Single-Molecule Tracking Photoactivated Localization Microscopy to Map Nano-Scale Structure and Dynamics in Living Spines.

Super-resolution microscopy has rapidly become an indispensable tool in cell biology and neuroscience by enabling measurement in live cells of structures smaller than the classical limit imposed by diffraction. The most widely applied super-resolution method currently is localization microscopy, which takes advantage of the ability to determine the position of individual fluorescent molecules with nanometer accuracy even in cells. By iteratively measuring sparse subsets of photoactivatable fluorescent proteins, protein distribution in macromolecular structures can be accurately reconstructed. Moreover, the motion trajectories of individual molecules within cells can be measured, providing unique ability to measure transport kinetics, exchange rates, and binding affinities of even small subsets of molecules with high temporal resolution and great spatial specificity. This unit describes protocols to measure and quantify the distribution of scaffold proteins within single synapses of cultured hippocampal neurons, and to track and measure the diffusion of intracellular constituents of the neuronal plasma membrane.