title: A visible-light-excited fluorescence method for imaging protein crystals without added dyes.
abstract: Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visible-light-inducible autofluorescence serves as a complementary method to bright-field microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the non-damaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays.
authors.affiliation: MacCHESS (Macromolecular Diffraction Facility at CHESS), Cornell University , 161 Synchrotron Drive, Ithaca, NY 14853, USA.
authors.forename: Tiit
authors.initials: T
authors.lastname: Lukk
authors.affiliation: MacCHESS (Macromolecular Diffraction Facility at CHESS), Cornell University , 161 Synchrotron Drive, Ithaca, NY 14853, USA.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Gillilan
authors.affiliation: MacCHESS (Macromolecular Diffraction Facility at CHESS), Cornell University , 161 Synchrotron Drive, Ithaca, NY 14853, USA.
authors.forename: Doletha M E
authors.initials: DM
authors.lastname: Szebenyi
authors.affiliation: Department of Biomedical Engineering, Cornell University , B41 Weill Hall, Ithaca, NY 14853, USA.
authors.forename: Warren R
authors.initials: WR
authors.lastname: Zipfel
keywords: crystal screening; crystal visualization; fluorescence microscopy; protein crystallography