Photoactivated Localization Microscopy (PALM) of Adhesion Complexes

Abstract

Key to understanding a protein's biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction‐limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (∼200 nm) have emerged. Here, both single‐ and dual‐color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein‐filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA‐FP) tags necessary to achieve PALM images at ∼20 nm resolution in 5 to 30 min in fixed cells. Curr. Protoc. Cell Biol. 41:4.21.1‐4.21.27. © 2008 by John Wiley & Sons, Inc.