The annealing behavior of twice-folded crystals of the long-chain alkane C294H590 is examined in situ, in real time, by atomic force microscopy (AFM). AFM is capable of following processes in real time provided that the time scale is sufficiently long for several images to be collected during the process. In this paper, we focus on the temperature dependence and the thickened morphology. We are able to investigate where the thickening starts and how this depends on temperature and how melting is influenced by morphology. By following the motion of holes within the crystal, a lower limit for the rate of diffusion of crystalline polyethylene is estimated. We also focus on the substrate effect on the crystal morphology and thickening, using mica, glass, and graphite.