TY - GEN
T1 - Quantitative measurement of calcium transients in perfused mouse heart using Rhod-2 fluorometry
AU - Du, Congwu
AU - MacGowan, Guy A.
AU - Farkas, Daniel L.
AU - Koretsky, Alan P.
PY - 1999
Y1 - 1999
N2 - We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye Rhod-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37°C, and is stimulated at 8 Hz, with 100μg of Rhod-2 bolused through the perfusate. After a 25-minute washout period, approx. 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O 2, thus minimizing interference due to changes in tissue oxygenation. Ca 2+-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33±9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of Rhod-2 through the perfusate, the reflected absorbance between 524 nm (Rhod-2 sensitive) and 589 nm (Rhod-2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATP ase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368±68nM and 654±164nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.
AB - We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye Rhod-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37°C, and is stimulated at 8 Hz, with 100μg of Rhod-2 bolused through the perfusate. After a 25-minute washout period, approx. 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O 2, thus minimizing interference due to changes in tissue oxygenation. Ca 2+-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33±9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of Rhod-2 through the perfusate, the reflected absorbance between 524 nm (Rhod-2 sensitive) and 589 nm (Rhod-2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATP ase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368±68nM and 654±164nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.
UR - https://www.scopus.com/pages/publications/0032643810
M3 - Conference contribution
AN - SCOPUS:0032643810
SN - 0819430722
SN - 9780819430724
T3 - Proceedings of SPIE - The International Society for Optical Engineering
SP - 214
EP - 219
BT - Proceedings of SPIE - The International Society for Optical Engineering
T2 - Proceedings of the 1999 Advances in Fluorescence Sensing Technology
Y2 - 24 January 1999 through 27 January 1999
ER -