Imaging Ca with a Fluorescent Rhodol.

Ca mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca-sensitive fluorophores has played an instrumental role in our understanding of the temporal and spatial dynamics of Ca. Coupling Ca-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca 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 Ca sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σ), making them excellent candidates for incorporation into Ca-sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca sensor 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca binding constant of 240 nM and a 10-fold turn response to Ca. RCS-1 effectively absorbs infrared light and has a σ of 76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca fluctuations and cultured neurons using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca sensing using two-photon illumination in neurons.