MINSTED tracking of single biomolecules

Here we show that MINSTED localization, a method whereby the position of a fluorophore is identified with precisely controlled beams of a STED microscope, tracks fluorophores and hence labeled biomolecules with nanometer/millisecond spatiotemporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within <250 μs using about 13 photons. The power of MINSTED tracking is demonstrated by resolving the stepping of the motor protein kinesin-1 walking on microtubules and switching protofilaments.