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# Open-Source Multiparametric Optocardiography

## Abstract

Since the 1970s fluorescence imaging has become a leading tool in the discovery of mechanisms of cardiac function and arrhythmias. Gradual improvements in fluorescent probes and multi-camera technology have increased the power of optical mapping and made a major impact on the field of cardiac electrophysiology. Tandem-lens optical mapping systems facilitated simultaneous recording of multiple parameters characterizing cardiac function. However, high cost and technological complexity restricted its proliferation to the wider biological community. We present here, an open-source solution for multiple-camera tandem-lens optical systems for multiparametric mapping of transmembrane potential, intracellular calcium dynamics and other parameters in intact mouse hearts and in rat heart slices. This 3D-printable hardware and Matlab-based RHYTHM 1.2 analysis software are distributed under an MIT open-source license. Rapid prototyping permits the development of inexpensive, customized systems with broad functionality, allowing wider application of this technology outside biomedical engineering laboratories.

## Introduction

Electrophysiology of excitable cells, such as cardiac myocytes and neurons, has been studied for more than a century using various electrode-based techniques to assess extracellular and transmembrane potentials, ionic currents and currents between two electrically coupled cells. However, these approaches are intrinsically limited in their ability to study excitable cells in relation to its surrounding multicellular environment with which it interacts, without compromising the spatial and temporal resolution of the techniques. Optical imaging was first developed to study axon electrophysiology as early as 1968 to overcome some of these limitations1,2 (Fig. 1a). Optical mapping utilizes either intrinsic fluorescent signals or fluorescent dyes that are sensitive to important physiological parameters such as NADH+, transmembrane potential, intracellular calcium concentration, etc., in order to study the related functions of the cell. Optical action potentials and NADH were first recorded from the heart in 19763,4. The development of ratiometric techniques using BAPTA-based compounds as a fluorescent intracellular calcium indicator allowed for correction of artifacts from bleaching or changes in illumination intensity and focus5. Over the next decade, progress in optocardiography was due to the development of CCD cameras6,7,8, which allowed dramatic increase in spatial resolution. In 1987, two parameters – voltage and NADH+, were recorded from the same heart9. Later developments in this methodology included increasing the signal-to-noise ratio (SNR) and spatiotemporal resolution using tandem lens systems10. In 1994, the simultaneous recording of voltage and calcium from the same heart was reported11. Further advancements included the implementation of LED light sources12, ratiometric techniques for measuring voltage13, panoramic imaging systems14, and CMOS cameras15. A more recent development eliminates the requirement for uncoupling agents by using a motion tracking technique to subtract motion artifact, permitting the study of the relationship between electrical and mechanical activity in intact hearts16. Due to these developments, optocardiography has become a key tool in understanding the mechanisms of cardiac arrhythmias17,18,19.

The complexity of an optical mapping system is determined by the range of applications required. Single or multiple parameters can be recorded simultaneously by splitting emitted light using dichroic mirrors and using multiple photodetectors. In its simplest form, a fluorescent probe is excited and the emitted light is collected using a single lens and directed through an emission filter to a photodetector (Fig. 1b). The excitation light delivery could be episcopic or epicentric. Episcopic light source delivers light at an angle to the field of view, in contrast to precise illumination of just the field of view by directing the excitation light through the objective lens in the epicentric source. In a more sophisticated version of the system, two or more fluorescent dyes are perfused through the tissue and excited simultaneously. The emitted light is then collected by a lens, split into separate paths using a dichroic mirror, filtered, and directed to individual photodetectors (Fig. 1c). This system can be further expanded to record multiple parameters using a tandem lens approach (Fig. 1d). A tandem lens system implements two lenses per light path, with the fronts of the lenses facing each other to achieve an infinity-corrected light path between them. The infinity-corrected light path then allows the addition of multiple photodetectors to record several parameters simultaneously (Fig. 1e). The use of a tandem lens macroscope system has also demonstrated reduction in photobleaching and phototoxicity, increase in SNR, and production of brighter fluorescent images10.

