In X-ray crystallography, a main concern of scientists is being able to distinguish between crystalline and aggregate protein to prevent unnecessary analysis of non-crystalline samples. Few methods exist that allow for analysis of protein samples to identify crystals prior to diffraction. Polarization dependent imaging using second harmonic generation (SHG) or two-photon excited-UV fluorescence (TPE-UV-F) imaging can be used as an alternative method. Protein crystals produce a different polarization dependence than aggregate due to the dependence on the orientation of the crystal. For crystalline material, each crystal will produce a different response depending upon the polarization state and the orientation of the crystal, and when averaged together results in a broadened distribution about the mean, termed heteroskedastic. Alternatively for aggregates, the orientation will be disordered throughout such that each particle produces the same response for different polarization states. Aggregates exhibit a narrow distribution about the mean, termed homoskedastic. This technique is demonstrated using snap-shot polarization dependent TPE-UV-F imaging to differentiate between protein aggregates and crystals.

Polarization dependent imaging for discrimination between protein aggregates and crystals

256th National Meeting (2018)

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