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Nanometre localization of single ReAsH molecules
Author(s) -
PARK H.,
HANSON G. T.,
DUFF S. R.,
SELVIN P. R.
Publication year - 2004
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.0022-2720.2004.01416.x
Subject(s) - total internal reflection fluorescence microscope , molecule , fluorescence , chemistry , fluorescence microscope , nanometre , photon , microscopy , crystallography , biophysics , materials science , nanotechnology , analytical chemistry (journal) , optics , physics , chromatography , biology , organic chemistry
Summary 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.