FLASH PHOTOLYTIC CONTROL OF THE CA-CONCENTRATION Understanding biological signal transduction systems requires detailed information of the ex- act mechanisms involved in such complex pro- cesses. In cardiac myocytes for example, the ac- tion potential is transduced into the mechanical activity of the contractile filaments, a process re- ferred to as excitation-contraction coupling (ec- coupling; for a recent review see [1]). A major step in this signal transduction cascade is the re- lease of Ca 2+ from intracellular stores, the sarco- plasmic reticulum (SR). The release itself is trig- gered by a Ca 2+ influx across the sarcolemma Photolysis of caged compounds characterized by ratiometric confocal microscopy: A new approach to homogeneously control and measure the calcium concentration in cardiac myocytes P.Lipp, C.Lüscher and E.Niggli Department of Physiology, University of Bern, Bern, Switzerland Abstract - Here we describe the subcellularly uniform control of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) by flash photolysis of caged Ca 2+ or a caged Ca 2+ buffer. A mixture of the two Ca 2+ indicators fluo-3 and Fura-Red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca 2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells with the indicator mixture together with either DM-Nitrophen or diazo-2 and to measure changes in the membrane current. An in-vivo calibration was performed to convert the fluo-3/Fura-Red-fluorescence ratios into [Ca 2+ ] values. The resulting calibration curve suggested an apparent k D of 1.6 μM, R max of 2.15, R min of 0.08 and a Hill-coefficient of 0.75 for the mixture. Controlled rup- ture of the cell membrane revealed a large fraction of immobile intracellular Fura-Red fluo- rescence that may account for the reduced in-vivo R max value when compared to the in- vitro value of 3.1. In cardiac myocytes flash photolytic release of Ca 2+ from DM-Nitrophen generated inwardly directed Na + /Ca 2+ exchange currents and Ca 2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca 2+ ] jumps regardless of the discharged flash energy. Ca 2+ signals evoked by L-type Ca 2+ currents (I Ca ) could be terminated rapidly in a spatially homogeneous manner by UV- flash photolysis of diazo-2. No side-effects of the photolytic products of DM-Nitrophen or diazo-2 with the mixture of fluo-3/Fura-Red were detectable in our experiments. The com- bination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca 2+ ] i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca 2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged com- pounds are available. CELL CALCIUM (1996) 255-266.