Using total fluorescence increase (signal mass) to determine the Ca$^2+$ current underlying localized Ca$^2+$ events.
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J. Gen. Physiol. 124 (3): 259--272 (September 2004)

The feasibility of determining localized Ca$^2+$ influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca$^2+$ fluorescence transients (SCCaFTs), due to Ca$^2+$ entry through single openings of Ca$^2+$-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca$^2+$ influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca$^2+$ influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca$^2+$ was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca$^2+$ current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca$^2+$ current underlying Ca$^2+$ sparks resulting from Ca$^2+$ release from intracellular stores via ryanodine receptors in the same preparation.
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