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

University Of California-Riverside, Riverside CA

Investigators

Abstract

0927297 Lyubovitsky The 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.