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

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