Due to the complexity and high cost of optical mapping systems, this technique was initially limited to a few laboratories. Even though this technology became more easily available in the 1990s, the cost of implementation and experimentation still poses a significant limitation. The majority of the price is due to electronic equipment, including illumination sources and photodetectors20. Another cost to consider in optical mapping systems is the continued expense of conducting experiments that arise from the need for expensive dyes and chemicals, such as electromechanical uncouplers. Despite the high cost, a commercially bought system is specific to one application, making it difficult to adapt the system for newly developed protocols. Some of these cost and customizability concerns of optical mapping systems can be mitigated through 3D-design and printing. The components that support and position the acquisition and filtering equipment can be fully customized using 3D-printing, to accommodate and optimize the use of a wide range of specialized setups and tissue types. For example, tissue chambers can be 3D-printed at low cost in a size specific to the species of interest, which optimizes the volume of chambers and baths that house the tissue and thereby reduces the amount of dyes and other chemicals required. In other attempts to mitigate optical mapping costs, one study demonstrates low-cost CMOS cameras for panoramic imaging20, while others utilize a single detector for multiple parameters21,22. 3D-printing technology accommodates these new options for photodetector setups through customization. With open-sourced 3D-desings, researchers can design, build, or expand a system of necessary optomechanical equipment around a protocol, to optimize the application of costly electrical components. The system and its individual components can be easily built, scaled, and adjusted according to the type of tissue preparation and equipment being used.

While 3D-printing has previously been used to print lab equipment such as lab jacks, equipment stands and holders, optics equipment, tissue perfusion chambers, incubators, and guide electrical components for recording and stimulation, we present, for the first time, a fully 3D-printable optomechanical system for optical mapping to support the optical, perfusion and electrical components23,24,25,26,27,28,29,30. We also provide RHYTHM 1.2, an updated version of our previously published open-source optocardiography data analysis software, RHYTHM 1.031, to analyze multiparametric optical mapping data, including voltage and calcium. On an open-source platform (https://github.com/optocardiography), we provide the designs of all system components as DWG and STL files for a 3D-printer and the Matlab code for data analysis. We demonstrate the use of our 3D-printed dual-camera tandem lens system by optically mapping voltage and calcium signals from intact mouse hearts and rat cardiac tissue slices.

## Results

This study implements and demonstrates the open-source optical mapping system illustrated in Fig. 1d. The system is comprised of three types of components: stage, optical, and perfusion components.

### Stage Components

The stage components include two lab jacks, a tilting platform, three hydraulic lifts, an upright bath lift, and two identical camera cages (Fig. 2). The purpose of these stage components is twofold: 1) to assemble and support the optical and perfusion components, and 2) to tilt the entire system from sideways to upright imaging mode, as required by experimental design.

The perfusion lab jack and optical lab jack that support the perfusion and optical components were printed with 27–34 cm and 15–23 cm height ranges, respectively, in order to align the optical and perfusion components during sideways as well as upright imaging. Both lab jacks were assembled in six steps (Fig. 2a), utilizing both a nut-and-bolt and a twist lock mechanism to secure pieces together. Featured mechanisms are illustrated in Supplementary Figs 1 and 2, accompanied by detailed assembly instructions. The lab jacks featured sliding rails on the top plate for securing perfusion and optical components. The lab jacks were able to support a load of up to 30 kg.

The tilting platform (Fig. 2b) allowed the optical components to be rotated 90° for upright imaging. The system was assembled in the sideways orientation, where the tilting platform was first secured onto the optical lab jack using sliding rails, followed by the optical components, which were secured onto the upright plate of the tilting platform in the same fashion. Next, the upright plate was rotated 90° from the horizontal position and the upright stabilizer was attached to the tilting platform to stabilize the optical components and prevent further tilting of the lab jack. Stoppers, shown in dark gray in Fig. 2b, provided additional support to the optical components. The tilting platform is further illustrated in Supplementary Figs 3 and 4, accompanied by detailed assembly instructions.

The upright bath lift (Fig. 2c, right), which consists of a vertically adjustable post and screw, allows for the positioning of a tissue bath within a 16–23 cm height range. This allows the tissue to be brought to the focal point of the objective lens in the upright orientation. The two cameras were supported by hydraulic lifts; two with a 25–35 cm height range for sideways imaging and one with 44–54 cm height range for upright orientation (Fig. 2c). The hydraulic lifts consisted of two 60 ml syringes (Cat# 13-689-8, Fisher Scientific) filled with water, separated by a 1-way stop cock (Cat# 120722, Radnoti). They could be positioned at any desired height by displacing the liquid in one syringe to the other. This additional support was required since the cameras were heavy and would otherwise not be in alignment with the rest of the system. The hydraulic lift positioning is further illustrated in Supplementary Fig. 5, accompanied by detailed instructions. While the hydraulic lifts supported the cameras in the vertical direction, a camera cage (Fig. 2d) secured each camera to the optical components using the twist lock mechanism. The hydraulic lifts were able to support a load of up to 15 kg.

