码成像探针，从根本上受到其物理、化学或遗传改变生物系统的必要性的限制。无标记非线性显微镜通过利用组织内固有的分子对比度来规避这些限制，提供活组织的实时代谢和结构成像而无需修改。然而，目前的无标记技术的范围受到分子灵敏度，特异性和成像深度之间的根本妥协的阻碍。 该项目旨在通过可见光到短波红外的创新实现无标签成像的变革能力（短波长红外）光产生和整形，具有三个方向：（1）开创用于深层组织无标记多路复用成像的高峰值功率、可调谐、可见光到短波红外光纤源;（2）利用超宽带光谱整形解决方案;（3）优化高通量超光谱无标记成像的激发和重建。实现这些成像能力将是我们长期努力提供无标签成像技术的一个重要里程碑，这些技术可以与基于标签的成像技术相媲美或超越基于标签的成像技术，从而推进了我们对动态生物过程的表征和理解，使扰动最小，并具有立即临床转化的潜力。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的学术价值和更广泛的影响审查标准。

Microscopy has revolutionized our understanding of the biological world since its inception centuries ago. Label-free microscopy stands out for its ability to investigate living systems by capitalizing on the inherent optical signatures of cells. This eliminates the need for staining agents, thereby preserving the natural state of biological specimens and enabling an authentic representation of dynamic cellular processes. However, the reach of current label-free techniques is hindered by a fundamental compromise between molecular sensitivity, specificity, and imaging depth. The objective of this CAREER proposal is to overcome these barriers through the development of innovative light sources, excitation methods, and algorithmic reconstruction techniques. This project will develop new fiber optic sources and imaging platforms tailored for intrinsic contrast extraction from deep within living tissues, mitigating perturbation and enhancing molecular contrast. Such advancements promise to enable breakthroughs across physics, biology, and biomedical engineering, with direct implications for the real-time exploration and diagnosis of diseases in their native environments. In addition, this project will leverage existing outreach programs at MIT to provide K-12 students and teachers with access to the micro-world and understanding of the working principles of imaging by developing LEGO-based microscope modules. The education and outreach program are designed to spark curiosity and deepen STEM literacy among students from diverse backgrounds, with particular emphasis on engaging groups traditionally underrepresented in the sciences. By connecting research with education, this project aims to not only expand the frontiers of label-free imaging but also to cultivate a more inclusive and well-rounded future scientific community.The goal of this CAREER proposal is to establish a research program focusing on advanced label-free imaging technologies, with the potential to enhance disease diagnosis and therapeutic assessment. Current gold-standard methods for evaluating cellular metabolism and tissue changes, including histochemistry, fluorescent antibody tags, and genetically encoded imaging probes, are fundamentally limited by their necessity to physically, chemically, or genetically alter the biosystems. Label-free nonlinear microscopy circumvents these limitations by harnessing intrinsic molecular contrast within the tissue, offering real-time metabolic and structural imaging of living tissue without modifications. However, the reach of current label-free techniques is hindered by a fundamental compromise between molecular sensitivity, specificity, and imaging depth. This project seeks to enable transformative capabilities in label-free imaging through innovations in visible-to-SWIR (short-wavelength infrared) light generation and shaping, with three directions: (1) pioneer a high-peak-power, tunable, visible-to-SWIR fiber source for deep-tissue label-free multiplex imaging; (2) leverage ultrabroadband spectral shaping solutions; (3) optimize excitation and reconstruction for high-throughput hyperspectral label-free imaging. Achieving these imaging capabilities will represent a major milestone in our long-term effort of delivering label-free imaging technologies that rival or surpass the capabilities of label-based imaging, thereby advancing our characterization and understanding of dynamic biological processes with minimal perturbation and immediate clinical translation potential.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.