Vr. Pratusevich et Cw. Balke, FACTORS SHAPING THE CONFOCAL IMAGE OF THE CALCIUM SPARK IN CARDIAC-MUSCLE-CELLS, Biophysical journal, 71(6), 1996, pp. 2942-2957
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.