This study seeks to expand the field of photonic crystal enhanced microscopy by extending simple proof-of-concept experiments to advanced biological applications. There are four distinct projects that are related to a central theme: sophisticated microscope instrumentation for photonic crystal biosensing. Also included in this study is a comprehensive list of software that I have written in order to control and optimize various instruments and data processing, including the band diagram transmission set-up, enhanced fluorescence label-free microscope and objective coupled label-free microscope. In the initial project, I demonstrate the first hybrid total internal reflection and photonic crystal microscope. This imaging system benefits from the low background signal of total internal reflection in addition to the enhanced fluorescence effect of photonic crystals at resonance. The noise of this system is shown to be dominated by the TiO$_2$ layer autofluorescence which increases exponentially with increasing illumination intensity because of the enhanced electric field in the TiO$_2$ layer. The response of the fluorophores with increasing laser power is measured, and a linear fluorescence response within 30\%-90\% of the maximum laser output power is observed, implying that the emission signal of the fluorophores could be intensified further by the photonic crystal enhanced fluorescence phenomena. Data acquired outside of this range did not create a statistically significant set of measurements as shown in the histograms, standard deviations and number of acquired traces.The second project is an angle-scanning technique that I developed that can be used with photonic crystal sensors in order to create 3D tomographic fluorescence and label-free images. Using photonic crystal enhanced fluorescence, this method measures the distance a fluorophore resides from the surface. For photonic crystal-based label-free biosensing, it constructs 3D refractive index maps. Simulations using rigorous coupled waveguide analysis reinforce the validity of the concept, and they are used as a visual aid when describing the decay of the evanescent field profile with angle. A microarray of fluorescent spots is used to demonstrate how to acquire and process the data for the technique. The procedure is applied to the processed spot array, which shows an unexpected nonlinear trend. The small molecule binding assumption that is used in one of the steps is validated with numerical simulations as well.The next project is the process I have developed to maximize the label-free sensitivity of transparent photonic crystal devices. The spectroscopic instrument that I built to acquire full band diagrams automatically is described, and included in this section is the software that handles the data acquisition and post-processing analysis. The band diagrams and corresponding wavelengths of minimum transmission for an ERv1 device in air, water and isopropyl alcohol are recorded and the label-free sensitivity, also known as the change in resonance wavelength per bulk refractive index unit, is plotted against angle. This analysis demonstrates that the highest sensitivities for both transverse electric and transverse magnetic polarizations occur at normal incidence. This trend is reinforced further with computational software simulations. The band diagram work expands to the angle of minimum transmission curves, which typically are employed when using high-resolution microscopes. The study indicates that the optimal placement of the source center wavelength is slightly red-shifted from the point of inflection of the band diagram and that a narrow band source yields slightly higher angle sensitivity. These results are used to direct the design of photonic crystal enhanced instruments.In the final phase of this study, I record the improvements that I have made to the current photonic crystal enhanced microscope, which is also known as the enhanced fluorescence and label-free microscope. These include the modifications of the optical design, software interface, data acquisition and post-acquisition data processing routines. Also, I characterize and address the current limitations of the system, such as spatial resolution, limit of detection, source of noise and image defects. Label-free images that are acquired with the current angle-scanning tool are shown. The protocol for the enhanced fluorescence and label-free microscope contributed to the design of the new objective coupled label-free microscope, which I have built specifically for the application of high-resolution label-free cell imaging.
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