Nanometre localization of single ReAsH molecules

H. Park, G. T. Hanson, S. R. Duff, P. R. Selvin

Research output: Contribution to journalArticle

Abstract

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 photo-stability 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 2 5 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.

Original languageEnglish (US)
Pages (from-to)199-205
Number of pages7
JournalJournal of Microscopy
Volume216
Issue number3
DOIs
StatePublished - Dec 2004

Keywords

  • Biomolecular motor
  • Localization
  • Nanometric stage
  • ReAsH
  • Step size
  • Total internal reflection fluorescence microscopy

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology

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