Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Abteilung NanoBiophotonik Max Planck Institut für biophysikalische Chemie Am Fassberg 11 37077 Göttingen Germany

Department of Chemistry University of Leuven Celestijnenlaan 200F B 3001 Heverlee Belgium

Department of Radioelectronics FEE Czech Technical University Prague Technická 2 166 27 Prague 6 Czech Republic

Laboratory of Nanoscale Biology and Laboratoire d'Optique Biomédicale STI IBI Ecole Polytechnique Fédérale de Lausanne Station 17 CH 1015 Lausanne Switzerland

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