@article {2078, title = {Photoactivated localization microscopy (PALM) of adhesion complexes. (With commentary)}, journal = {Current Protocols in Cell Biology}, volume = {Chapter 4}, year = {2008}, month = {2008 Dec}, pages = {21}, abstract = {

Key to understanding a protein{\textquoteright}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 (approximately 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 approximately 20 nm resolution in 5 to 30 min in fixed cells.

Commentary: A paper spearheaded by Hari which gives a thorough description of the methods and hardware needed to successfully practice PALM, including cover slip preparation, cell transfection and fixation, drift correction with fiducials, characterization of on/off contrast ratios for different photoactivted fluorescent proteins, identifying PALM-suitable cells, and mechanical and optical components of a PALM system.

}, keywords = {Animals, Cell Line, Cell-Matrix Junctions, Cells, Fluorescence, Humans, Microscopy, Photobleaching, Transfection}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0421s41}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19085989}, author = {Shroff, Hari and White, Helen and Betzig, Eric} }