An infrared optical pacing system for screening cardiac electrophysiology in human cardiomyocytes

Figures

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  Fig 1. System diagram. Fully optical high-throughput platform for measuring electrophysiological parameters in hCM monolayers with beating rate control. A 96-well plate sits atop an XYZ stage. Infrared laser light (1465 nm or 1860 nm) is directed to the center of a single well through a long pass dichroic mirror (DM1) and uncoated custom objective. Light from a high power LED (470 nm or 530 nm) is reflect by a second dichroic mirror (DM2) and DM1 to the same well. Fluorescence from the well is reflected by DM1 and passed by DM2 to filter F1 (535±30nm) and focused onto a CCD camera.

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  Fig 2. Demonstration of optical pacing and activation recording. Optical pacing in an hCM monolayer from a single well of a 96-well plate. Top shows an action potential recording (Vm) and a Ca2+ transient recording from a single pixel in separate wells at room temperature (RT). Middle shows action potentials recorded at 34°C with faster pacing rates. Bottom shows an activation map during optical pacing at 0.5 Hz indicating the local time of maximum Vm derivative (dVm/dt) during the action potential upstroke. Vectors superimposed on contours represents local impulse conduction velocity. The laser spot (red, 400 μm) was imaged while temporarily using a visible wavelength to shows the location of pacing.

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  Fig 3. Measurement of threshold energies for optical pacing. (A) Assessment of threshold by a search pattern through a fixed number of monolayers, N = 30 total. To compare threshold under different conditions, 50% capture probability was calculated (see text for details). (B) Cell density at three different values (3.1 x 102, 1.0 x 103 and 2.0 x 103 cells/mm2), N = 30 for each point. (C) Pulse widths were varied between 5 ms and 40 ms, N = 18 for each point. (D) Spot size was varied between 4 x 10−4 and 2.9 x 10−3 cm2, N = 30 for each point. Thresholds required to achieve 50% pacing probability are plotted against radiant exposure per pulse (B, D) and irradiance (C). Error bars reflect the standard error (A) and standard deviation (B-D). In panels C and D, * indicates a statistically significance (p < 0.001) decrease compared to the smallest pulse width and spot size.

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  Fig 4. Comparison between electrical and optical measurements of action potentials. Comparison of action potential measured using FluoVolt (cyan) and a sharp microelectrode (orange) simultaneously (top) and at a much higher resolution showing the action potential upstroke (middle). All action potential recordings are normalized to a 100 mV amplitude. In both plots, action potentials recorded with FluoVolt and a sharp microelectrode are highly correlated. The action potential upstroke derivative (dVm/dt) for these examples (bottom left) and over all recordings were identical.

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  Fig 5. Effect of FluoVolt on electrical activity. Left panel shows phototoxic effects of FluoVolt when using various excitation intensities. Top shows that at 40% (178 mW/cm2, red) and 24% (109 mW/cm2, purple) of maximum excitation light, significant prolongation of APD is observed over 20 seconds of continuous excitation. In contrast, at 10% (50 mW/cm2, blue) of maximum, no APD changes are observed. Action potential recordings (inset) from separate wells at each light level including baseline (black, first action potential recorded) show that signal quality is preserved. The left bottom graph shows the phototoxic effect on depolarization, as quantified by activation time delay (time from a fiducial point to maximum dVm/dt of each consecutive action potential). Only 40% excitation light had an effect on depolarization time as indicated by a delay in activation time by about 2000 ms. Right panel (top) shows the effect of FluoVolt washout on the action potential over a 90 minute time period, and the superior signal fidelity of FluoVolt compared to di-4-ANEPPS averaged from the same number of pixels (bottom).

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  Fig 6. Effect of optical pacing on action potential upstroke. Top shows action potential upstrokes normalized to a 100 mV action potential that were recorded within 0.5 mm of the stimulation site, before pacing began (baseline, 0 min) and then after continuous pacing at 5 min, 10 min, 15min, 20 min, and 25 min. Bottom shows maximum dVm/dt during the upstroke for sites within 0.5 mm of the stimulation site (n = 4). No significant differences were observed between baseline (0 min) and all other time points.

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  Fig 7. System validation using Flecainide and Quinidine. Activation time (top) and APD (bottom) contour maps measured at baseline (left) and after 0.3 μM Flecainide (right) in a single well of a 96-well plate. Mean local conduction velocity and APD for each map are shown below. Site of optical pacing is shown by red spot in activation contours. Flecainide decreased conduction velocity and increased APD. To the right are summary data for mean local conduction velocity (top) and APD (bottom) before (CNTL) and after Flecainide (n = 7, n = 6) and Quinidine (1.0 μM, n = 7).