The formation of dislocations is central to our understanding of yield, work hardening, fracture, and fatigue1 of crystalline materials. While dislocations have been studied extensively in conventional materials, recent results have shown that colloidal crystals offer a potential model system for visualizing their structure and dynamics directly in real space 2. Although thermal fluctuations are thought to play a critical role in the nucleation of these defects, it is difficult to observe them directly. Nano-indentation, during which a small tip deforms a crystalline film, is a common tool for introducing dislocations into a small volume that is initially defect-free3-10. Here, we show that an analogue of nano-indentation performed on a colloidal crystal provides direct images of defect formation in real time and on the single particle level, allowing us to probe the effects of thermal fluctuations. We implement a new method to determine the strain tensor of a distorted crystal lattice and we measure the critical dislocation loop size and the rate of dislocation nucleation directly. Using continuum models, we elucidate the relation between thermal fluctuations and the applied strain that governs defect nucleation. Moreover, we estimate that although bond energies between particles are about fifty times larger in atomic systems, the difference in attempt frequencies makes the effects of thermal fluctuations remarkably similar, so that our results are also relevant for atomic crystals. © 2006 Nature Publishing Group.