The interpretation of confocal line-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 Ca(2+)-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 Ca(2+)-release sites and 2) fixed position of the scan line going through a set of possible SR Ca(2+)-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 Ca(2+)-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 Ca(2+)-release sites in mammalian ventricular heart cells.
Animals Calcium