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Live cell imaging of focal adhesions requires a sufficiently high temporalresolution, which remains a challenging task for super-resolution microscopy.We have addressed this important issue by combining photo-activatedlocalization microscopy (PALM) with super-resolution optical fluctuationimaging (SOFI). Using simulations and fixed cell focal adhesion images, weinvestigated the complementarity between PALM and SOFI in terms of spatial andtemporal resolution. This PALM-SOFI framework was used to image focal adhesionsin living cells, while obtaining a temporal resolution below 10 s. Wevisualized the dynamics of focal adhesions, and revealed local mean velocitiesaround 190 nm per minute. The complementarity of PALM and SOFI was assessed indetail with a methodology that integrates a quantitative resolution andsignal-to-noise metric. This PALM and SOFI concept provides an enlargedquantitative imaging framework, allowing unprecedented functional explorationof focal adhesions through the estimation of molecular parameters such as thefluorophore density and the photo-activation and photo-switching rates.

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions