Strong cytoskeleton activity on millisecond timescales upon particle binding revealed by ROCS microscopy

Cells change their shape within seconds, cellular protrusions even on subsecond timescales enabling various responses to stimuli of approaching bacteria, viruses or pharmaceutical drugs. Typical response patterns are governed by a complex reorganization of the actin cortex, where single filaments and molecules act on even faster timescales. These dynamics have remained mostly invisible due to a superposition of slow and fast motions, but also due to a lack of adequate imaging technology. Whereas fluorescence techniques require too long integration times, novel coherent techniques such as ROCS microscopy can achieve sufficiently high spatiotemporal resolution. ROCS uses rotating back-scattered laser light from cellular structures and generates a consistently high image contrast at 150 nm resolution and frame rates of 100 Hz-without fluorescence or bleaching. Here, we present an extension of ROCS microscopy that exploits the principles of dynamic light scattering for precise localization, visualization and quantification of the cytoskeleton activity of mouse macrophages. The locally observed structural reorganization processes, encoded by dynamic speckle patterns, occur upon distinct mechanical stimuli, such as soft contacts with optically trapped beads. We find that a substantial amount of the near-membrane cytoskeleton activity takes place on millisecond timescales, which is much faster than reported ever before.