Direct optical resolution in the intra-molecular distance range

Estimations of molecular distances <10 nm have long been the almost exclusive domain of Förster resonance energy transfer (FRET) and related spectroscopic techniques. By site-specifically labeling molecular subunits with (at least) two fluorophores, single-molecule FRET has also allowed for the monitoring of distances within macromolecules. In this presentation, we report on the direct determination of such intra-molecular distances based on the MINFLUX localization concept. We measured intra-macromolecular spacings of photoactivatable small-molecule fluorophores in well-characterized uni- or few-molecular systems, with simple polypeptides and proteins serving as initial examples. Our experiments indicate that the widely employed STORM-type dark-state photoblinking of cyanines reaches an impasse for dye separations below ∼6-10 nm, as the dyes couple and no longer behave independently. The complex photophysics of such dye-dye communication impedes the reliable, isolated detection of their signals. As we show however, sequential photoactivation - providing the required off-on-off transitions in a more orthogonal way - is a viable strategy to read out dye coordinates even for separations as low as few nm, beyond any perceived remaining barriers of resolution. Our results, with direct resolution of intra-molecular distances and conformations, herald the possibility of fluorescence-based analyses of structural biology at room or physiological temperature relying on direct positional measurements rather than on indirect FRET readouts.