Extracting quantitative parameters from images in multiphoton microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy allows for fast, three-dimensionally resolved detection of molecules based on vibrational contrast. In CARS, the generated signal is nonlinearly dependent upon the concentration of the vibrational mode of interest. This makes it challenging to extract quantitative parameters (such as the concentration or orientation) from CARS images of biological and synthetic samples. Because of this, many investigations which employ CARS microscopy generally only report qualitative information extracted from these images. In this thesis, three methods have been developed to extract the quantitative concentration information from CARS images.;In the first, the ratio of the forward-propagating and back-reflected CARS signal generated in tissue is used to monitor the percolation of DMSO into excised human cadaver skin. Through this, we find that the maximum clearing of skin with DMSO occurs at 40% v/v. We also combine CARS with second harmonic generation (SHG) to investigate the effects of DMSO on collagen. Up to a 20% v/v concentration of DMSO in the skin, the collagen becomes disrupted, resulting in a significant drop in the generated SHG.;In the second method, the ratio between the CARS resonance peak and dip is correlated with the concentration to measure the concentration of water and deuterated glycine in hair. Both molecules are found to distribute throughout the hair fiber homogenously, water at a 34% v/v concentration, and d-glycine with a 0.22 M concentration.;In the final method, CARS spectra over one vibrational mode are used to extract the imaginary part of the third-order nonlinear susceptibility. This quantity is linearly dependent upon the concentration of the vibrational mode of interest. This procedure is used to determine the degree of conversion of two-photon polymerized microstructures synthesized with varying writing powers. A sigmoidal relationship is observed between the applied intensity and the degree of conversion.;The last chapter investigates another quantitative parameter, the orientation, of the CH2 vibrational mode in natural (cotton) and synthetic (rayon) cellulose fibers. Coupled with SHG, this method gives insight into the origins of observed optical nonlinearities in dry and hydrated cellulose fibers.