Simultaneous widefield voltage and interferometric dye-free optical mapping quantifies electromechanical waves in human iPSC-cardiomyocytes

Abstract

Coupled electro-mechanical waves define heart's function in health and disease. Genetic abnormalities, drug-triggered or acquired pathologies can disrupt and uncouple these waves with potentially lethal consequences. Optical mapping of electrical waves using fluorescent dyes or genetically-encoded sensors in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) offers mechanistic insights into cardiac conduction abnormalities. Interferometric dye-free/label-free wave mapping (without specific sensors) presents an alternative, likely capturing the mechanical aspects of cardiac conduction. Because of its non-invasive nature and spectral flexibility (not restricted to a specific excitation wavelength), it is an attractive chronic imaging tool in iPSC-CMs, as part of all-optical high-throughput platforms. In this study, we developed simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used: 1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-CMs; 2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature CMOS cameras; 3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence imaged) and mechanical (dye-free imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wave-front tortuosity. During regular pacing, the dye-free imaged conduction velocity and the electrical wave velocity are correlated; both modalities being sensitive to pharmacological uncoupling and both dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover strong frequency-dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CM inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology

“…The total cost of the system, including two cameras, all optical components, all light sources and drivers, and the mechanical scaffolding is less than <$15,000, substantially cheaper compared to existing optical mapping solutions. Key enabling technologies for building such a system are as follows: (1) improved low-cost machine-vision CMOS cameras 28 , 30 , 31 over the last 5 years, offering sufficient sensitivity, suitable speed, spatial resolution, and spectral response to work in the demanding low-light conditions of fast fluorescence imaging in cell culture; (2) developments in LEDs, that allow easy on-off control and provide a lower-cost, stable alternative to incandescent lamps and lasers, so that they entered the optical mapping field almost 20 years ago; 32 , 33 (3) improved voltage-sensitive probes in the near-infrared region developed recently, that offer sufficient SNR at high speed; 27 , 34 and (4) development of optogenetic actuation tools, which offer optical control of cell function 20 , 35 …”

“…In future studies, we intend to combine the two measurements using temporal multiplexing onto the same camera, as we have done in a microscopic version of all-optical electrophysiology. 18 Comprehensive electromechanical responses obtained in human iPSC-CMs 28 over space and time under various pacing conditions can be used to develop better in silico tools for prediction of drug action. 55 , 56 …”

“…Camera 1 collects the emitted fluorescence to image or , whereas simultaneously camera 2 collects the dye-free signal visualizing the mechanical contraction waves. 28 The main imaging lens is a 50-mm camera lens (0.95 F-stop, Navitar Inc., Ottawa, Canada); two additional 25-mm aspherical condenser lenses (focal length 20 mm, NA 0.6, Thorlabs) are placed in front of each camera to focus the image. The system is based on oblique transillumination for the multimodal imaging of cardiac waves.…”

“…Figures 1(a) and 1(b) show the system, which uses two low-cost cameras, both Basler acA720-520um (Basler AG, Germany), having 720×540 pixels, 6.9 μm per pixel, capable of running at up to 525 frames per second (fps). Camera 1 collects the emitted fluorescence to image Vm or [Ca2+false]i, whereas simultaneously camera 2 collects the dye-free signal visualizing the mechanical contraction waves 28 . The main imaging lens is a 50-mm camera lens (0.95 F-stop, Navitar Inc., Ottawa, Canada); two additional 25-mm aspherical condenser lenses (focal length 20 mm, NA 0.6, Thorlabs) are placed in front of each camera to focus the image.…”

“…Here we focus exclusively on the Vm and [Ca2+false]i records obtained under various conditions. Custom software 19 , 28 , 29 in MATLAB, MathWorks, Natick, Massachusetts, was used to preprocess the data (baseline correction, Bartlett spatial filter, and locally weighted temporal regression filter) before event detection. Activation maps were constructed based on the detected times of activation, and the analysis software quantified the following functional parameters of interest: APD at 80% repolarization (APD80), calcium transient duration (CTD) at 80% recovery (CTD80), and conduction velocity (CV) (cm/s).…”