Single Molecule Localization Microscopy

The strategy for fluorescence microscopy with currently the highest resolution involves limiting the number of fluorophores active in each frame of a long series of acquisitions and then combining the entire data set into one image.  Photo-Activation Localization Microscopy (PALM) accomplishes this with fluorescent proteins with particular characteristics, such as PA-GFP, while Stochastic Optical Reconstruction Microscopy (STORM) employs chemical fluorophores to perform essentially the same thing.  In each case the centroid of each fluorophore is identified in each frame creating an image which is essentially devoid of the point spread functions (PSFs) that limit resolution.  The maximum resolution gain that can be realized can be over ten-fold, and 3D and live-cell variants of these techniques have been developed but can be complex and not well suited to some particular applications.

See our chapter on super-resolution microscopy for more details.