Reversible Photoactivatable Fluorophores                 2008–2015

Reversible Photoactivatable FluorophoresThe advent of super-resolution fluorescence imaging prompted Professor Raymo to realize that his mechanisms to switch fluorescence for all-optical processing can be adapted to reconstruct images with spatial resolution at the nanometer level. As a result, he aimed his research program at the development of photoswitchable fluorophores, based on the reversible ring opening and closing of his photochromic oxazines, with the ultimate goal of perfecting synthetic dyes for sub-diffraction imaging. Indeed, diffraction prevents the differentiation of fluorescent probes separated by sub-wavelength distances and restricts the spatial resolution of conventional fluorescence images to length scales that are, at least, two orders of magnitude greater than the physical dimensions of molecules. This fundamental limitation can be overcome with the sequential detection of closely-spaced fluorophores at the single-molecule level, if their emissions are engineered to switch on at different times. In search of optimal structural designs to satisfy the stringent photochemical and photophysical requirements for this transformative sub-diffraction-imaging strategy, his research group developed a family of compounds integrating a fluorescent chromophore and a photochromic oxazine in the same molecular skeleton, over the course of two funding cycles from the National Science Foundation. The resulting fluorophore–photochrome dyads convert from a nonemissive to an emissive state under irradiation and revert thermally to the original form for multiple switching cycles (a). Furthermore, their emissive state is sufficiently bright for detection at the single-molecule level (b). This unique combination of photochemical and photophysical properties enables the detection of closely-spaced dyads individually at different intervals of time and the sequential reconstruction of fluorescence images with sub-diffraction resolution. His research group demonstrated that his compounds allow the visualization of individual polymer nanoparticles positioned only a few tens of nanometers away from each other (c. Additionally, he devised a general experimental protocol to conjugate these photoswitchable compounds to antibodies, label intracellular targets selectively and visualize the immunolabeled structures at the nanometer level (d). In fact, these photoswitchable probes permit the acquisition of fluorescence images with a spatial resolution that is simply not accessible with conventional fluorophores and can evolve into invaluable analytical tools for imaging applications demanding the visualization of nanostructures.

Reversible Photoactivatable Fluorophores


  • “Luminescent Switches for Fluorescence Nanoscopy”: National Science Foundation, CHE-0749840 (PI: F. M. Raymo), $ 489,450, 02/01/08–01/31/12
  • “Blinking Fluorophores by Design”: National Science Foundation, CHE-1049860 (PI: F. M. Raymo), $ 493,705, 09/01/11–08/31/15

Selected Articles

  • “Fast Fluorescence Photoswitching in a BODIPY–Oxazine Dyad with Excellent Fatigue Resistance”: E. Deniz, S. Sortino, F. M. Raymo, J. Phys. Chem. Lett., 2010, 1, 1690–1693
  • “Photoswitchable Fluorescent Dyads Incorporating BODIPY and [1,3]Oxazine Components”: E. Deniz, S. Ray, M. Tomasulo, S. Impellizzeri, S. Sortino, F. M. Raymo, J. Phys. Chem. A, 2010, 114, 11567–11575
  • “Fluorescence Switching with a Photochromic Auxochrome”: E. Deniz, S. Sortino, F. M. Raymo, J. Phys. Chem. Lett., 2010, 1, 3506–3509
  • “Photoactivatable Fluorophores for Super-Resolution Imaging Based on Oxazine Auxochromes”: E. Deniz, M. Tomasulo, J. Cusido, I. Yildiz, M. Petriella, M. L. Bossi, S. Sortino, F. M. Raymo, J. Phys. Chem. C, 2012, 116, 6058−6068
  • “Fast Fluorescence Switching within Hydrophilic Supramolecular Assemblies”: J. Cusido, M. Battal, E. Deniz, I. Yildiz, S. Sortino, F. M. Raymo, Chem. Eur. J., 2012, 18, 10399–10407
  • “A Photochromic Bioconjugate with Photoactivatable Fluorescence for Superresolution Imaging”: J. Cusido, S. Shaban Ragab, E. R. Thapaliya, S. Swaminathan, J. Garcia-Amorós, M. J. Roberti, B. Araoz, M. M. A. Mazza, S. Yamazaki, A. M. Scott, F. M. Raymo, M. L. Bossi, J. Phys. Chem. C, 2016, 120, 12860–12870

Selected Reviews

  • “Fluorescence Modulation with Photochromic Switches in Nanostructured Constructs”: I. Yildiz, E. Deniz, F. M. Raymo, Chem. Soc. Rev., 2009, 38, 1859–1867
  • “Fast and Stable Photochromic Oxazines for Fluorescence Switching”: E. Deniz, M. Tomasulo, J. Cusido, S. Sortino, F. M. Raymo, Langmuir, 2011, 27, 11773–11783
  • “Photoactivatable Synthetic Dyes for Fluorescence Imaging at the Nanoscale”: F. M. Raymo, J. Phys. Chem. Lett., 2012, 3, 2379–2385
  • “Photoactivatable Synthetic Fluorophores”: F. M. Raymo, Phys. Chem. Chem. Phys., 2013, 15, 14840–14850