Fig. 1: Visualization of substeps in primary rat hippocampal neurites.

a Fluorophore-labeled kinesin motor walking into the stationary confocal volume (top) creating an increased signal and triggering a MINFLUX tracking measurement (bottom). b Schematic of the MINFLUX tracking process. For each time step t_j the x- and y-position of the minimum are updated to the newly estimated emitter position. c Exemplary on-axis position-time traces of construct K28C (inset) labeled at the back of the head domain recorded at 50 µM (purple), 500 µM (orange) and 5 mM (green) ATP concentration. The raw position data (semi-transparent lines) are overlaid with a step-fit (solid lines). Zoom-ins highlight ~16 nm regular steps and pairs of similar-sized ~8 nm substeps. d Population-normalized histograms of the measured step sizes recorded at the three ATP concentrations showing two populations of step sizes; 8 nm substeps and 16 nm regular steps (N_50µM = 1699, N_500µM = 1931, N_5mM = 1818). e Average duration of the one-head-bound (1HB) state between substeps (purple) and the two-head-bound (2HB) state (orange) plotted over the average velocity at 50 µM (\(\bar{V}=232\pm 10\) nm s⁻¹ s.e.m), 500 µM (\(\bar{V}=541\pm 13\) nm s⁻¹ s.e.m) and 5 mM (\(\bar{V}=297\pm 11\) nm s⁻¹ s.e.m). The solid black line shows a fit modeling the 1HB duration to velocity dependence by a simple rational function with parameters \({{{{{\rm{dx}}}}}}=9.1\pm 5.8\,{{{{{\rm{nm}}}}}}\) and \({{{{{{\rm{t}}}}}}}_{2{{{{{\rm{HB}}}}}}}=8.8\pm 19.4\,{{{{{\rm{ms}}}}}}\)_. Dwell times and velocity were averaged from in total 348 traces of 12 biological replicates. Error bars denote the standard deviation. Scale bar: 1 µm (a).