title: Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy.
abstract: Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we demonstrate label-free tracking of microtubules with nanometer precision at kilohertz frame rates using interferometric scattering microscopy (iSCAT). With microtubules tethered to a glass substrate using low-density kinesin, we readily detect sequential 8 nm steps in the microtubule center of mass, characteristic of a single kinesin molecule moving a microtubule. iSCAT also permits dynamic changes in filament length to be measured with <5 nm precision. Using the arbitrarily long observation time enabled by label-free iSCAT imaging, we demonstrate continuous monitoring of microtubule disassembly over a 30 min period. The ability of iSCAT to track microtubules with nm precision together with its potential for label-free single protein detection and simultaneous single molecule fluorescence imaging represent a unique platform for novel approaches to studying microtubule dynamics.
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: Andrecka
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega Arroyo
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Katie
authors.initials: K
authors.lastname: Lewis
authors.affiliation: Warwick Medical School, Coventry, United Kingdom.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Cross
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom. Electronic address: philipp.kukura@chem.ox.ac.uk.
authors.forename: Philipp
authors.initials: P
authors.lastname: Kukura
mesh_terms: D000818:Animals; D008857:Microscopy, Interference / Q000379:methods*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D013552:Swine; D013997:Time Factors
chemical_list: D017239:Paclitaxel