Bayesian localisation microscopy reveals nanoscale podosome dynamics

We demonstrate a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and Xenon arc lamp illumination. Our Bayesian analysis of blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores which may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame, and unifies the analysis of localization from blinking and bleaching events. By modeling the entire dataset we are able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allows us to reveal the nanoscale dynamics of podosome formation and dissociation with a resolution of 50 nm on a four second timescale.