PT - JOURNAL ARTICLE AU - Scheiderer, Lukas AU - von der Emde, Henrik AU - Hesselink, Mira AU - Weber, Michael AU - Hell, Stefan W. TI - MINSTED tracking of single biomolecules AID - 10.1101/2023.09.15.557902 DP - 2023 Jan 01 TA - bioRxiv PG - 2023.09.15.557902 4099 - http://biorxiv.org/content/early/2023/09/15/2023.09.15.557902.short 4100 - http://biorxiv.org/content/early/2023/09/15/2023.09.15.557902.full AB - 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 spatio-temporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within < 250 microseconds using about 13 photons. The power of MINSTED tracking is demonstrated by quantifying side-steps of the motor protein kinesin-1 walking on microtubules and switching protofilaments.Competing Interest StatementThe Max Planck Society holds patents on selected procedures and embodiments of MINSTED, benefitting S. W. H., H. v. d. E., and M. W..