Separation of calcium transients and motion artifact using frequency filtering of fluorescent signals arising from the perfused mouse heart

To eliminate the influence of motion artifacts on measurements of intracellular calcium, a technique was developed using frequency domain analysis which filters motion artifact from the calcium transient signal arising from the perfused mouse heart using the calcium sensitive, fluorescent dye Rhod-2. The perfused mouse heart was stimulated at 8 Hz and placed in a water-jacketed chamber at 37°C. After a washout period following Rhod-2 loading, ∼6-fold increase in fluorescence above background was detected spectrofluorimetrically at 589 nm when excited at 524 nm. Calcium dependent fluorescence transients mixed with motion artifacts and system noise were measured. Simultaneously, heart motion was monitored by recording the reflected excitation light from the heart. A Fourier transform was utilized to separate signals arising from the fluorescence transients and those resulting from motion in the frequency domain. Several major steps were adopted to implement the algorithm for elimination of motion as well as system instability from the transient signals. These included 1) extracting the fluorescence calcium transient signal from the raw data in the frequency domain by subtracting the motion recorded using the reflectance of excitation light, 2) digitally filtering out the random noise using multiple bandpass filters centralized at harmonic frequencies of the 8 Hz signal, and 3) extracting high frequency noise with the Kernel method. Comparing the processed signal of transients acquired with excessive motion artifact to transients acquired with minimal motion obtained by immobilizing the heart against the detection window demonstrated that thefiltering techniques helped minimize the effects of motion.