Laser Seeding for Biomolecular Crystallization

Crystal seeding, which permits the decoupling of crystal nucleation and growth, is a potentially powerful tool for biomolecular crystallization. However, its usefulness is severely limited by the inability of current techniques to manipulate single seeds and it is often considered as a last resort in protein crystal growth trials. Remarkably, current practice relies on the cumbersome technique of entraining seed crystals on animal whiskers. Seeding by whisker entrainment is not reproducible and does not allow the observation, selection or individual transfer of seeds. Here, we present a novel approach in which optical forces generated by a focused laser beam are used to select and transfer single microscopic seed crystals in a growth solution. The technique permits the non-mechanical, in situ manipulation of individual seed crystals as small as one micron. The seed transfer is simple and efficient, and can be completed in a few minutes. To demonstrate the procedure we have successfully seeded and grown crystals of tomato bushy stunt virus (TBSV). Applications to other situations which require the selection and manipulation of delicate microcrystals are discussed briefly.