Direct observation of chain folding in polymer single crystals using single molecule Förster resonance energy transfer

We use single-molecule Förster resonance energy transfer (SM-FRET) microscopy to directly quantify the number of folds adopted by individual poly(ethylene glycol) (PEG) chains within single crystals. Low FRET efficiencies correspond to unfolded (n = 0) chains, while high efficiencies correspond to singly folded (n = 1) chains, demonstrating SM-FRET’s ability to resolve single chain conformations inside crystalline domains. Colocalized SM-FRET and atomic force microscopy imaging across thousands of single chains showed strong agreement between FRET efficiencies and lamellar heights. This provides direct evidence that a crystal’s lamellar thickness is intrinsically linked to its corresponding chain fold number, validating a long-assumed relationship. SM-FRET also revealed spatial heterogeneity where n = 0 and n = 1 regions coexist within the same crystal. We also identified a narrowly distributed FRET population with intermediate efficiency appearing exclusively in crystalline regions. Although not yet fully resolved, this population is consistent with chains adopting an n = 4 folded conformation. These results establish SM-FRET as a powerful, quantitative tool for connecting single chain conformation to lamellar structure, providing crucial molecular detail to the study of polymer crystallization.