Emission Ratiometry for Simultaneous Calcium and Action Potential Measurements with Coloaded Dyes in Rabbit Hearts: Reduction of Motion and Drift

Simultaneous Ca and AP Emission Ratiometry. Introduction: Optical measurements of the cardiac calcium transient (Ca) and transmembrane action potential (AP) may be performed simultaneously with emission ratiometry to lessen motion artifacts and photobleaching effects. We examined changes in emission spectrum in perfused rabbit hearts coloaded with Rh237 and a green‐emitting Ca dye (Fluo‐4 or Oregon Green BAPTA 1) to determine wavelength bands for emission ratiometry and to test whether ratiometry reduces motion artifacts and drift. Methods and Results: A 488‐nm laser illuminated hearts while a spectrofluorometer collected fluorescence from 489 to 838 nm at 1 kHz. Ratiometry with the Ca‐ and AP‐insensitive emission band 663 to 685 nm (IS) as denominator and the Ca‐sensitive band 510 to 532 nm as numerator lessened motion artifacts, which was quantified as a 1.4‐fold increase in relative amplitude of the Ca (P < 0.05) . Ratiometry with the AP‐sensitive band 772 to 794 nm as denominator and the IS as numerator produced a 1.7‐fold increase in relative amplitude of the AP (P < 0.05) . The ratiometry decreased photobleaching‐dependent drift by a factor of 0.6 (P < 0.05) for Ca and 0.45 (P < 0.05) for AP. Conclusion: Simultaneous Ca and AP emission ratiometry reduces motion artifacts and drift in hearts with coloaded dyes.