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Biophys J, January 2001, p. 549-561, Vol. 80, No. 1
¶
*Center for Light Microscope Imaging & Biotechnology,
Dept. of Biological Sciences, ¶Pittsburgh
NMR Center for Biomedical Research, Carnegie Mellon University,
Pittsburgh, Pennsylvania and Cardiovascular Institute,
§Dept. of Bioengineering, University of Pittsburgh,
Pittsburgh, Pennsylvania 15213 USA
Both theoretical and experimental results are presented
for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2.
Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of
the changes in quantum yield of Rhod-2 upon binding calcium in the
heart in which multiple scattering effects are predominant. Theoretical
modeling and experimental results demonstrate that both reflected
absorbance and fluorescence emission are attenuated linearly with
Rhod-2 washout. According to this relation, a ratiometric method using
fluorescence and absorbance is validated as a measure of the quantum
yield of calcium-dependent fluorescence, enabling determination of the
dynamics of cytosolic calcium in the perfused mouse heart. The
feasibility of this approach is confirmed by experiments quantifying
calcium transients in the perfused mouse heart stimulated at 8 Hz. The
calculated cytosolic calcium concentrations are 368 ± 68 nM and
654 ± 164 nM in diastole and systole, respectively. Spectral
distortions induced by tissue scattering and absorption and errors
induced by the geometry of the detection optics in the calcium
quantification are shown to be eliminated by using the ratio method.
Methods to effectively minimize motion-induced artifacts and to monitor
the oxygenation status of the whole perfused heart are also discussed.
Biophys J, January 2001, p. 549-561, Vol. 80, No. 1
© 2001 by the Biophysical Society 0006-3495/01/01/549/13 $2.00
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