INTRODUCTION Light-sensitive caged compounds allow rapid concentra- tion jumps of biologically-active constituents inside and outside cells by UV-light flash photolysis [1]. A range of caged compounds has been developed and used to probe the kinetics of various biological signaling systems. Combined with spatially confined two-photon excitation photolysis (TPP) of the caged compound, the technique can also be used to deliver biologically-active con- stituents in a spatially-controlled manner (this is with dif- fraction-limited precision), and then detect the biological response with a confocal laser-scanning microscope. So far this combination of techniques has been applied to map the distribution of receptors on cell surfaces [2] and to generate spatially-confined artificial Ca 2+ sparks inside cardiac myocytes [3]. When using available caged compounds for the libera- tion of Ca 2+ two problems have surfaced: first, the absorbance spectrum for two-photon absorption is not simply the single-photon absorbance spectrum with the wavelength divided by two [4]; and second, the two-pho- ton absorption cross-section of fluorescent indicators and caged compounds is generally much smaller than for single-photon excitation. These features result in ineffi- cient excitation of available caged Ca 2+ compounds at 690 nm, the shortest available wavelength of the Ti:Sapphire lasers most frequently used for two-photon excitation. This means that, for example, caged indicators that work well with UV-laser flash photolysis at 355 nm show very little photolysis with TPP at 710 nm. So far, DM-nitrophen [5,6] has turned out to be the most suit- able photolabile Ca 2+ chelator for TPP, although its sig- nificant affinity for Mg 2+ (K d = 2.5 μM) limits its usefulness when investigating signaling systems that may be Mg 2+ sensitive or that require Mg 2+ -ATP. Research Two-photon and UV-laser flash photolysis of the Ca 2+ cage, dimethoxynitrophenyl-EGTA-4 F. DelPrincipe, 1 M. Egger, 1 G. C. R. Ellis-Davies, 2 E. Niggli 1 1 Department of Physiology, University of Bern, Bern, Switzerland 2 Department of Physiology, MCP Hahnemann University, Philadelphia, USA Summary We report efficient two-photon and UV-laser flash photolysis of dimethoxynitrophenyl-EGTA-4 (DMNPE-4), a newly-developed photolabile Ca 2+ -specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg 2+ affinity (≈7 mM), a K d for Ca 2+ of 19 nM, a quantum yield of 0.20 and changes its Ca 2+ affinity by 21 000- fold upon photolysis. Two-photon excitation photolysis (TPP) experiments were performed with a Ti:Sapphire laser in solutions containing DMNPE-4 with either 0 or 10 mM Mg 2+ and compared to that of the widely used Ca 2+ cage, DM- nitrophen (K d for Ca 2+ 5 nM, K d for Mg 2+ 2.5 μM, quantum yield 0.18, affinity change 600 000-fold). The resulting Ca 2+ signals were recorded with the fluorescent Ca 2+ indicator fluo-3 and a laser-scanning confocal microscope in the line- scan mode. In vitro, photolysis of DMNPE-4:Ca 2+ produced Ca 2+ -release signals that had comparable amplitudes and time courses in the presence and absence of Mg 2+ . However, photorelease of Ca 2+ from DM-nitrophen was obviated by the presence of Mg 2+ . In patch-clamped isolated cardiac myocytes, equivalent TPP results were obtained in analogous experiments. Single-photon excitation of DMNPE-4 by Nd:YAG laser flashes produced Na–Ca exchange currents of comparable amplitude in the absence and presence of Mg 2+ . However, only very small currents were observed in DM- nitrophen solution containing 10 mM Mg 2+ . In conclusion, both DMNPE-4 and DM-nitrophen undergo TPP, however, only DMNPE-4 exhibits efficient release of Ca 2+ in the presence of Mg 2+ . 85 Cell Calcium (1999) 25 (1), 85–91 © Harcourt Brace & Co. Ltd 1999 Article no. ceca.1998.0009 Received 29 June 98 Revised 12 October 98 Accepted 13 October 98 Correspondence to: Ernst Niggli, Department of Physiology, University of Bern, Bühlplatz 5, CH-3012 Bern, Switzerland. Tel.: +41 31 631 87 17; fax: +41 31 631 46 11; e-mail: niggli@pyl.unibe.ch