Cardiomyocytes derived from human induced pluripotent stem cells (hiPS-HCM) have the potential to provide individualized therapies for patients and to test drug candidates for cardiac toxicity. In order for hiPS-CM to be useful for such applications, there is a need for high-throughput technology to rapidly assess cardiac electrophysiology parameters. Here, we designed and tested a fully contactless optical mapping (OM) and optical pacing (OP) system capable of imaging and point stimulation of hiPS-CM in small wells. OM allowed us to characterize cardiac electrophysiological parameters (conduction velocity, action potential duration, etc.) using voltage-sensitive dyes with high temporal and spatial resolution over the entire well. To improve OM signal-to-noise ratio, we tested a new voltage-sensitive dye (Fluovolt) for accuracy and phototoxicity. Stimulation is essential because most electrophysiological parameters are rate dependent; however, traditional methods utilizing electrical stimulation is difficult in small wells. To overcome this limitation, we utilized OP (λ = 1464 nm) to precisely control heart rate with spatial precision without the addition of exogenous agents. We optimized OP parameters (e.g., well size, pulse width, spot size) to achieve robust pacing and minimize the threshold radiant exposure. Finally, we tested system sensitivity using Flecainide, a drug with well described action on multiple electrophysiological properties.
Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart.
The role of hemodynamics in early heart development is poorly understood. In order to successfully assess the impact of hemodynamics on development, we need to monitor and perturb blood flow, and quantify the resultant effects on morphology. Here, we have utilized cardiac optical pacing to create regurgitant flow in embryonic hearts and OCT to quantify regurgitation percentage and resultant morphology. Embryonic quail in a shell-less culture were optically paced at 3 Hz (well above the intrinsic rate or 1.33-1.67 Hz) on day 2 of development (3-4 weeks human) for 5 minutes. The pacing fatigued the heart and led to a prolonged period (> 1 hour) of increased regurgitant flow. Embryos were kept alive until day 3 (cardiac looping - 4-5 weeks human) or day 8 (4 chambered heart - 8 weeks human) to quantify resultant morphologic changes with OCT. All paced embryos imaged at day 3 displayed cardiac defects. The extent of regurgitant flow immediately after pacing was correlated with cardiac cushion size 24-hours post pacing (p-value < 0.01) with higher regurgitation leading to smaller cushions. Almost all embryos (16/18) surviving to day 8 exhibited congenital heart defects (CHDs) including 11/18 with valve defects, 5/18 with ventricular septal defects and 5/18 with hypoplastic right ventricles. Our data suggests that regurgitant flow leads to smaller cushions, which develop into abnormal valves and septa. Our model produces similar phenotypes as found in our fetal alcohol syndrome and velo-cardio-facial/DiGeorge syndrome models suggesting that hemodynamics plays a role in these syndromes as well. Utilizing OCT and optical pacing to understand hemodynamics in development is an important step towards determining CHD mechanisms and ultimately developing earlier treatments.
KEYWORDS: Heart, Microscopy, Signal to noise ratio, 3D metrology, Electrophysiology, 3D image processing, Action potentials, Integrated optics, System integration, Velocity measurements
Optical mapping (OM) using fluorescent voltage-sensitive dyes (VSD) to measure membrane potential is currently the most effective method for electrophysiology studies in early embryonic hearts due to its noninvasiveness and large field-of-view. Conventional OM acquires bright-field images, collecting signals that are integrated in depth and projected onto a 2D plane, not capturing the 3D structure of the sample. Early embryonic hearts, especially at looping stages, have a complicated, tubular geometry. Therefore, conventional OM cannot provide a full picture of the electrical conduction circumferentially around the heart, and may result in incomplete and inaccurate measurements. Here, we demonstrate OM of Hamburger and Hamilton stage 14 embryonic quail hearts using a new commercially-available VSD, Fluovolt, and depth sectioning using a custom built light-sheet microscopy system. Axial and lateral resolution of the system is 14µm and 8µm respectively. For OM imaging, the field-of-view was set to 900µm×900µm to cover the entire heart. 2D over time OM image sets at multiple cross-sections through the looping-stage heart were recorded. The shapes of both atrial and ventricular action potentials acquired were consistent with previous reports using conventional VSD (di-4-ANNEPS). With Fluovolt, signal-to-noise ratio (SNR) is improved significantly by a factor of 2-10 (compared with di-4-ANNEPS) enabling light-sheet OM, which intrinsically has lower SNR due to smaller sampling volumes. Electrophysiologic parameters are rate dependent. Optical pacing was successfully integrated into the system to ensure heart rate consistency. This will also enable accurately gated reconstruction of full four dimensional conduction maps and 3D conduction velocity measurements.
