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

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 ∼ω