title: Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains.
abstract: Polarization-resolved second-harmonic generation (PR-SHG) microscopy is described and applied to identify the presence of multiple crystallographic domains within protein-crystal conglomerates, which was confirmed by synchrotron X-ray diffraction. Principal component analysis (PCA) of PR-SHG images resulted in principal component 2 (PC2) images with areas of contrasting negative and positive values for conglomerated crystals and PC2 images exhibiting uniformly positive or uniformly negative values for single crystals. Qualitative assessment of PC2 images allowed the identification of domains of different internal ordering within protein-crystal samples as well as differentiation between multi-domain conglomerated crystals and single crystals. PR-SHG assessments of crystalline domains were in good agreement with spatially resolved synchrotron X-ray diffraction measurements. These results have implications for improving the productive throughput of protein structure determination through early identification of multi-domain crystals.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA.
authors.forename: Emma L
authors.initials: EL
authors.lastname: DeWalt
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Begue
authors.forename: Judith A
authors.initials: JA
authors.lastname: Ronau
authors.forename: Shane Z
authors.initials: SZ
authors.lastname: Sullivan
authors.forename: Chittaranjan
authors.initials: C
authors.lastname: Das
authors.forename: Garth J
authors.initials: GJ
authors.lastname: Simpson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D002861:Chromobacterium / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D008859:Microscopy, Polarization / Q000295:instrumentation / Q000379:methods*; D025341:Principal Component Analysis / Q000379:methods*; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D017356:Synchrotrons / Q000295:instrumentation; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D001708:Biopterins; C003402:sapropterin