Calcium measurements in perfused mouse heart: Quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory

Both theoretical and experimental results are presented for the quantitativ e detection of calcium transients in the perfused mouse heart loaded with t he calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detect ed from the mouse heart. These models allow correlation of the measured spe ctral intensities with the relative quantity of Rhod-2 in the heart and mea surement 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 absorban ce and fluorescence emission are attenuated linearly with Rhod-2 washout. A ccording to this relation, a ratiometric method using fluorescence and abso rbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium i n the perfused mouse heart. The feasibility of this approach is confirmed b y experiments quantifying calcium transients in the perfused mouse heart st imulated 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 mi nimize motion-induced artifacts and to monitor the oxygenation status of th e whole perfused heart are also discussed.