Suppression of motion artifacts in fluorescence spectroscopy of perfused hearts

Fluorescence spectroscopy of beating hearts has been used previously to measure intracellular regulators of function. Unfortunately, heart motion could introduce a spurious motion artifact (MA), influencing the measured fluorescence intensity ('signal'). To suppress MA, a ratio (or difference) has been calculated previously between the signal and an intensity reference detected at a different wavelength ('reference'). However, no studies have attempted to evaluate or optimize the efficiency of MA suppression. MA suppression was evaluated using reflected excitation light or fluorescence as reference. In addition, the MA contribution to the intensity ratio from a fluorescent dye, indo-1, was quantified. A reflected light reference resulted in poor suppression of MA. The use of a fluorescence reference resulted in suppression that was inversely related to the detection wavelength difference (Δ) between the reference and signal. Therefore optimal MA suppression was obtained using a fluorescence reference at a wavelength close to the signal. For Δ = 60 nm, MA was suppressed from ~10 to <2%. Finally, suppressed MA (Δ = 60 nm) accounted for <10% of the indo-1 ratio fluctuations.