Multiphoton instrumentation and applications for protein crystal detection

Elucidation of the protein structure represents a critical step in the determination of the key functions of the biomolecule, with this goal being conventionally accomplished through X-ray diffraction. However, the major cost in terms of time and expense associated with the deduction of a protein structure at high resolution is the identification of the conditions for generating diffraction quality protein crystals. This thesis focuses on the detection and analysis of protein crystals across a variety of novel nonlinear multiphoton instruments and corresponding applications. Utilizing the advantages offered by second harmonic generation, reduction of the bottlenecks associated with protein structure determination was accomplished through the development of SHG instrumentation. These novel instruments permitted both a decrease in the time and increase in the measurement confidence associated with the screening of crystallization trials, with the extension to on-axis detection and crystal centering at a synchrotron beamline. The information content from these studies was supplemented through the detection of protein crystals using two-photon excited fluorescence, offering both chemical and structural information when collected complementary to the SHG signal. Further application of these techniques for polarization-dependent fluorescence measurements and pharmaceutical analysis not only solidified the use of these techniques for sensitive protein crystal detection but illustrated their application across a wide variety of fields