Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein

we use the combined techniques of atomic force microscopy (AFM) imaging and force spectroscopy to pull a single polyethylene oxide (PEO) chain out of its spiral crystal in amyl acetate. From these data

the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area

leading to the decrease in the apparent lamellar height. While in the area of screw dislocation

the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.