1. Members of a family of fluorescent calcium-sensitive indicator dyes including Calcium Green, Calcium Orange, Calcium Crimson, Flo-3 and Rhod- 2 have been transferred to retinal rods of frogs by the silylated-dye method described in previous reports in 1993-4. Dye levels exceeding 1 micromolar in the outer segments of rods have been achieved with one hour incubations. The dyes partially emerge from the labeled cells when the cell membranes are ruptured osmotically. 2. Studies by image-intensified fluorescence microscopy of the responses of th dyes to calcium ions show that the indicators are almost completely bound to the rod disk membranes and are almost totally insensitive to changes in calcium ion activity in the bathing solutions. Analysis of the polarization of the fluorescence emitted by the dyes Calcium Green, Calcium Crimson, and Calcium Orange show that the E-vectors of the emitting oscillators are parallel to the surfaces of the disk membranes, while that of the membrane responsive fluorophore N, N'Bis(dansyl cystine) is parallel to the fatty acid side chains in the membranes. (Yoshikami, Hagins, Sahu). 3. A family of precursors for chelation of divalent ions, including Ca++, have been synthesized with functional groups ready for coupling to fluorescent chromophores.
The aim i s to produce a group of ned Calcium- sensitive fluorophores that are not inactivated by binding to cell organelles as are the above-studied dyes available commercially. A total of eight chelators have been made and studies of the coupling reactions are under way. (Hagins, Mani). 4. A pyroelectric photocalorimeter that uses evanescent-waves from stripped optical fibers to introduce photo excitation into small fluid samples is under construction. (Hagins)??5. A pyroelectric calorimeter wit improved signal-to-noise properties is under construction for studying phototransduction in frog retinal rods at very low light intensities. Improvements will make it possible to measure heat bursts from nucleotide hydrolysis within 1-2 minutes after the tissue is introduced into the calorimeter. (Yoshikami, Hagins).
