Photobleaching of fura-2 and its effect on determination of calcium concentrations.

This study was performed to determine the effect of photobleaching on the spectral properties of the calcium-sensitive fluorescent dye fura-2. Fura-2, whether in cells or in calibrating solutions, was found to be bleached when exposed to excitation light. In contrast to the widely held belief, photobleaching altered the spectral properties of the dye. Decomposition of the excitation spectra of partially bleached fura-2 solutions revealed an intermediate that is still fluorescent and is not sensitive to calcium over the same range as fura-2, but which can bind calcium in the millimolar range. The presence of this intermediate violates one of the assumptions on which the ratio method of calibration is based; that is, that the only fluorescent species present are the calcium-bound and the free anion forms of fura-2. Thus, if photobleaching occurs, the ratio method will not give accurate calcium concentration values. We calculate that as little as an 8% loss of total fluorescence intensity is sufficient to produce a large error. Photobleaching of fura-2-loaded cells and fura-2 containing calibrating solutions can be minimized by reducing the oxygen concentration and by reducing the excitation light intensity. Strategies are presented to help maintain a high signal-to-noise ratio in fura-2 fluorescence detection systems, despite a lower excitation intensity so that photobleaching, and the resulting inaccuracies in calculated [Ca2+], can be largely avoided.