Nonlinear Optical Methods for Noninvasive Analytics.

Abstract

To date, many of the current tools and technologies used to explore biophysical processes and phenomena are limited in part by their inability to probe without external perturbation. Factors such as size restrictions, samples used for non-terminal studies, and small molecule dynamics cannot be addressed by these techniques. This has not only driven the field of noninvasive analytics, but has had a direct impact on shaping the next generation of biological and clinical assays. Highlights of my thesis work herein focus on the development of nonlinear optical spectroscopy and imaging modalities, sum frequency generation (SFG) spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy. Each has their own unique qualities that make them ideal as noninvasive tools and techniques.

Using these two techniques, my studies address fundamental questions about: (1) the orientation and behavior of chemically immobilized peptides on abiotic surfaces for the rational design of improved biosensors and bioactive textiles, (2) understanding the relations between cytosolic lipids, cellular energy homeostasis/consumption, and developmental biology in female reproductive cells, and (3) the ability to classify and measure male reproductive health as it relates to acrosome integrity.

Results from SFG, a surface-sensitive spectroscopy, demonstrate that surface tethering mechanisms govern both the orientation and activity of antimicrobial peptide MSI-78 on surfaces. Attachment of the n-terminus of this peptide results in an orientation perpendicular to the surface normal (lying down) and higher antimicrobial activity whereas c-terminus attachment leads to a parallel orientation (standing up) and lower activity. Other methods complementary to SFG including circular dichroism (CD) spectroscopy and coarse-grained simulation molecular dynamics simulations also support this conclusion.

CARS, a live cell noninvasive microscopy, is used to evaluate the contributions of lipid in oocyte growth, development, and for metabolic disease. Results show that lipid content fluctuates as oocytes progress through meiosis, indicated by an increase in content as the oocytes grow and a decrease as oocytes resume meiosis. Lipid content is also higher for oocytes from females who exhibit metabolic disease. As for male reproductive cells, CARS microscopy is beginning to be used for the identification of acrosome reaction, an important predictor of male infertility.