FACTORS SHAPING THE CONFOCAL IMAGE OF THE CALCIUM SPARK IN CARDIAC-MUSCLE-CELLS

The interpretation of confocal line-scan images of local [Ca2+](i) tra nsients (such as Ca2+ sparks in cardiac muscle) is complicated by unce rtainties in the position of the origin of the Ca2+ spark (relative to the scan line) and by the dynamics of Ca2+-dye interactions. An inves tigation of the effects of these complications modeled the release, di ffusion, binding, and uptake of Ca2+ in cardiac cells (producing a the oretical Ca2+ spark) and image formation in a confocal microscope (aft er measurement of its point-spread function) and simulated line-scan i mages of a theoretical Ca2+ spark (when it was viewed from all possibl e positions relative to the scan line). In line-scan images, Ca2+ spar ks that arose in a different optical section or with the site of origi n displaced laterally from the scan line appeared attenuated, whereas their rise times slowed down only slightly. These results indicate tha t even if all Ca2+ sparks are perfectly identical events, except for t heir site of origin, there will be an apparent variation in the amplit ude and other characteristics of Ca2+ sparks as measured from confocal line-scan images. The frequency distributions of the kinetic paramete rs (i.e., peak amplitude, rise time, fall time) of Ca2+ sparks were ca lculated 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 l ine 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 involve d in the evaluation of Ca2+-spark image parameters from noisy experime ntal recordings. The mean value of the resulting amplitude distributio ns underestimates the brightness of in-focus Ca2+ sparks because large numbers of out-of-focus Ca2+ sparks are detected (as small Ca2+ spark s). The distribution of peak amplitudes may split into more than one s ubpopulation even when one is viewing stereotyped Ca2+ sparks because of the discrete locations of possible SR Ca2+-release sites in mammali an ventricular heart cells.