Photolysis of caged calcium using a low-cost flash unit: efficacy analysis with a calcium selective electrode

Photolysls of caged calcium (Nitr!B, Calbiochem) can be used to study calcium dependent processes such as excitation-contraction coupling and muscular mechanics. Expensive high energy light sources are routinely used for UV light exposure, but this study describes an ahernatfve low cost xenon flash unit constrwted in our labor@tory. A 3W J short arc xenon flash lamp (Heimann) was mounted in an e#ptfcal reflecW and driven by a modiffed Metz 60 CT 4 photoflash unit up to 240 J Input energy and 4 ms 20 fl cuvette containing a test solution was placed In a complementary An ion selectfve calcium electrode was used to measure the free calcium conwWsWn [c$*J before and after flash in test solutions containing 1.00 mM Nitr5 in combinutkm with different added [Ca2$. Using this technique we eWmatedthatlflash nl.00mW@tr!5 increased the free [ca2+) from lo-’ to 1.1 x la6 M. When the added [ 2.3 x lo”’ M, the used Nit6 behaved as a strong cafclum cheiator & was less than untoaded with calcium. It is concluded that a phystokqfc@y releva k& can be evoked by photolysis of Nlt6 using a low cost (approximately $fMw) xenon ftash unit, and that ion selective Ca electrodes can be adeqwWy used to monitor the reauitfng changes in [Ca2’]. Photolabile calcium chelators have been designed to calcium compound Nitr5 is appmximately 250 study intracellular calcium dependent processes mJ/cm2 in a wavelength band from 320-370 run 11.21. These ‘caged’ calcium compounds are useful [9,101. The commonly used frequency doubled ruby tools to bypass rate limiting steps in muscular excilaser [6,7] meets these specifications in a flash duratationcontraction coupling 13-51. When using the tion of 25 ns, but is very expensive. When a longer chelators intracellularly [6-g], it is possible to evoke flash duration. f 4 ms, is acceptable, the commera stepwise increase in the intracellular calcium concentration [Ca2’] by exposing the cells to high incially available xenon flashlamp developed by Rapp and oath [ll] can be used Even at one fourth of ten&y ultra-violet (W) light. The minimum enthepriceofth:laser,thisisstillnotaveryeconergy density required for photolysis of the caged omical alternative. We decided to develop a UV