Volume 111, Issue 2, Pages 438-451 (July 2016) Optical Mapping of Membrane Potential and Epicardial Deformation in Beating Hearts  Hanyu Zhang, Kenichi Iijima, Jian Huang, Gregory P. Walcott, Jack M. Rogers  Biophysical Journal  Volume 111, Issue 2, Pages 438-451 (July 2016) DOI: 10.1016/j.bpj.2016.03.043 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Selection of emission band. (A and B) Emission spectra recorded during action potential plateau and during rest for blue (450 nm) and cyan (505 nm) excitation, respectively. (C and D) Difference spectra obtained by subtracting the resting from plateau spectra from (A) and (B), respectively. Horizontal bars in (C) and (D) indicate the passband of the emission filter (635 ± 27.5 nm). Plots in (C) and (D) have the same vertical scale. The emission spectra are averages; the gray bands show the 95% confidence intervals for the spectra. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Optical mapping system with two geometry cameras. MC, mapping camera; GC1 and GC2, geometry cameras; F1 and F3, emission filter (590-nm long-pass filter); F2, emission filter (635 ± 27.5-nm band-pass filter). EX, excitation light source units (see Figs. S1 and S2). “Fire” is a digital pulse marking the start of each MC frame. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Evaluation of motion tracking with different marker spacings. (A) Pattern of marker placement. (B) Histograms of distance error with different marker spacings. Distance error is defined as the distance (in pixels) between the estimated image coordinates of site R and the actual image coordinates of site R. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Vm reconstruction on a Langendorff-perfused beating heart. The heart was paced at 500 ms BCL from the LV subepicardium near the apex. (A) Triangulation of trackable markers and location of epicardial sites. The signals in (B) are from epicardial site 1. Blue/cyan: deinterlaced blue- and cyan-elicited signals from the pixel where site 1 is located at the first frame, after spatial filtering. Blue tracked/cyan tracked: deinterlaced blue- and cyan-elicited signals, respectively, after motion tracking and spatial filtering. The bars are CCD signal counts (proportional to fluorescence intensity). Ratio: the motion-corrected Vm signal is the ratio of the blue- to cyan-elicited signals. The bar is dF/F. (C) Reconstructed Vm signals and dF/F from sites 2–5, respectively. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 APDs of monophasic action potential (APDMAP) and reconstructed optical Vm (APDOAP). In three Langendorff-perfused hearts, MAP and Vm signals were simultaneously recorded at different spontaneous or paced activation rates. One-hundred-thirty activations from a total of 23 MAP-Vm site-pairs were analyzed. MAP-Vm site separation was <2 cm in all cases. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Reconstructed Vm signals in the setting of manually induced motion in a heart treated with BDM. Residual motion artifacts are apparent as shifts of Vm baseline. Angular displacement indicates the angle between the local surface normal vector and the optical axis of the mapping camera. Linear displacement indicates 3D displacement of the recording site. (Column A) The heart was rotated about its vertical axis. (Column B) The heart was swung from left-to-right relative to the camera. (Column C) The heart was swung forward and backward relative to the camera. (Column D) The heart was translated forward and backward in the direction parallel to optical axis of the camera. The calibration bars represent 2.5°, 5 mm, and 5%, respectively. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Simultaneously recorded Vm and mechanical contraction from an LV working heart. (A) Action potential propagation (upper rows) and the resulting mechanical contraction (lower rows) from a single beat. Regions in which Vm is unavailable are colored gray. (B) Mechanical contraction (fine line) and the simultaneously recorded Vm signal (bold line) from sites 1 and 2 in (A). The spatial average of shortening is reported at the bottom of each shortening map. Shortening is defined as the most negative eigenvalue of the stretch tensor, U. The heart was paced from the LV apex at 700 ms BCL, and LV preload and afterload pressures were set to 15 and 30 mmHg, respectively. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 8 Altered electromechanical function after coronary artery occlusion. (A) Shortening (negative deformation) or stretch (positive deformation) in the major deformation direction (thin lines) and Vm (thick lines) before and 5 min after coronary artery occlusion. Site 1 is in a perfused region adjacent to the ischemic zone and site 2 is in the ischemic zone. Numbers under Vm show the APDs in milliseconds. (B) Spatial maps of strains before and after occlusion. This is an anterior view of the LV; the base is at the top and the LAD runs along the top-left edge of the mapped region. The bold circle indicates the LAD occlusion site. The strain maps were computed for the times (end-systole) indicated by the arrows in (A). White bars indicate positive strains (stretch); black bars indicate negative strains (shortening). Bar length is proportional to stretch or shortening relative to end-diastole. The bars are oriented in the major/minor directions. End-diastole was taken as the time immediately before action potential upstroke. The heart was paced at 600 ms BCL, and LV preload and afterload pressure values were set to 15 and 30 mmHg, respectively. Biophysical Journal 2016 111, 438-451DOI: (10.1016/j.bpj.2016.03.043) Copyright © 2016 Biophysical Society Terms and Conditions