Figure 3.

Calibration of Ca-dependent fluorescence in the presence of caged Ca. (A) Increasing fluorescence during progressive photolysis of DM-nitrophen at λ = 340 nm (both for excitation and photolysis). Spectra were obtained from 50 μM DM-nitrophen dissolved in pipette solution. (B) Fluorescence quenching of Ca-saturated fluo-5F by increasing concentrations of caged (DM-N, left) or photoreleased (DM-N*, center) DM-nitrophen (0, 1, 3, 10, 30, 100, 300, 1,000, 3,000, 5,000 μM). The diagram illustrates the higher quenching efficacy of DM-N* at concentrations >100 μM. λ_exc = 475 nm. (C) Decline of the fluorescence of Ca-saturated fluo-5F during a series of 50-ms flashes due to the increasing ratio of DM-N*/DM-N. The same flash protocol was used for the determination of Ca sensitivity in this study. Fluorescence quenching causes a ∼15% reduction of the signal in cells (black traces) and in cell-free pipette solution (red trace). (D) Calibration of fluo-5F fluorescence. The fluorescence of 20 μM fluo-5F was measured in a series of Ca-standard solutions (•), which was also used to calibrate a Ca electrode. The fluorescence was then measured in two sets of solutions containing either 5 mM DM-N (♦) or 5 mM DM-N* (◯), as well as different concentrations of total Ca²⁺, resulting in the indicated free Ca concentrations ([Ca²⁺]), which were determined using the Ca electrode. The apparent K_D of fluo-5F (0.80–0.84 μM) was not significantly changed by DM-N or DM-N*.