Open-Source Multiparametric Optocardiography

www.nature.com/scientificreports OPEN Open-Source Multiparametric Optocardiography Brianna Cathey1, Sofian Obaid1, Alexander M. Zolotarev2, Roman A. Pryamonosov2,3, Roman A. Syunyaev2,3, Sharon A. George1 & Igor R. Efimov 1,2 Received: 15 May 2018 Accepted: 27 November 2018 Published: xx xx xxxx 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. 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 cameras6–8, 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 arrhythmias17–19. 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 1 Department of Biomedical Engineering, George Washington University, Washington, DC, 20052, USA. Laboratory of Human Physiology, Moscow Institute of Physics and Technology, Moscow, Russia. 3Institute of Personalized Medicine, Sechenov University, Moscow, Russia. Brianna Cathey and Sofian Obaid contributed equally. Correspondence and requests for materials should be addressed to S.A.G. (email: ) or I.R.E. (email: ) 2 ScIentIfIc REPOrTS | (2019) 9:721 | DOI:10.1038/s41598-018-36809-y 1 www.nature.com/scientificreports/ Figure 1. History of Optical Mapping. (a) Timeline of advances in optical mapping technology. (b–e) Optical mapping system setups with colored lines representing light path. Single-camera imaging is the simplest implementation. (b) Dual-camera imaging allows the simultaneous measurement of two parameters. (c) Tandem-lens arrangement (d) permits extension of the system for multi-parametric imaging (e). 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 conc (...truncated)