DOI: 10.1002/asia.201301334 Energy Transfer in Aminonaphthalimide-Boron-Dipyrromethene (BODIPY) Dyads upon One- and Two-Photon Excitation: Applications for Cellular Imaging Daniel Collado, [a] Patricia Remón, [b] Yolanda Vida, [a] Francisco Najera, [a] Pratik Sen, [c] Uwe Pischel,* [b] and Ezequiel Perez-Inestrosa* [a] Introduction Boron dipyrromethene (BODIPY) dyes [1–3] are nowadays widely commercialized chromophores that have attracted immense interest as versatile tools in biological chemistry, for example, as pH sensors in intracellular environments, [4] as probes in fluorescence imaging, [5–9] or as agents in photo- dynamic therapy. [10–14] Furthermore, they have been used in chemosensing, [8, 15–17] molecular switching, [18, 19] and for the re- alization of logic operations. [12, 13, 15] These dyes are character- ized by large molar absorption coefficients (e) and high fluo- rescence quantum yields (F fl ), resulting in extraordinary bright emitting (e  F fl ) fluorophores. [1–3] However, the gen- erally very small Stokes shifts of BODIPY dyes may cause re-absorption or effects from excitation-light scattering, which is a potential drawback for their application, especial- ly in bioimaging. One strategy to bypass this problem is the integration of BODIPY dyes as acceptors in bi- or multi- chromophoric arrays, some of them qualifying as energy transfer cassettes where donor and acceptor units are elec- tronically non-conjugated. [8, 17, 20–26] The electronic energy transfer (EET) from a donor with adequate photophysical characteristics to the BODIPY moiety may proceed through space (often Fçrster resonance energy transfer) [21, 22, 26] or through a bond mechanism; [7, 8, 23, 24] actually, both mecha- nisms may act in parallel. [21] This yields an artificial increase in the Stokes shift, also known as virtual or pseudo-Stokes shift. A complementary approach that evolved from techni- cal advances in multiphoton techniques [27, 28] builds on the two-photon excitation of BODIPY dyes, which facilitates the use of near-infrared laser sources and separates the exci- tation spectrally from the emission through upconver- sion. [5, 6, 29, 30] In this context, two-photon excitation (TPE) fluorescence laser-scanning microscopy has opened a steadily expanding field of in vivo imaging of tissues, being a tool for the diagnosis and therapy monitoring of pathological condi- [a] Dr. D. Collado, Dr. Y. Vida, Dr. F. Najera, Prof. E. Perez-Inestrosa Department of Organic Chemistry University of Malaga 29071 Malaga (Spain) and Andalusian Center for Nanomedicine and Biotechnology - BION- AND 29590 Malaga (Spain) Fax: (+ 34) 952137565 E-mail : inestrosa@uma.es [b] P. Remón, Dr. U. Pischel CIQSO - Center for Research in Sustainable Chemistry and Department of Chemical Engineering, Physical Chemistry, and Organic Chemistry University of Huelva 21071 Huelva (Spain) Fax: (+ 34) 959219983 E-mail: uwe.pischel@diq.uhu.es [c] Dr. P. Sen Department of Chemistry Indian Institute of Technology Kanpur Kanpur 208 016, U.P. (India) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301334. Abstract: Aminonaphthalimide– BODIPY energy transfer cassettes were found to show very fast (k EET  10 10 –10 11 s 1 ) and efficient BODIPY fluorescence sensitization. This was ob- served upon one- and two-photon exci- tation, which extends the application range of the investigated bichromo- phoric dyads in terms of accessible ex- citation wavelengths. In comparison with the direct excitation of the BODIPY chromophore, the two- photon absorption cross-section d of the dyads is significantly incremented by the presence of the aminonaphthali- mide donor [d  10 GM for the BODIPY versus 19–26 GM in the dyad at l exc = 840 nm ; 1 GM (Goeppert– Mayer unit) = 10 50 cm 4 s molecule 1 photon 1 ]. The electronic decoupling of the donor and acceptor, which is a precondition for the energy transfer cassette concept, was demonstrated by time-dependent density functional theory calculations. The applicability of the new probes in the one- and two- photon excitation mode was demon- strated in a proof-of-principle approach in the fluorescence imaging of HeLa cells. To the best of our knowledge, this is the first demonstration of the merg- ing of multiphoton excitation with the energy transfer cassette concept for a BODIPY-containing dyad. Keywords: bioimaging · boron · energy transfer · fluorescence · two- photon absorption Chem. Asian J. 2014, 9, 797 – 804  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 797 FULL PAPER