A Photoactivatable Push-Pull Fluorophore for Single-Molecule Imaging in Live Cells

Recent advances in optical imaging with single molecules beyond the diffraction limit (e.g., PALM, FPALM, STORM)1–3 have introduced a new requirement for fluorescent labels: fluorophores must be actively controlled (usually via photoswitching or photo-activation) to ensure that only a single emitter is switched on at a time in a diffraction-limited region. The location of each of these sparse molecules is precisely determined, and a super-resolution image is obtained from the summation of many successive rounds of photoactivation. The ultimate spatial resolution is determined by a number of factors, most importantly the total number of photons detected from each individual molecule.4 Super-resolution imaging by these methods, and cell imaging in general, often use photoswitchable fluorescent proteins, which have the advantage of being genetically targeted;5,6 however, they provide 10-fold fewer photons before photobleaching than good small-molecule emitters.