3D-MINSTED nanoscopy and protein tracking in densely labelled living cells

Abstract Investigating the movements and conformational changes of proteins in living cells is essential for understanding their function. The recently introduced fluorescence nanoscopy method called MINSTED has successfully tracked single fluorophore-labelled proteins, albeit in fixed cells and two dimensions only. Here we introduce a MINSTED setup for live-cell tracking of individual proteins in three dimensions (3D) with a localization precision σ down to < 1 nm. Applied to the motor protein kinesin-1, our MINSTED nanoscope follows single proteins in 3D as they process along microtubules in 16 nm steps. Individual steps are resolved amid substantial intracellular background. The unique capability of the 3D stimulated emission depletion (STED) beam to carve out the signal of the protein label of interest enables efficient single-molecule investigations at hitherto unpractically high concentrations of labelled proteins and thus in crowded live-cell environments.