Confocal microscopy on the beamline: Novel three-dimensional imaging and sample positioning

Confocal microscopy, a technique that has been extensively applied in cellular biological studies, may also be applied to the visualization and three-dimensional imaging of protein crystals at high resolution on synchrotron beamlines. Protein crystal samples are examined using a commercially available confocal microscope adapted for cryogenic use. A preliminary test using a custom confocal design adapted for beamline use is also presented. The confocal optics configuration is compatible with nonlinear imaging techniques such as two-photon excited fluorescence imaging and second harmonic generation. The possibilities of this method are explored using two modes: fluorescence and reflection confocal. In fluorescence mode, small amounts of dye are introduced into the crystal through soaking or growth conditions. Under such conditions, protein crystals are easily resolved from salts and amorphous precipitates, which do not generally take up dye. Reflection mode, which does not require dye, still exhibits greater resolution and sensitivity to surface detail than conventional wide-field microscopy as a result of the confocal optics configuration. The inherent three-dimensional nature of the method means that on-axis sample views (along the direction of the X-ray beam) can be reconstructed from an off-axis configuration, simplifying the beamline setup and providing uniquely detailed views of cryogenically cooled crystals.