Imaging Ca2+ with a Fluorescent Rhodol

Ca2+ mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca2+-sensitive fluorophores has played an instrumental role in our understanding of the temporal and spatial dynamics of Ca2+. Coupling Ca2+-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca2+ affinities suited to a variety of cellular contexts. One underdeveloped area is the use of hybrid rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca2+ sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σTPA), making them excellent candidates for incorporation into Ca2+-sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca2+ sensor 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca2+ binding constant of 240 nM and a 10-fold turn response to Ca2+. RCS-1 effectively absorbs infrared lig...