Single Molecule Analysis

Roderick P. Tas, Trusanne G. A. A. Bos, Lukas C. Kapitein, Tas, Roderick P., Bos, Trusanne G. A. A., Kapitein, Lukas C.

The cytoskeleton is involved in many cellular processes. Over the last decade, super-resolution microscopy has become widely available to image cytoskeletal structures, such as microtubules and actin, with great detail. For example, Single-Molecule Localization Microscopy (SMLM) achieves resolutions of 5-50 nm through repetitive sparse labeling of samples, followed by Point-Spread-Function analysis of individual fluorophores. Whereas initially this approach depended on the controlled photoswitching of fluorophores targeted to the structure of interest, alternative techniques now depend on the transient binding of fluorescently labeled probes, such as the small polypeptide lifeAct that can transiently interact with polymerized actin. These techniques allow for simple multicolor imaging and are no longer limited by a fluorophore's blinking properties. Here we describe a detailed step-by-step protocol to purify, label, and utilize the lifeAct fragment for SMLM. This purification and labeling strategy can potentially be extended to a variety of protein fragments compatible with SMLM.