Lights, camera, path splitter: a new approach for truly simultaneous dual optical mapping of the heart with a single camera

Abstract

Background: Optical mapping of transmembrane voltage and intracellular calcium is a powerful tool for investigating cardiac physiology and pathophysiology. However, simultaneous dual mapping of two fluorescent probes remains technically challenging. We introduce a novel, easy-to-use approach that requires a path splitter, single camera and excitation light to simultaneously acquire voltage and calcium signals from whole heart preparations, which can be applied to other physiological models - including neurons and isolated cardiomyocytes. Results: Complementary probes were selected that could be excited with a single wavelength light source. Langendorff-perfused hearts (rat, swine) were stained and imaged using a sCMOS camera outfitted with an optical path splitter to simultaneously acquire two emission fields at high spatial and temporal resolution. Voltage (RH237) and calcium (Rhod2) signals were acquired concurrently on a single sensor, resulting in two 384 × 256 images at 814 frames per second. At this frame rate, the signal-to-noise ratio was 47 (RH237) and 85 (Rhod2). Imaging experiments were performed on small rodent hearts, as well as larger pig hearts with sufficient optical signals. In separate experiments, each dye was used independently to assess crosstalk and demonstrate signal specificity. Additionally, the effect of ryanodine on myocardial calcium transients was validated - with no measurable effect on the amplitude of optical action potentials. To demonstrate spatial resolution, ventricular tachycardia was induced - resulting in the novel finding that spatially discordant calcium alternans can be present in different regions of the heart, even when electrical alternans remain concordant. The described system excels in providing a wide field of view and high spatiotemporal resolution for a variety of cardiac preparations. Conclusions: We report the first multiparametric mapping system that simultaneously acquires calcium and voltage signals from cardiac preparations, using a path splitter, single camera and excitation light. This approach eliminates the need for multiple cameras, excitation light patterning or frame interleaving. These features can aid in the adoption of dual mapping technology by the broader cardiovascular research community, and decrease the barrier of entry into panoramic heart imaging, as it reduces the number of required cameras.

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

“…A wide-pupil 50 mm/F0.95l lens is attached to the front of the image splitter. This configuration results in adequate emission light separation, as previously validated 43,44 . 4.…”

“…Accordingly, high spatial resolution images (in the described setup 1240 x 1024 total, or 620 x 512 per channel, 6.5 µm pixel size) are often spatially binned during or post-acquisition ( Figure 5C). Image processing can be performed to generate activation and repolarization maps using custom algorithms 23,33,43,45 (Figure 3D), with the activation time of each pixel on the heart was defined as the maximum derivative of the action potential or calcium transient upstroke. Example of epicardial pacing at cycle length of 400 ms (S1−S1), which was used for imaging experiments.…”

“…6. After temporal parameters are calculated, generate isochronal maps to depict aspects of a single action potential or calcium transient across the entire imaged epicardial surface using custom algorithms23,33,43,45,46…”

Age-dependent changes in electrophysiology and calcium handling: implications for pediatric cardiac research

“…The path splitter was configured with a dichroic mirror (660ϩ nm, Chroma Technologies) that passed RH237 emission and Signal processing. Following image acquisition, signal processing and data analysis were performed using a custom MATLAB script, as previously described (23)(24)(25). A region of interest (ROI) was selected in identical locations on the split image of the heart for each raw image.…”

KairoSight: Open-Source Software for the Analysis of Cardiac Optical Data Collected From Multiple Species

“…Cardiovascular research has been advanced by the development of parameter-sensitive dyes, which can be used to monitor transmembrane voltage (V m ) and intracellular calcium (Ca 2+ ) within cardiac tissue (Jaimes et al, 2019a). Fluorescent reporters allow researchers to monitor action potentials and calcium transients in excitable cells and identify underlying alterations in ionic currents.…”