Engineering Studies towards Simultaneous Imaging of Membrane Potential and Calcium Transients in Beating Hearts

Abstract

Optical mapping uses fluorescent reporters to image propagation of signals like transmembrane potential (Vm) and/or calcium transients (CaT) in the heart with high spatiotemporal resolution. Simultaneous optical mapping of transmembrane potential and intracellular free calcium has been described in motionless ex vivo heart preparations, but simultaneous optical mapping of voltage and calcium in beating hearts is complicated by motion artifact and has yet to be reported. Studying these two parameters in beating hearts could produce invaluable information about the interactions between them under normal and pathophysiologic conditions. This thesis presents hardware development and design studies to demonstrate the feasibility of simultaneous Vm and CaT mapping inbeating hearts. Our overall design is to load reporters for both voltage and calcium. Dyes are excited with two bands on alternating camera frames and two emission bands are recorded from each cardiac site. This produces four signals every two frames, and, with judicious filter choice, those signals include Vm + motion, CaT + motion, and motion only. The motion only signal can be used to cancel motion artifact in the other two. An optical splitter is needed to relay fluorescence emission to two cameras that record different wavelengths. In addition, color mixing is critical to avoid artifacts from spatial heterogeneity of excitation light. Two optical splitters were developed and optimized to record the above signals and a color mixing device was built that enabled independent filtering of two different LED wavelengths. A series of experiments in organotypic slices of ventricular tissue and ex vivo swine hearts suggest two promising designs: (1) Di-4-ANEQ(F)PTEA (Vm dye)/Rhod-4 (CaT dye); green (525 nm) and amber (590 nm) excitation light; 540–560 nm and 700 nm longpass emission bands. (2) Di-4-ANBDQBS (Vm dye)/Rhod-2 (CaT dye); green and amber excitation; 550–580 nm and 700 nm longpass emission bands. With the optimized optical splitters, the promising spectral designs, and the color mixing device, simultaneous imaging of Vm and CaT in beating hearts should soon be realized.