Photolysis of caged compounds characterized by ratiometric confocal microscopy: a new approach to homogeneously control and measure the calcium concentration in cardiac myocytes.
Here we describe the subcellularly uniform control of the intracellular Ca2+ concentration ([Ca2+]i) by flash photolysis of caged Ca2+ or a caged Ca2+ buffer. A mixture of the two Ca2+ indicators Fluo-3 and Fura-red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca2+ 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 to [Ca2+] values. The resulting calibration curve suggested an apparent KD of 1.6 microM, Rmax of 2.15, Rmin of 0.08 and a Hill-coefficient of 0.75 for the indicator mixture. Controlled rupture of the cell membrane revealed a large fraction of immobile intracellular Fura-red fluorescence that may account for the reduced in vivo Rmax value when compared to the in vitro value of 3.1. In cardiac myocytes, flash photolytic release of Ca2+ from DM-nitrophen generated inwardly directed Na+/Ca2+ exchange currents and Ca2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca2+] jumps regardless of the discharged flash energy. Ca2+ signals evoked by L-type Ca2+ currents (ICa) 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 combination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca2+]i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged compounds are available.
Lipp
P
P
Department of Physiology, University of Bern, Switzerland. LIPP@PYL.UNIBE.CH
Lüscher
C
C
Niggli
E
E
D000085:Acetates; D000814:Aniline Compounds; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents; D003979:Diazonium Compounds; D005029:Ethylenediamines; D005260:Female; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D007091:Image Processing, Computer-Assisted; D007093:Imidazoles; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D009206:Myocardium / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D010642:Phenoxyacetates; D010782:Photolysis; D051381:Rats; D017208:Rats, Wistar; D014466:Ultraviolet Rays; D014966:Xanthenes
D000085:Acetates; D000814:Aniline Compounds; D001572:Benzofurans; D002614:Chelating Agents; D003979:Diazonium Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D007093:Imidazoles; D010642:Phenoxyacetates; D014966:Xanthenes; C063685:diazo-2; C082877:fura red; C057014:DM-nitrophen; C059715:Fluo-3; D002118:Calcium