Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

By Jeremy T. Madden, Scott J. Toth, Christopher M. Dettmar, Justin A. Newman, Robert A. Oglesbee, Hartmut G. Hedderich, R. Michael Everly, Michael Becker, Judith A. Ronau, Susan K. Buchanan, Vadim Cherezov1, Marie E. Morrow, Shenglan Xu, Dale Ferguson, Oleg Makarov, Chittaranjan Das, Robert Fischetti, Garth J. Simpson

1. Bridge Institute - University of Southern California

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journal-article

Author

Jeremy T. Madden and Scott J. Toth and Christopher M. Dettmar and Justin A. Newman and Robert A. Oglesbee and Hartmut G. Hedderich and R. Michael Everly and Michael Becker and Judith A. Ronau and Susan K. Buchanan and Vadim Cherezov and Marie E. Morrow and Shenglan Xu and Dale Ferguson and Oleg Makarov and Chittaranjan Das and Robert Fischetti and Garth J. Simpson

Citation

Madden, J.T. et al., 2013. Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline. Journal of Synchrotron Radiation, 20(4), pp.531–540. Available at: http://dx.doi.org/10.1107/s0909049513007942.

Abstract

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase fromChromobacterium violaceumcPAH,Trichinella spiralisdeubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.

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