Super-resolution imaging by localization microscopy.

Photoactivated localization microscopy (PALM) and the related technique of Stochastic optical reconstruction microscopy (STORM) are super-resolution imaging methods based on the precise localization of single molecules. Instruments based on these techniques are now commercially available and are capable of generating images with lateral resolutions in the tens of nanometers range. Here, we give an overview of the current state of this technology including live-cell and 3D PALM and provide an in-depth protocol for performing PALM experiments in a fixed cell monolayer. This includes both the instrumentation/acquisition aspects and the data analysis required for generating quantitative, super-resolution data of molecular distributions. In this example, the system under investigation will be fixed HeLa cells transfected with the photo-switchable fluorescent protein PS-CFP2 targeted to the plasma membrane by fusion to the N-terminus of the protein kinase Lck.