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Keywords:

  • actin–myosin motility;
  • biophysics;
  • photophysics;
  • single-molecule studies;
  • superresolution fluorescence microscopy

Abstract

Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of ∼20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of ∼100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of ∼30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to ∼0.18 μm s−1, observed strong bending and disruption of filaments as well as locally immobile fragments.