Nonlinear optical microscopy for ultrastructural bioimaging

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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.