In Vitro reconstitution and imaging of microtubule dynamics by fluorescence and label-free microscopy

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hirst, W. G., Kiefer, C., Abdosamadi, M. K., Schäffer, E., & Reber, S. In Vitro reconstitution and imaging of microtubule dynamics by fluorescence and label-free microscopy. STAR Protocols, 1(3), (2020): 100177, doi:10.1016/j.xpro.2020.100177.Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy.We thank the AMBIO imaging facility (Charité, Berlin) and Nikon at MBL for imaging support. We thank all former and current members of the Reber lab for discussion and helpful advice, in particular Christoph Hentschel and Soma Zsoter for technical assistance. S.R. acknowledges funding by the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra co-funded by a grant from the Deutsche Forschungsgemeinschaft (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. C.K. thanks the Deutsche Forschungsgesellschaft (DFG, JA 2589/1-1). C.K. and M.A. thank Steve Simmert and Tobias Jachowski former and current members of the Schäffer lab