Uncaging

Polychrome: A powerful light source for uncaging

High time resolution measurement of [Ca²⁺]_i and simultaneous release of calcium from DM-Nitrophen. "Because the fluorescence excitation wavelength (of fura-2) also covers the UV range that can photolyze DMN (DM-Nitrophen), we can use the fluorescence excitation light to measure [Ca²⁺]_i and photorelease Ca²⁺ simultaneously. .... The R p (rate of Ca²⁺ release) for 380 nm and 390nm excitation in our setup was found ... to be 1.497 +/- 0.005 s^-1 and 0.966 +/- 0.050 s^-1 (mean +/- S.D.), respectively." (See Reference 2 below, Xu et al.)

Comment: From the rate constants above you can calculate that 50% of the DM-Nitrophen will be photolyzed in 460 ms (380 nm) and 715ms(390 nm). Higher rates of photolysis of DM-Nitrophen are reached at shorter wavelength (e.g. 350 nm). Prof. Robert S. Zucker (Univ. of California at Berkeley) reported that the Polychrome II (a predecessor of the Polychrome V) at his setup could photolyse 50% of DM-Nitrophen in about 200 ms.

Figure 1: An example experiment using a Polychrome II for photolysis of DM-Nitrophen. fura-2 was the Ca²⁺ indicator. At the Ca²⁺ sensitive wavelength (here 380 nm) of fura-2 the intensity of the fura excitation light is still so bright that Ca²⁺ was substantially released from DM-Nitrophen. This way you can elevate Ca²⁺ and measure its concentration simultaneously.

Read more about photolysis with steady light sources:

• Zucker R.S. 1993. The calcium concentration clamp: spikes and reversible pulses using the photolabile chelator DM-nitrophen. Cell Calcium 14 :87-100
• Xu T., Naraghi M., Kang H., Neher E. 1997. Kinetic studies of Ca²⁺ binding and Ca²⁺ clearance in the cytosol of adrenal chromaffin cells. Biophys. J. 73 :532-545