Certain super-resolution fluorescence microscopy techniques used for biological imaging rely on photoswitching of cyanine dyes. Understanding the mechanism of this photoswitching, in which red laser light switches a dye from a fluorescent to a dark state and subsequent ultraviolet illumination turns the fluorescence back on, could help scientists design improved photoswitchable probes. Using single-molecule imaging and mass spectrometry, Harvard University chemistry professor Xiaowei Zhuang and coworkers tested the role that primary thiols play in facilitating the reaction (J. Am. Chem. Soc., DOI: 10.1021/ja904588g). They propose that the thiol forms a nonfluorescent adduct with the dye, a switching mechanism originally hypothesized by Roger Y. Tsien of the University of California, San Diego, one of the coauthors. Mass spectrometry confirms that the mass difference between the fluorescent and dark states matches the mass of the thiol (β-mercaptoethanol shown). Fragmentation patterns in the mass spectra are consistent with the addition of the thiol to the dye’s polymethine bridge. Such reactivity helps explain why the cyanine dye Cy3, which has a shorter polymethine bridge than Cy5 or Cy7, is unable to switch to the dark state.