Principal Results: I: Synthesis and study of improved fluorescence indicators tailored for laser confocal microscopy: (Hagins, Yoshikami, Rice, Whittaker) A chromophore based on a known laser dye has been developed with the following useful properties: a) Narrow absorption with high extinction centered at 532 nm, the emission line of frequency-doubled Nd-YAG lasers. b) High fluorescence efficiency with a small Stokes shift, narrow fluorescence emission bandwith and great resistence to photobleaching and metabolic degradation. c) Ready adaptability to chemical coupling with a variety of functional groups that can facilitate probing of cellular environments and binding sites. d) Small size, weak non-specific binding to cellular components and ready permeation through lipid portions of cell membranes. So far, four derivatives have been prepared to function as intracellular potential probes, probes of hydrophobic regions, and precursors for labels to study drug-binding sites in cells and tissues. II: Construction of a confocal microscope for study of living cells and tissues with good time resolution: (Yoshikami, Hagina) A confocal microscope has been built on a standard optical table using standard modular optical components with the following useful features: a) A low-power diode-pumped laser light source is suitably dispersed by refractive and diffractive optics elements to give controlled illumination of cell cultures or thin tissues. b) Taking advantage of the coherent nature of the laser source together with diffracting plates, illumination patterns consisting of multiple slits or arrays of spots are formed in the specimen plane that are conjugate with selected pixels of a CCD camera. Using programmed selection of responses downloaded from the camera pixels and sweeping the illumination pattern over the specimen plane with a galvanometer mirror, a confocal image can be had in a small fraction of the time required by a single-beam confocal microscope. A holographic filter eliminates the backscattered exciting light from the fluorescent image. c) The use of readily available optical components has made the cost of the microscope a small fraction of standard commercial confocals, probably within the budgets of high schools and college teaching laboratories. d) Preliminary experiments have been undertaken to measure permeability and binding of our new fluorophores to cells in tissue culture and to estimate membrane potentials.
