Optics are a powerful probe of chemical structure that can often be linked to theoretical predictions, providing robustness as a measurement tool. Not only do optical interactions like second harmonic generation (SHG), single and two-photon excited fluorescence (TPEF), and infrared absorption provide chemical specificity at the molecular and macromolecular scale, but the ability to image enables mapping heterogeneous behavior across complex systems such as biological tissue. This thesis will discuss nonlinear and linear optics, leveraging theoretical predictions to provide frameworks for interpreting analytical measurement. In turn, the causal mechanistic understanding provided by these frameworks will enable structurally specific quantitative tools with a special emphasis on application in biological imaging. The thesis will begin with an introduction to 2nd order nonlinear optics and the polarization analysis thereof, covering both the Jones framework for polarization analysis and the design of experiment. Novel experimental architectures aimed at reducing 1/f noise in polarization analysis will be discussed, leveraging both rapid modulation in time through electro-optic modulators (Chapter 2), as well as fixed-optic spatial modulation approaches (Chapter 3). In addition, challenges in polarization-dependent imaging within turbid systems will be addressed with the discussion of a theoretical framework to model SHG occurring from unpolarized light (Chapter 4). The application of this framework to thick tissue imaging for analysis of collagen local structure can provide a method for characterizing changes in tissue morphology associated with some common cancers (Chapter 5). In addition to discussion of nonlinear optical phenomena, a novel mechanism for electric dipole allowed fluorescence-detected circular dichroism will be introduced (Chapter 6). Tackling challenges associated with label-free chemically specific imaging, the construction of a novel infrared hyperspectral microscope for chemical classification in complex mixtures will be presented (Chapter 7). The thesis will conclude with a discussion of the inherent disadvantages in taking the traditional paradigm of modeling and measuring chemistry separately and provide the multi-agent consensus equilibrium (MACE) framework as an alternative to the classic meet-in-the-middle approach (Chapter 8). Spanning topics from pure theoretical descriptions of light-matter interaction to full experimental work, this thesis aims to unify these two fronts.