With increasing demand for synchrotron X-ray beam time, the need for high sample throughput has driven the development of methods for rapid and reliable crystal centering on synchrotron sources. Currently, the most reliable crystal centering method rasters the X-ray beam across the sample and centers on the region of strongest diffraction. However, rastering is time consuming and exposes the protein to damaging X-rays.A nonlinear optical (NLO) microscope for combined second harmonic generation (SHG) and two-photon excited ultraviolet fluorescence (TPE-UVF) has been integrated with a synchrotron X-ray diffraction (XRD) beamline for rapid centering of protein crystals. SHG, the frequency doubling of light, is selective to non-centrosymmetric material, and forbidden for symmetric crystals and disordered media. The inherent chirality of proteins yields SHG generation for 84% of proteins. TPE-UVF, accessing fluorescence of aromatic amino acids, can distinguish protein crystals from SHG-active salts and locate protein crystals where the symmetry generates weak SHG. NLO microscopy was found to be a fast and reliable method for centering protein crystals. Good agreement between NLO images and XRD raster scans was found. The NLO instrument achieved high spatial resolution images (2�m) with fast acquisition times (<10s) compared to XRD rasters (5�m resolution and 3-60min acquisition times). Once fully developed, NLO microscopy may replace X-ray rastering as a method for centering, increase throughput of synchrotron beamlines, and eliminate damage from X-ray exposure during rastering.

Integrated nonlinear optical microscope for crystal centering on a synchrotron X-ray beamline

246th National Meeting (2013)

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