Photoactivated localization microscopy (PALM) of adhesion complexes.
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 (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.
Shroff
Hari
H
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
White
Helen
H
Betzig
Eric
E
D000818:Animals; D002460:Cell Line; D021982:Cell-Matrix Junctions / Q000378:metabolism*; D002477:Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D014162:Transfection