Factors shaping the confocal image of the calcium spark in cardiac muscle cells

The interpretation of confocal fine-scan images of local [Ca²⁺](i) transients (such as Ca²⁺ sparks in cardiac muscle) is complicated by uncertainties in the position of the origin of the Ca²⁺ spark (relative to the scan line) and by the dynamics of Ca²⁺-dye interactions. An investigation of the effects of these complications modeled the release, diffusion, binding, and uptake of Ca²⁺ in cardiac cells (producing a theoretical Ca²⁺ spark) and image formation in a confocal microscope (after measurement of its point-spread function) and simulated line-scan images of a theoretical Ca²⁺ spark (when it was viewed from all possible positions relative to the scan line). In line-scan images, Ca²⁺ sparks that arose in a different optical section or with the site of origin displaced laterally from the scan line appeared attenuated, whereas their rise times slowed down only slightly. These results indicate that even if all Ca²⁺ sparks are perfectly identical events, except for their site of origin, there will be an apparent variation in the amplitude and other characteristics of Ca²⁺ sparks as measured from confocal line-scan images. The frequency distributions of the kinetic parameters (i.e., peak amplitude, rise time, fall time) of Ca²⁺ sparks were calculated for repetitive registration of stereotyped Ca²⁺ sparks in two experimental situations: 1) random position of the scan line relative to possible SR Ca²⁺-release sites and 2) fixed position of the scan line going through a set of possible SR Ca²⁺-release sites. The effects of noise were incorporated into the model, and a visibility function was proposed to account for the subjective factors that may be involved in the evaluation of Ca²⁺-spark image parameters from noisy experimental recordings. The mean value of the resulting amplitude distributions underestimates the brightness of in-focus Ca²⁺ sparks because large numbers of out-of-focus Ca²⁺ sparks are detected (as small Ca²⁺ sparks). The distribution of peak amplitudes may split into more than one subpopulation even when one is viewing stereotyped Ca²⁺ sparks because of the discrete locations of possible SR Ca²⁺-release sites in mammalian ventricular heart cells.