### Optical Components

The optical components consisted of the excitation and emission filter cubes, stationary and adjustable optics holders, objective and projection lens sleeves, and excitation light adaptor (expanded view in Fig. 3). These components housed the lenses, filters and dichroic mirrors and guided the excitation and emission light to and from the tissue preparation. Filters were fit into circular slots in the optics holder sets, while dichroic mirrors slid into rectangular slots in both optics holders.

The stationary optics holder was inserted into the excitation filter cube which positioned the dichroic mirror at 45° to the excitation light. The adjustable optics holder with the emission filters and dichroic mirror was inserted into the emission filter cube. The filter cubes were attached to each other using the twist lock mechanism and then to the tilting platform using the sliding rails and twist locks (Supplementary Fig. 1). The lenses were inserted into the objective and projection lens sleeves and were attached to the excitation and emission filter cubes, respectively. The projection lens sleeves feature a focal adjustor that was manually rotated to focus the cameras. The excitation light adaptor allowed the attachment of a light guide from the excitation light source to the excitation filter cube. Once the optical components were assembled and the cameras were attached to the projection lens sleeves, the images on the two cameras were aligned. This was done by adjusting the angle of the dichroic mirror inside the emission filter cube along the axis shown in blue in Fig. 3, which allows the dichroic mirror to be adjusted ±5° from the diagonal. The images were further aligned by finely adjusting the height of the cameras using the hydraulic lifts. The system achieved 99.07% spatial alignment, as illustrated in Supplementary Fig. 6, accompanied by detailed instructions and the alignment quantification method.

### Perfusion Components

The perfusion components include the sideways bath, the sideways bath stage, the upright bath and the upright bath stage (Fig. 4). The baths house the tissue preparations in temperature-controlled, oxygenated perfusate and the bath stages allow for xy-plane adjustment of the baths using sliding rails. The baths feature inlets and outlets to which silicone tubing is attached, that circulates the bath perfusate through a heat exchanger to keep the bath at an optimal temperature (37 °C). The inlets and outlets are placed at diagonally opposite ends of the bath to maintain uniform perfusate temperature and flow.

The sideways bath designed for a Langendorff-perfused mouse heart included an electrode paddle to stabilize the heart against the optical window and to hold the pseudo-ECG electrodes in place. Additionally, the sideways bath stage featured a cannula holder next to the tissue bath that held a cannula in place using a three-prong extension clamp (Cat# 05-769-6Q, Fisher Scientific). The sideways bath stage with its bath was mounted onto the perfusion lab jack.

In the upright bath designed for tissue slices or other flat preparations, PDMS gel in the inner bottom surface allowed for securing ECG electrodes and insect pins that hold the tissue preparation in place. The upright bath stage with its bath was mounted on the upright bath lift.

### Additional Preparation of 3D-Printed Components

To prevent leakage, the porous surface of each tissue chamber was treated with a 100% acetone vapor bath. The chamber was fully submerged in acetone vapor (~1 inch above boiling surface) for 3–6 second bursts until the surface appeared shiny and smooth. After a drying time of 24 hours, the chamber was tested for leakage using DI water. If leakage was present, the waterproofing process was repeated.

To allow the securing of tissue in place, a 0.5cm-thick layer of PDMS gel (DC 184 SYLGARD 0.5KG 1.1LB KIT, Krayden) was added to the inner bottom surface of the upright bath. To make the PDMS gel, first, a 10:1 ratio of the elastomer and curing agent was mixed together using a tongue depressor attached to an electric mixer for 10 minutes. Next, the mixture was centrifuged for 5 minutes to remove bubbles. The mixture was then slowly poured into the upright bath and left at room temperature for 24 hours to dry.

The sideways bath features a 37 × 41.44 mm optical glass window (Stock #43-927, Edmund Optics) that was adhered using a 1:1 epoxy resin (Model #20945, Devcon) mixture after the adjustable paddle was attached. To fit the slot on the chamber and leave room for glue, the glass was cut 1/32” shorter than the slot on each side. After a drying time of 24 hours, the sideways bath was tested for leakage using DI water. If leakage was present, glue was re-applied accordingly.

