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.

Full text

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Open-Source Multiparametric
Optocardiography
Brianna Cathey1, Soan Obaid1, Alexander M. Zolotarev2, Roman A. Pryamonosov2,3,
Roman A. Syunyaev2,3, Sharon A. George1 & Igor R. Emov
1,2
Since the 1970s uorescence imaging has become a leading tool in the discovery of mechanisms of
cardiac function and arrhythmias. Gradual improvements in uorescent probes and multi-camera
technology have increased the power of optical mapping and made a major impact on the eld 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 lim-
ited 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
rst developed to study axon electrophysiology as early as 1968 to overcome some of these limitations1,2 (Fig.1a).
Optical mapping utilizes either intrinsic uorescent signals or uorescent 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 rst recorded from
the heart in 19763,4. e development of ratiometric techniques using BAPTA-based compounds as a uores-
cent intracellular calcium indicator allowed for correction of artifacts from bleaching or changes in illumina-
tion 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 imple-
mentation 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.
e 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 uorescent probe is excited and the emitted light is col-
lected using a single lens and directed through an emission lter to a photodetector (Fig.1b). e excitation light
delivery could be episcopic or epicentric. Episcopic light source delivers light at an angle to the eld of view, in
contrast to precise illumination of just the eld of view by directing the excitation light through the objective
1Department of Biomedical Engineering, George Washington University, Washington, DC, 20052, USA.
2Laboratory of Human Physiology, Moscow Institute of Physics and Technology, Moscow, Russia. 3Institute of
Personalized Medicine, Sechenov University, Moscow, Russia. Brianna Cathey and Soan Obaid contributed equally.
Correspondence and requests for materials should be addressed to S.A.G. (email: sharonag@email.gwu.edu) or
I.R.E. (email: emov@gwu.edu)
Received: 15 May 2018
Accepted: 27 November 2018
Published: xx xx xxxx
OPEN
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lens in the epicentric source. In a more sophisticated version of the system, two or more uorescent dyes are
perfused through the tissue and excited simultaneously. e emitted light is then collected by a lens, split into
separate paths using a dichroic mirror, ltered, and directed to individual photodetectors (Fig.1c). is sys-
tem 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
innity-corrected light path between them. e innity-corrected light path then allows the addition of multiple
photodetectors to record several parameters simultaneously (Fig.1e). e use of a tandem lens macroscope sys-
tem has also demonstrated reduction in photobleaching and phototoxicity, increase in SNR, and production of
brighter uorescent 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 signicant limitation. e 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 specic to one application,
making it dicult to adapt the system for newly developed protocols. Some of these cost and customizability con-
cerns of optical mapping systems can be mitigated through 3D-design and printing. e components that support
and position the acquisition and ltering 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 specic 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 imag-
ing20, 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. e 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 rst time, a fully 3D-printable optomechanical system for optical mapping to
support the optical, perfusion and electrical components23–30. We also provide RHYTHM 1.2, an updated version
of our previously published open-source optocardiography data analysis soware, RHYTHM 1.031, to analyze mul-
tiparametric 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 les 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.
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).
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Results
is study implements and demonstrates the open-source optical mapping system illustrated in Fig.1d. e sys-
tem is comprised of three types of components: stage, optical, and perfusion components.
Stage Components. e stage components include two lab jacks, a tilting platform, three hydraulic lis, an
upright bath li, and two identical camera cages (Fig.2). e 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.
e 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. e lab jacks featured sliding rails
on the top plate for securing perfusion and optical components. e lab jacks were able to support a load of up
to 30 kg.
e tilting platform (Fig.2b) allowed the optical components to be rotated 90° for upright imaging. e sys-
tem was assembled in the sideways orientation, where the tilting platform was rst 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 compo-
nents. e tilting platform is further illustrated in Supplementary Figs3 and 4, accompanied by detailed assembly
instructions.
e upright bath li (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. is allows the tissue to be brought to the focal point
of the objective lens in the upright orientation. e two cameras were supported by hydraulic lis; two with a
25–35 cm height range for sideways imaging and one with 44–54 cm height range for upright orientation (Fig.2c).
e hydraulic lis consisted of two 60 ml syringes (Cat# 13-689-8, Fisher Scientic) lled with water, separated
by a 1-way stop cock (Cat# 120722, Radnoti). ey could be positioned at any desired height by displacing the
liquid in one syringe to the other. is additional support was required since the cameras were heavy and would
otherwise not be in alignment with the rest of the system. e hydraulic li positioning is further illustrated in
Figure 2. Stage Components. (a) Lab jack assembly. (b) Tilting platform for upright imaging mode. Emission
and excitation lter cubes are mounted onto the upright plate. (c) Lis. Two hydraulic camera lis (le and
middle) support the cameras in both system orientations. e upright bath li (right) permits height adjustment
of tissue preparation during upright imaging. (d) Camera cage secures cameras to projection lens sleeves.
