BRIGE: Towards the in-situ analysis of the assembly of structural proteins by multi-photon optical image guided spectroscopy

0927297 LyubovitskNSF Brige项目的目的是启动一项综合研究和教育计划，以了解复合生物材料开发过程中纤维蛋白就地组装和分解反应的机制。研究目标是发展一种新的多光子光学显微镜方法(MPM)，该方法将二次谐波(SHG)和双光子荧光(TPF)信号的非线性光学现象与拉曼光谱相结合。这种能力可以对生物学研究产生变革性的影响--特别是对生物制造中蛋白质组装的理解。智力上的优点。了解蛋白质组装的机制在各种生物医学应用中具有重要的意义和实用价值。蛋白质组装的复杂过程包括形成从埃到微米和更高维度的结构元素。研究分子尺寸等小尺度结构的最佳技术是利用与这些结构大小相当的波长，如X射线和电子。由形成高阶组装的纤维组成的蛋白质可以有效地使用光学显微镜方法进行研究，这种方法使用光谱学作为对比源。蛋白质组装知识已经存在于化学和生物领域。然而，在这些学科中，它仅限于理解分子现象。非线性光学和光谱方法与无损显微镜相结合，非常适合于三维定量跟踪蛋白质的原位组装和拆解，具有高的空间分辨率和实时对比度。由于对光信号的分子来源及其灵敏度的认识不足，这些技术的实用性受到限制。PI正在发展一种新的多光子光学显微镜方法(MPM)，该方法将二次谐波(SHG)和双光子荧光(TPF)信号的非线性光学现象与拉曼光谱相结合来研究蛋白质的自组装。TPF是通过吸收蛋白质中的氨基酸和交联键等UV/Vis部分而产生的。二次谐波产生信号(SHG)是由没有对称中心的结构产生的，例如纤维蛋白胶原蛋白。拉曼光谱的集成提供了与蛋白质基材料自组装相关的属性的更多信息，如化学成分、结晶度、构象、密度、单体含量、降解和应力-应变分布。更广泛的影响。这项研究和教育计划的绩效成果将包括设计和实施多模式光学显微镜和图像引导光谱平台，作为一种非侵入性工具，实时、原位地监测和了解蛋白质衍生生物材料和粘合剂的结构、组成和功能。开发的实验方法和获得的知识可以作为反馈系统的一部分，为活的工程组织、植入物和新型粘合剂的设计、构建、修改、生长和维护建立科学原则。因此，该项目将使组织工程、生物材料和生物医学工程社区受益。NSF布里奇奖将使PI能够将一些教育和外展活动与研究相结合。她将为选定的K-12学生和教师以及本科生和研究生提供实验室研究机会。她将为研究生和高级本科生开发新课程，要求学生深入了解与光学技术及其生物应用相关的主题。不同年龄段的学生和普通公众将更好地了解不同领域如何相互作用，以产生对蛋白质材料组装机制的高影响工程研究。此外，PI将扩大初步努力，接触年轻女性，并展示她们如何追求非传统的科学和技术职业，以及如何平衡苛刻的工作和家庭需求，以成功地生活并为公众福祉做出重要贡献。

0927297LyubovitskyThe purpose of this NSF BRIGE project is to initiate an integrated research and educational program to understand the mechanisms of fibrous protein assembly and disassembly reactions in-situ during composite biomaterial development. The research objective is the development of a new multi-photon optical microscopy method (MPM) that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals integrated with Raman spectroscopy. Such a capability can have a transformative impact on biological research - specifically the understanding of protein assembly for biomanufacturing. INTELLECTUAL MERIT. Understanding the mechanisms of protein assembly is of fundamental interest and practical importance in diverse biomedical applications. The complex process of protein assembly involves formation of structural elements that range from Angstroms to microns and higher dimensions. Structures on a small scale such as molecular dimensions are best studied with techniques that utilize wavelengths comparable to the size of those structures, such as X-rays and electrons. Proteins composed of fibrils that form higher order assemblies can be effectively investigated using optical microscopy methods that use spectroscopy as a source of contrast. Protein assembly knowledge already exists in chemical and biological fields. In these disciplines, however, it is limited to understanding the molecular phenomena. Non-linear optical and spectroscopic methods combined with non-destructive microscopy are ideally suited to quantitatively follow in situ assembly and disassembly of proteins in three dimensions, with high spatial resolution and contrast in real time. The usefulness of these techniques is limited by deficient comprehension of the molecular sources of the optical signals and their sensitivities.The PI is developing a new multi-photon optical microscopy method (MPM) that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals integrated with Raman spectroscopy to study protein self-assembly. TPF is generated by UV/VIS absorbing moieties in proteins such as amino acids and cross-links. Second harmonic generation signals (SHG) are produced by structures without a center of symmetry, such as fibrous protein collagen. Integration of Raman spectroscopy provides additional information about properties relevant to self-assembly of protein-based materials, such as chemical composition, crystallinity, conformation, density, monomer content, degradation and stress-strain distribution. BROADER IMPACTS. Performance outcomes of this research and educational program will include design and implementation of the multi-modality optical microscopy and image guided spectroscopy platform as a tool to non-invasively monitor and understand the structure, composition and function of protein-derived biological materials and adhesives in real time, in-situ. The experimental methods developed and the knowledge obtained can be incorporated as part of a feedback system to establish scientific principles for the design, construction, modification, growth, and maintenance of living engineered tissues, implants and novel adhesives. The project will therefore benefit tissue engineering, biomaterial, and biomedical engineering communities. The NSF BRIGE Award will enable the PI to integrate a number of education and outreach activities with the research. She will provide laboratory research opportunities to selected K-12 students and teachers, as well as undergraduates and graduates. She will develop new courses for graduate students and advanced undergraduates that require students' in-depth understanding of subject matters related to optical technologies and their biological applications. Students of various ages and the general public will gain a greater appreciation of how diverse fields interact to produce high impact engineering research on the assembly mechanisms of protein-based materials. Additionally, the PI will expand preliminary efforts to reach out to young women and show how they can pursue non-traditional science and technology careers, and balance demanding work and family needs, to live successfully and make important contributions to public well-being.