The role of hemodynamics in early heart development is poorly understood. In order to successfully assess the impact of hemodynamics on development, we need to monitor and perturb blood flow, and quantify the resultant effects on morphology. Here, we have utilized cardiac optical pacing to create regurgitant flow in embryonic hearts and OCT to quantify regurgitation percentage and resultant morphology. Embryonic quail in a shell-less culture were optically paced at 3 Hz (well above the intrinsic rate or 1.33-1.67 Hz) on day 2 of development (3-4 weeks human) for 5 minutes. The pacing fatigued the heart and led to a prolonged period (> 1 hour) of increased regurgitant flow. Embryos were kept alive until day 3 (cardiac looping - 4-5 weeks human) or day 8 (4 chambered heart - 8 weeks human) to quantify resultant morphologic changes with OCT. All paced embryos imaged at day 3 displayed cardiac defects. The extent of regurgitant flow immediately after pacing was correlated with cardiac cushion size 24-hours post pacing (p-value < 0.01) with higher regurgitation leading to smaller cushions. Almost all embryos (16/18) surviving to day 8 exhibited congenital heart defects (CHDs) including 11/18 with valve defects, 5/18 with ventricular septal defects and 5/18 with hypoplastic right ventricles. Our data suggests that regurgitant flow leads to smaller cushions, which develop into abnormal valves and septa. Our model produces similar phenotypes as found in our fetal alcohol syndrome and velo-cardio-facial/DiGeorge syndrome models suggesting that hemodynamics plays a role in these syndromes as well. Utilizing OCT and optical pacing to understand hemodynamics in development is an important step towards determining CHD mechanisms and ultimately developing earlier treatments.
Currently, cardiac radiofrequency ablation is guided by indirect signals. We demonstrate an integrated radiofrequency ablation (RFA) and optical coherence tomography (OCT) probe for directly monitoring of the RFA procedure with OCT images in real time. The integrated RFA/OCT probe is modified from a standard commercial RFA catheter, and a newly designed and fabricated miniature forward-viewing cone-scanning OCT probe is integrated into the modified probe. The OCT system is verified with the human finger images, and the results show the integrated RFA/OCT probe can acquire high quality OCT images. The radiofrequency energy delivering function of the integrated probe is verified by comparing the RFA lesion sizes with standard commercial RFA probe. For the standard commercial probe, the average width and depth of the 10 lesions were 3.5 mm and 1.8 mm respectively. For the integrated RFA/OCT probe, the average width and depth of the 10 lesions were 3.6 mm and 1.7 mm respectively. The lesions created by the two probes are indistinguishable in size. This demonstrates that our glass window in the integrated probe has little effect on the RF energy delivery. And the integrated probe is used to monitoring the cardiac RFA procedure in real time. The results show that the RFA lesion formation can be confirmed by the loss of birefringence in the heart tissue. The system can potentially in vivo image of the cardiac wall to aid RFA therapy for cardiac arrhythmias.
Sensory information is conveyed to the central nervous system via small diameter unmyelinated fibers. In general, smaller diameter axons have slower conduction velocities. Selective control of such fibers could create new clinical treatments for chronic pain, nausea in response to chemo-therapeutic agents, or hypertension. Electrical stimulation can control axonal activity, but induced axonal current is proportional to cross-sectional area, so that large diameter fibers are affected first. Physiologically, however, synaptic inputs generally affect small diameter fibers before large diameter fibers (the size principle). A more physiological modality that first affected small diameter fibers could have fewer side effects (e.g., not recruiting motor axons). A novel mathematical analysis of the cable equation demonstrates that the minimum length along the axon for inducing block scales with the square root of axon diameter. This implies that the minimum length along an axon for inhibition will scale as the square root of axon diameter, so that lower radiant exposures of infrared light will selectively affect small diameter, slower conducting fibers before those of large diameter. This prediction was tested in identified neurons from the marine mollusk Aplysia californica. Radiant exposure to block a neuron with a slower conduction velocity (B43) was consistently lower than that needed to block a faster conduction velocity neuron (B3). Furthermore, in the vagus nerve of the musk shrew, lower radiant exposure blocked slow conducting fibers before blocking faster conducting fibers. Infrared light can selectively control smaller diameter fibers, suggesting many novel clinical treatments.
Nerve block can eliminate spasms and chronic pain. Kilohertz frequency alternating current (KHFAC) produces a safe and reversible nerve block. However, KHFAC-induced nerve block is associated with an undesirable onset response. Optical inhibition using infrared (IR) laser light can produce nerve block without an onset response, but heats nerves. Combining KHFAC with IR inhibition [alternating current and infrared (ACIR)] produces a rapidly reversible nerve block without an onset response. ACIR can be used to rapidly and reversibly provide onset-free nerve block in the unmyelinated nerves of the marine mollusk Aplysia californica and may have significant advantages over either modality alone. ACIR may be of great clinical utility in the future.
KEYWORDS: Heart, Optical coherence tomography, 3D image processing, Imaging systems, Velocity measurements, 3D metrology, System integration, Electrophysiology, Image segmentation, Signal to noise ratio
Optical mapping (OM) of cardiac electrical activity conventionally collects information from a three-dimensional (3-D) surface as a two-dimensional (2-D) projection map. When applied to measurements of the embryonic heart, this method ignores the substantial and complex curvature of the heart surface, resulting in significant errors when calculating conduction velocity, an important electrophysiological parameter. Optical coherence tomography (OCT) is capable of imaging the 3-D structure of the embryonic heart and accurately characterizing the surface topology. We demonstrate an integrated OCT/OM imaging system capable of simultaneous conduction mapping and 3-D structural imaging. From these multimodal data, we obtained 3-D activation maps and corrected conduction velocity maps of early embryonic quail hearts. 3-D correction eliminates underestimation bias in 2-D conduction velocity measurements, therefore enabling more accurate measurements with less experimental variability. The integrated system will also open the door to correlate the structure and electrophysiology, thereby improving our understanding of heart development.
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