Ultrafast and Single Molecule spectroscopy of Photoswitchable Fluorophores containing an Oxazine Auxochrome

ARAOZ, BEATRIZ; CADRANEL, ALEJANDRO; CUSIDO, JANET; SWAMINATHAN,SUBRAMANI; RAYMO, FRANCISCO M.; HODAK, JOSE; BOSSI, MARIANO L.

Cordoba

Congreso; 16th International Congress of Photobiology; 2014

ULTRAFAST AND SINGLE MOLECULE SPECTROSCOPY OF PHOTOSWITCHABLE FLUOROPHORES CONTAINING AN OXAZINE AUXOCHROME Beatriz Araoz,a Alejandro Cadranel, a Janet Cusido, b Subramani Swaminathan,b Françisco M. Raymo,b José H. Hodak,a and Mariano L. Bossia a INQUIMAE-DQIAyQF, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina b Department of Chemistry, University of Miami, Coral Gables, Florida, USA. Advances in far field fluorescence microscopy have pushed spatial resolution below the diffraction limit and into the scale of single biomolecules. Such techniques require probes having states with different emission properties. In particular, photochromic compounds possessing dark and bright states can act as molecular photoswitches, thus becoming useful fluorescent probes. A reliable switching mechanism, high brightness contrast between states, and detection at the single molecule level, are key properties for achieving high resolution meaningful images. We have introduced a family of photochromic molecular switches whose emission can be modulated by optical stimulations, through the opening of an oxazine ring on the core of the chromophore.[1-2] Intracellular imaging in fixed cells was achieved by the inclusion of such markers into polymeric micelles, that were endocyted by the cells before the fixation step.[3] However, the utilization of such compounds using general labeling protocols such as immunolabeling has resulted elusive due to poor solubility in water, and a strong dependence of the photochemical properties on solvent polarity that challenges photoswitching in aqueous solutions. We present recent advances in establishing these compounds as valuable markers for fluorescence nanoscopies, aiming to overcome the solubility/polarity issues. On one hand, we show the spectroscopic properties of photoswitch-IgG adducts at the single molecule level. We observe a strong dependency on the environment both on the average burst time (residing time in the emissive isomer) and in the brightness. Adducts with different labeling ratios are being explored. On the other hand, ultrafast spectroscopic measurements shed light on the mechanism involved in signal modulation. A fast photo-conversion with a single characteristic time of ca. 20 ps was observed in acetonitrile solutions, confirming the fast reaction of the switches under illumination with 375 nm light. The ultimate aim of this study is to feed back the design of the fluorescent markers. Acknowledgments JHH and MLB thanks grants from CONICET, ANPCYT and UBA. [1] E. Deniz, M. Tomasulo, J. Cusido, I. Yildiz, M. Petriella, M. L. Bossi, S. Sortino, and F. M. Raymo, J. Phys. Chem. C, 2012, 116, 6058. [2] S. Swaminathan, M. Petriella, E. Deniz, J. Cusido, J. Baker, M. Bossi, F. Raymo, J. Phys. Chem. A, 2012, 116, 9928. [3] M. Petriella, E. Deniz, S. Swaminathan, M. J. Roberti, F. M. Raymo, M. L. Bossi, Photochem. Photobiol., 2013, 89, 1391.