Determination of intracellular free calcium ion distribution with fluorescent calcium reagent Fura-2 using video image processing

We constructed an instrument for determining the distribution of intracellular free Ca2+ concentration( [Ca2+]i) using fluorescent Ca indicator Fura-2. This instrument consists of an inverted vertical light fluorescence microscope attached to a 75 W xenon lamp and automatic changer of excitation filters. Image data, recorded by high sensitive SIT camera, are processed by personal computer, and displayed on VDT with pseudocolor. We improved the drug applicator for rapid stimulation of cells and accuracy measurement. The [Ca2+]i estimated with Fura-2 resulted in a lower concentration in comparison with other methods as bioluminescent protein Aequorin, Ca microelectrode, etc. It appears that Fura-2 may interact with intracellular proteins. We used a new Ca concentration buffer containing 80 mg/ml bovine serum albumin (BSA). As for the influence of BSA, fluorescence excitation spectra of Fura-2 alters reduction at 340 nm peak intensity, enlargement at 380 nm, and constant at 360 nm. Also, the appearance dissociation constant of Fura-2 for Ca2+ changed from 109 nM to 138 nM by adding 80 mg/ml BSA. When using the calibration curve calculated by BSA containing the buffer, [Ca2+] i NG108-15 cells with stimulation of 80 mM KCl were increased 185 nM to 1072 nM. These [Ca2+]i almost agreed with the estimated concentration from other methods. Increased [Ca2+]i with KCl stimulation is due to opening voltage sensitive Ca channels present on cell membranes. Fluorescence intensity excited at 340 nm from the KCl stimulated cells increase whole aspect of the cells. But the pseudocolor image showed the peripheral part of the cells to rapidly increase, and then central part to do so slowly. These results due to fluorescence intensity differed with cell thickness that central part of cell show an averaged [Ca2+]i change and peripheral part are a nearly real.