Nanometre localization of single ReAsH molecules

ReAsH is a red-emitting dye that binds to the unique sequence Cys-Cys-Xaa-Xaa-Cys-Cys (where Xaa is a noncysteine amino acid) in the protein. We attached a single ReAsH to a calmodulin with an inserted tetracysteine motif and immobilized individual calmodulins to a glass surface at low density. Total internal reflection fluorescence microscopy was used to image individual ReAsH molecules. We determined the centre of the distribution of photons in the image of a single molecule in order to determine the position of the dye within 5 nm precision and with an image integration time of 0.5 s. The photostability of ReAsH was also characterized and observation times ranging from several seconds to over a minute were observed. We found that 2-mercaptoethanesulphonic acid increased the number of collected photons from ReAsH molecules by a factor of two. Individual ReAsH molecules were then moved via a nanometric stage in 25 or 40 nm steps, either at a constant rate or at a Poisson-distributed rate. Individual steps were clearly seen, indicating that the observation of translational motion on this scale, which is relevant for many biomolecular motors, is possible with ReAsH.