FlAsH-PALM: super-resolution pointillist imaging with FlAsH-tetracysteine labeling.
Abstract
Super-resolution light microscopy including pointillist methods based on single molecule localization (e.g., PALM/STORM) allow to image protein structures much smaller than the diffraction limit (200-300 nm). However, commonly used labeling strategies such as antibodies or protein fusions have several important drawbacks, including the risk to alter the function or distribution of the imaged proteins. We recently demonstrated that pointillist imaging can be performed using the alternative labeling technique known as FlAsH, which better preserves protein function, is compatible with live cell imaging, and may help reach single nanometer resolution. We applied FlAsH-PALM to visualize HIV integrase in isolated virions or infected cells, allowing us to obtain sub-diffraction resolution images of this enzyme's spatial distribution and analyze HIV morphology without altering viral replication. The technique should also prove useful to image delicate proteins in intracellular vesicles and organelles at high resolution. Here, we present a detailed protocol in order to facilitate the application of FLAsH-PALM to other proteins and biological structures.