In Situ Observation of Dislocations in Protein Crystals during Growth by Advanced Optical Microscopy

We attempted to visualize defects in a tetragonal lysozyme crystal in situ by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). Birefringence microscopy (BM) and phase-contrast microscopy (PCM) were also employed for the in situ observations. LCM-DIM enabled us to observe the {1 1 0} surfaces of the crystals and visualize the strain fields around the dislocations normal to a light beam with sufficient contrast during growth for the first time. Relatively large inclusions (60 to 300 μm) inside the crystal could also be visualized during growth, with the use of BM and PCM. We found that the dislocations appeared in bundles and were probably generated at the periphery of the relatively large inclusions inside the crystal. The existence of the dislocations was confirmed by etching experiments, in which we observed two kinds of etch pits: those with a point bottom and those with a flat bottom; the former corresponded to the dislocations inside the crystal and the latter corresponded to microdefects. Two different critical undersaturations existed above which the point-bottomed and flat-bottomed etch pits started to appear. The critical undersaturation for the point-bottomed etch pits was definitely less than that for the flat-bottomed ones. In the central region of the spiral growth hillock were many point-bottomed etch pits corresponding to the dislocations but not contributing to the formation of spiral growth steps. Although the point-bottomed etch pits were formed in only the central region of the spiral growth hillock, the flat-bottomed ones were randomly formed all over the {1 1 0} surfaces.