Nonlinear optical microscopy for ultrastructural bioimaging

介绍了非线性光学显微镜技术及其在生物成像中的应用。新的光学显微镜模式将被开发用于在成像结构的每个焦点体积内有序分子结构的超微结构研究，超出衍射限制的空间分辨率。基于纠缠光子与样品相互作用的非线性量子显微镜模式将被开发。显微成像也将使用经典的超短脉冲激光器来获得振动和频率产生和受激拉曼散射图像对比，为基于所选分子振动的成像提供化学特异性。偏振显微镜测量将采用我们小组最近开发的非线性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。淀粉颗粒、结晶纤维素和光合色素聚集体的超微结构也将进行研究。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结构决定了组织的光学和机械性能，肌球蛋白纳米马达在横纹肌中组装成肌丝，循环地改变其构象，以产生生物体运动的收缩，淀粉颗粒以复杂的晶体结构组织，以有效地储存和释放碳水化合物，有序的色素聚集体用于有效地收集太阳能。因此，开发无创、化学特异性和无标记的成像工具来可视化体内生物结构和揭示分子组装的纳米级超微结构组织是很重要的。结构信息对生物物理学、生物医学和植物生物学研究人员、病理学家和生物纳米技术研究人员很重要。关于生物组织中特定分子群组织的超微结构信息将验证从以前的非线性显微镜研究中得出的结构模型。这些新技术将促进新的体内组织成像工具的发展，为研究肌肉疾病提供新的方法，促进植物太阳能转换和储存的研究，并应用于人工光合作用、太阳能电池的开发和仿生纳米技术。

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 in vivo biological structures and reveal the nanoscopic ultrastructural organization of the molecular assemblies. The structural information is important for biophysics, biomedical and plant biology researchers, pathologists, and bio-nanotechnology investigators. The ultrastructural information about the organization of specific molecular groups in biological tissue will validate the structural models drawn from previous nonlinear microscopy studies. The novel techniques will stimulate the development of new in vivo tissue imaging tools, provide new methods to study muscular disorders, and facilitate investigations in solar energy conversion and storage in plants, with applications to artificial photosynthesis, solar cell development and biomimetic nanotechnology.