Using Total Fluorescence Increase (Signal Mass) to Determine the Ca²⁺ Current Underlying Localized Ca²⁺ Events

The feasibility of determining localized Ca²⁺ influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca²⁺ fluorescence transients (SCCaFTs), due to Ca²⁺ entry through single openings of Ca²⁺-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 (ΔF_total or “signal mass”) associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca²⁺ 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 ΔF_total is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca²⁺ influx and ΔF_total was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ current underlying Ca²⁺ sparks resulting from Ca²⁺ release from intracellular stores via ryanodine receptors in the same preparation.