Purdue University Graduate School
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posted on 2020-07-29, 16:04 authored by Hilary M FlorianHilary M Florian

Understanding the function and structure of a protein is crucial for informing on rational drug design and for developing successful drug candidates. However, this understanding is often limited by the protein pipeline, i.e. the necessary steps to go from developing protein constructs to generating high-resolution structures of macromolecules. Because each step of the protein pipeline requires successful completion of the prior step, bottlenecks are often created and therefore this process can take up to several years to complete. Addressing current limitations in the protein pipeline can help to reduce the time required to successfully solve the structure of a protein.

The field of nonlinear optical (NLO) microscopy provides a potential solution to many issues surrounding the detection and characterization of protein crystals. Techniques such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) have already been shown to be effective methods for the detection of proteins with high selectivity and sensitivity. Efforts to improve high throughput capabilities of SHG microscopy for crystallization trials resulted in development of a custom microretarder array (μRA) for depth of field (DoF) extension, therefore eliminating the need for z-scanning and reducing the overall data acquisition time. Further work was done with a commercially available μRA to allow for polarization dependent TPE-UVF. By placing the μRA in the rear conjugate plane of the beam path, the patterned polarization was mapped onto the field of view and polarization information was extracted from images by Fourier analysis to aid in discrimination between crystalline and aggregate protein.

Additionally, improvements to X-ray diffraction (XRD), the current gold standard for macromolecular structure elucidation, can result in improved resolution for structure determination. X-ray induced damage to protein crystals is one of the greatest sources of loss in resolution. Previous work has been done to implement a multimodal nonlinear optical (NLO) microscope into the beamline at Argonne National Lab. This instrument aids in crystal positioning for XRD experiments by eliminating the need for X-ray rastering and reduces the overall X-ray dosage to the sample. Modifications to the system to continuously improve the capabilities of the instrument were done, focusing on redesign of the beam path to allow for epi detection of TPE-UVF and building a custom objective for improved throughput of 1064 nm light. Furthermore, a computational method using non-negative matrix factorization (NMF) was employed for isolation of unperturbed diffraction peaks and provided insight into the mechanism by which X-ray damage occurs. This work has the potential to improve the resolution of diffraction data and can be applied to other techniques where X-ray damage is of concern, such as electron microscopy.


Degree Type

  • Doctor of Philosophy


  • Chemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Garth Simpson

Additional Committee Member 2

Mary Wirth

Additional Committee Member 3

Chittaranjan Das

Additional Committee Member 4

Adam Wasserman