Subdiffraction-resolution fluorescence microscopy of myosin-actin motility.
Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of approximately 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 approximately 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of approximately 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 approximately 0.18 microm s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.
Endesfelder
Ulrike
U
Applied Laser Physics and Laser Spectroscopy, Physics Department, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
van de Linde
Sebastian
S
Wolter
Steve
S
Sauer
Markus
M
Heilemann
Mike
M
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D018482:Muscle, Skeletal / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009218:Myosins