The spectroscopic ruler revisited at 77 K

We report on single-molecule FRET measurements on the classical molecular ruler polyproline immersed in amorphous ice at 77 K. Confocal scanning microscopy allows for observation of the fluorescence of the dyes Alexa Fluor 488 as the donor and Alexa Fluor 594 as the acceptor in the FRET experiment. The dipole orientations are fixed in amorphous ice leading to a broad distribution of transfer efficiencies. The measured transfer efficiency distributions are mainly in accordance with simulated distributions, with a significant deviation at low transfer efficiencies, which are absent in the experimental distribution, in stark contrast to the theory.