Polarization-dependent nonlinear optical microscopy methods for the analysis of crystals and biological tissues
Abstract
The ability to solve a high-resolution protein structure is largely dependent on the successful generation and identification of protein crystals prior to X-ray diffraction (XRD). For novel protein targets, high-throughput crystallography often involves generation of multiple targets and thousands of crystallization trials per target to generate diffraction-quality crystals. Second harmonic generation (SHG) imaging has been developed as a fast, non-destructive and sensitive method for the selective identification of protein crystals, even in highly scattering environments. Polarization-dependent SHG microscopy methods were developed to assess the presence of multidomain crystals to provide a handle on crystal quality. In addition, polarization-dependent two-photon excited fluorescence (TPEF) microscopy was developed as a complementary method to SHG, providing selectivity based on the presence of protein and crystalline order, thereby reducing the potential for false negatives and positives that can arise with SHG and conventional TPEF imaging. Novel instrumentation, data acquisition methods, and data analysis techniques were developed for quantitative polarization-modulated SHG microscopy at imaging speeds up to video rate, offering significantly greater signal to noise ratios compared to polarization modulation through the manual rotation of wave plates. Quantitative polarization-dependent SHG imaging was extended to the analysis of collagen structures in biological tissues, where local-frame second order susceptibility tensors were solved for every pixel within an image of collagenous tissue and combined with ab initio modeling to assess internal ordering of collagen fibers in different tissue types.
Degree
Ph.D.
Advisors
Simpson, Purdue University.
Subject Area
Analytical chemistry
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