Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these measurements have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, we resolved the precise stepping motion of the motor protein kinesin-1 as it walked on microtubules in living cells. Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable future in vivo studies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.

Dendronized oligoethylene glycols (dendron OEGs) with two phosphonate groups (phosphonate tweezers) have been drawing significant attention as a new class of coating materials for superparamagnetic iron oxide surfaces. However, despite dendron OEGs showing outstanding stability in physiological fluids in previous studies, little is understood about their structure and mechanical properties. Herein we report the surface and internal structures and mechanical properties of dendron OEGs, and quantitatively determine their ability to avoid non-specific adhesion of blood platelets. To gain insight into the interfacial force interactions, we measured the coarse-scale surface force acting on cell-sized particles and mapped the nanoscopic pinning centers by fast force mapping.

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.