3 years ago

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping
Matthias D. Koch, Alexander Rohrbach

Abstract

Mechanical manipulation of single cytoskeleton filaments and their monitoring over long times is difficult because of fluorescence bleaching or phototoxic protein degradation. The integration of label-free microscopy techniques, capable of imaging freely diffusing, weak scatterers such as microtubules (MTs) in real-time, and independent of their orientation, with optical trapping and tracking systems, would allow many new applications. Here, we show that rotating-coherent-scattering microscopy (ROCS) in dark-field mode can also provide strong contrast for structures far from the coverslip such as arrangements of isolated MTs and networks. We could acquire thousands of images over up to 30 min without loss in image contrast or visible photodamage. We further demonstrate the combination of ROCS imaging with fast and nanometer-precise 3D interferometric back-focal-plane tracking of multiple beads in time-shared optical traps using acoustooptic deflectors to specifically construct and microrheologically probe small microtubule networks with well-defined geometries. Thereby, we explore the frequency-dependent elastic response of single microtubule filaments between 0.5 Hz and 5 kHz, which allows for investigating their viscoelastic response up to the fourth-order bending mode. Our spectral analysis reveals constant filament stiffness at low frequencies and frequency-dependent stiffening following a power law ∼ωp with a length-dependent exponent p(L). We find further evidence for the dependence of the MT persistence length on the contour length L, which is still controversially debated. We could also demonstrate slower stiffening at high frequencies for longer filaments, which we believe is determined by the molecular architecture of the MT. Our results shed new light on the nanomechanics of this essential, multifunctional cytoskeletal element and pose new questions about the adaptability of the cytoskeleton.

Publisher URL: http://www.cell.com/biophysj/fulltext/S0006-3495(17)31163-3

DOI: 10.1016/j.bpj.2017.10.036

You might also like
Never Miss Important Research

Researcher is an app designed by academics, for academics. Create a personalised feed in two minutes.
Choose from over 15,000 academics journals covering ten research areas then let Researcher deliver you papers tailored to your interests each day.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.