### Software

Utilizing previously described methods for signal processing of optical mapping data31, RHYTHM 1.2 was developed using Matlab 2017 and is compatible with .gsh/.gsd and .rsh/.rsd file formats. The open-source platform includes the code and user manual to analyze voltage and calcium data simultaneously collected using the 3D-printed system.

### Tissue Preparations

The system was tested to optically map Langendorff-perfused mouse hearts and rat cardiac organotypic slice preparations. Briefly, mice were anesthetized using isoflurane vapors, hearts were excised following thoracotomy, and the aorta was cannulated as previously described46. Mouse heart studies utilized the sideways system orientation. For the rat heart slices, approximately 1.5 × 1.5 cm section from the LV was collected and sliced into 400 μm thick sections using a precision vibrating microtome (Campden Instruments), as previously described47. Rat cardiac slice studies utilized the upright system orientation.

### Optical Mapping

The mouse hearts (n = 4) and rat slice (n = 1) were optically mapped as previously described46,48,49. Briefly, tissue was perfused/superfused with Tyrodes’s solution (130 mM NaCl, 24 mM NaHCO3, 1.2 mM NaH2PO4, 4 mM KCL, 1 mM MgCl2, 5.6 mM Glucose, 1.8 mM CaCl2, 15 mM BDM, pH ~7.4 with carbogen bubbling) The tissue was stained with RH237, a voltage sensitive dye, and Rhod2-AM, a calcium indicator46. The tissue was then paced with 2 ms stimuli at 1.5x threshold of stimulation and a basic cycle length (BCL) of 150 ms. Dyes were excited by halogen light (UHP-Mic-LED-520, Prizmatix) that first passed through an excitation filter (ET500/40×, Chroma) before reflecting off of a dichroic mirror (T550LPXR-UF1, Chroma) towards the tissue. The emitted light was collected using a 1X lens (Part #10450028, Leica) and then split into the voltage and calcium signals by a second dichroic mirror (T630LPXR-UF1, Chroma). Each light path contained an emission filter (590 ± 33 nm for calcium and 690 ± 50 nm nm for voltage, ET590/33 m and ET690/50 m, Chroma) and was then recorded using a MiCam Ultima L-type CMOS camera with 100 × 100 pixel resolution. Temperature was continuously monitored during experiments using a temperature probe (Part #MLT1401 and #ML312, AD Instruments) and maintained at ~37 °C by varying the flow rate of the circulation of bath perfusate.

Prior to each experiment, the cameras were aligned by the manual adjustment of the adjustable optics holder and camera lifts to ensure the spatially accurate alignment of the signals (see Supplementary text and Supplementary Fig. 6). After each experiment, the tubing and tissue chamber were rinsed with dilute HCl (0.1 M).

### Statistical Analysis

All data are reported as mean ± standard error. Student’s t-tests were performed to detected significant difference between groups. P < 0.05 was reported as significant.

### Code Availability

The Matlab code developed to analyze optical mapping data and sample optical data is available under open-source MIT license at: (https://github.com/optocardiography).

## Data Availability

The optical mapping and ECG data used for system demonstration are available from the corresponding author upon request. The 3D-printed hardware designs will be available in both drawing (.dwg) and printing (.stl) format at: (https://github.com/optocardiography).

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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## Acknowledgements

We gratefully acknowledge support by a grant from the Foundation Leducq and NIH grants R21 EB023106, R01 HL126802, and R01 HL114395. We also gratefully acknowledge Russian Foundation for Basic Research grant 18-00-01524. R.S. research was supported by Russian Science Foundation grant 18-71-10058. We would like to thank the following people who helped conduct this study: Mark Wagner, Jaclyn Brennan, and Zexu Lin. Mark Wagner provided access and assistance in utilizing the university’s 3D-printers. Jaclyn Brennan performed the experiment to collect ECG data. Zexu Lin provided cardiac rat slices used in system demonstration.

## Author information

I.E. and S.G. conceived the project. B.C. and S.O. designed and constructed the 3D-printed optical mapping system. R.S. developed Rhythm 1.2 software architecture and optocardiography data analysis modules. S.G., R.S., R.P. and A.Z. developed the Matlab GUI for data analysis. S.G. demonstrated use of the 3D-printed system by conducting experiments on whole mouse hearts and rat cardiac slices. S.O. made the 3D-renderings for figures. B.C. and S.G. wrote the manuscript. All authors reviewed the manuscript.

### Competing Interests

The authors declare no competing interests.

Correspondence to Sharon A. George or Igor R. Efimov.