Live-cell PALM of intracellular proteins in neurons

Synaptic communication is a tightly regulated process requiring the coordinated assembly and regulation of a multitude of scaffolding and signaling molecules critical to synaptic maintenance and plasticity. The small spatial scales of the synapse and the dendritic spines which house the postsynaptic machinery have limited the direct visualization of their structure in living cells. A number of imaging methods have evolved in recent years with the capability to resolve structures within living neurons at resolution far exceeding that achievable by confocal microscopy. We will focus in this chapter on this laboratory's experience with Photoactivated Localization Microscopy (PALM) over the last several years and attempt to provide a useful and pragmatic guide to its utilization in the study of neuronal structures and their dynamic reorganization within living neurons. PALM offers several advantages over other imaging modalities. First, it permits the localization of single molecules with precision on the order of tens of nanometers and the rendering of neuronal structures at high resolution. As this technique utilizes expressed proteins tagged with photocon-vertible fluorescent proteins, it permits time-resolved measurement of both the behavior of single molecules and the reshaping of protein networks within living neurons. We provide a framework to aid the reader through the initial process of designing or selecting an appropriate optical system, optimizing acquisition and hardware settings, and the analysis of single-molecule data. © 2014 Springer Science+Business Media New York.