Nonlinear optical microscopy for ultrastructural bioimaging

本发现计划提出了非线性光学显微镜技术的进步及其在生物成像中的应用。将开发新的光学显微镜模式来研究成像结构的每个焦点体积内的有序分子结构的超微结构，超出衍射限制的空间分辨率。将发展基于纠缠光子与样品相互作用的非线性量子显微镜模式。显微成像也将使用经典的超短脉冲激光来获得振动和频率产生和受激拉曼散射图像对比，这为基于选定的分子振动的成像提供了化学特异性。偏光显微镜测量将使用我们团队最近发展的非线性Stokes-Mueller形式来研究生物样品的超微结构组织。超微结构显微镜将用于研究生物组织细胞外基质中的胶原组织、肌肉细胞中的肌球蛋白纳米运动组织以及肌肉收缩过程中纳米运动活动的同步性。还将研究淀粉颗粒、结晶纤维素和光合色素聚集体的超微结构。蛋白质、多糖和色素的有序分子组装在生物体中起着重要的结构和功能作用。例如，细胞外基质中胶原纤维的组织决定了组织的光学和机械性质，在横纹肌中组装成肌丝的肌球蛋白纳米运动器周期性地改变其构象以产生生物体运动的收缩，淀粉颗粒被组织在复杂的晶体结构中以有效地储存和释放碳水化合物，有序的色素聚集体被用于有效地收集太阳能。因此，开发非侵入性、化学特异性和无标记的成像工具来可视化是很重要的

Advancement of nonlinear optical microscopy technology and application for biological imaging is proposed in this Discovery program. Novel optical microscopy modalities will be developed for ultrastructural investigations of ordered molecular structures within each focal volume of the imaged structure, beyond the diffraction limited spatial resolution. Nonlinear quantum microscopy modalities based on the interactions of entangled-photons with the sample will be developed. The microscopic imaging will also be employed using classical ultrashort pulsed lasers for obtaining vibrational sum-frequency generation and stimulated Raman scattering image contrasts that provide chemical specificity for imaging based on the selected molecular vibrations. Polarimetric microscopy measurements will be employed using nonlinear Stokes-Mueller formalism recently developed by our group, to investigate ultrastructural organization of biological samples. The ultrastructural microscopy will be employed to study collagen organization in the extracellular matrix of biological tissue, myosin nanomotor organization in muscle cells and synchronization of nanomotor activity during muscle contraction. The ultrastructure of starch granules, crystalline cellulose and photosynthetic pigment aggregates will also be investigated. Ordered molecular assemblies of proteins, polysaccharides and pigments play important structural and functional role in the living organisms. For example, the organization of collagen fibers in the extracellular matrix determines optical and mechanical properties of the tissue, myosin nanomotors assembled into myofilaments in the striated muscles cyclically change their conformation to produce contractions for movement of the organisms, starch granules are organized in intricate crystalline structures for efficient storage and release of carbohydrates, and ordered pigment aggregates are used for efficient solar energy collection. Therefore, it is important to develop non-invasive, chemically-specific and label-free imaging tools to visualize