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Supplementary Fig.5, accompanied by detailed instructions. While the hydraulic lis supported the cameras in
the vertical direction, a camera cage (Fig.2d) secured each camera to the optical components using the twist lock
mechanism. e hydraulic lis were able to support a load of up to 15 kg.
Optical Components. e optical components consisted of the excitation and emission lter cubes, station-
ary and adjustable optics holders, objective and projection lens sleeves, and excitation light adaptor (expanded
view in Fig.3). ese components housed the lenses, lters and dichroic mirrors and guided the excitation and
emission light to and from the tissue preparation. Filters were t into circular slots in the optics holder sets, while
dichroic mirrors slid into rectangular slots in both optics holders.
e stationary optics holder was inserted into the excitation lter cube which positioned the dichroic mir-
ror at 45° to the excitation light. e adjustable optics holder with the emission lters and dichroic mirror was
inserted into the emission lter cube. e lter 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). e lenses were
inserted into the objective and projection lens sleeves and were attached to the excitation and emission lter
cubes, respectively. e projection lens sleeves feature a focal adjustor that was manually rotated to focus the
cameras. e excitation light adaptor allowed the attachment of a light guide from the excitation light source to
the excitation lter 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. is was done by adjusting the angle of the
dichroic mirror inside the emission lter cube along the axis shown in blue in Fig.3, which allows the dichroic
mirror to be adjusted ±5° from the diagonal. e images were further aligned by nely adjusting the height of the
cameras using the hydraulic lis. e system achieved 99.07% spatial alignment, as illustrated in Supplementary
Fig.6, accompanied by detailed instructions and the alignment quantication method.
Perfusion Components. e perfusion components include the sideways bath, the sideways bath stage, the
upright bath and the upright bath stage (Fig.4). e 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. e 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). e inlets and outlets are placed at diagonally
opposite ends of the bath to maintain uniform perfusate temperature and ow.
e sideways bath designed for a Langendor-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 Scientic). e sideways bath stage with its bath was mounted onto the
perfusion lab jack.
In the upright bath designed for tissue slices or other at preparations, PDMS gel in the inner bottom surface
allowed for securing ECG electrodes and insect pins that hold the tissue preparation in place. e upright bath
stage with its bath was mounted on the upright bath li.
System Assembly and Cost Comparison. e tandem lens optical mapping system was assembled as
shown in Fig.5. e system was designed to be capable of imaging in the sideways and upright orientation as
Figure 3. Optical Components. Expanded view of components housing lters, dichroic mirrors, and lenses
(transparent gray). e objective lens sleeve (1) secures the objective lens to the excitation lter cube (2) that
guides excitation light to the tissue preparation. e excitation light adaptor (3) secures the excitation light
guide to the excitation lter cube. e stationary optics holder (4), placed in the excitation lter cube in the
orientation shown, houses a dichroic mirror and features a circular emission lter holder for single-camera
imaging. e emission lter cube (5) houses an adjustable wall (6) holding a second dichroic mirror that split
the emitted light from the tissue preparation to two cameras (not shown). e projection lens sleeves (7) houses
the projection lenses and secures one camera at the end of each.
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illustrated in Fig.5a–d. A manual of parts is provided in Supplementary Tables1–3. e total cost of a fully
3D-printable optomechanical system was compared to commercially available equivalents and is detailed in
Supplementary Tables4–6. e commercial costs recorded were gathered from quotes provided by companies
that sell optical mapping equipment and from other laboratory technology manufacturers. e total cost of
3D-printing customizable optomechanical parts necessary to support recording, illumination, and light ltering
components is $1341 (Supplementary Table5).
Software. Custom Matlab soware RHYTHM 1.2 was developed to analyze simultaneously collected voltage
and calcium data. e open-source platform includes the soware, a user manual, and sample data sets. e GUI
provides the user the ability to condition signals, perform the subsequent analysis on a selected region of interest,
and extract visuals. Voltage parameters calculated include action potential rise time (RT), action potential dura-
tion (APD), and conduction velocity (CV) using the Bayly method32. Calcium signal analyses include calcium
transient RT, calcium transient duration (CaTD), and decay time constants (Tau). Four windows allow the user
to view the optical recording, frame-by-frame, of up to four les independently and view their analysis maps.
Statistical results are also displayed in the GUI for the analysis chosen.
Functional Demonstration. Functional demonstration of the 3D-printed dual-camera tandem-lens sys-
tem was performed using intact Langendor-perfused mouse hearts in the sideways system orientation, and
rat organotypic cardiac slices in the upright system orientation. Figure6 and Table1 display the experimen-
tal data. Representative voltage and calcium activation maps obtained from the mouse hearts are illustrated in
Fig.6a. Representative action potential (AP) and calcium transient (CT) traces from mouse hearts and a rat slice,
during control conditions and aer treatment with 300 μM pinacidil, are superimposed and shown in Fig.6b,c,
respectively. In whole mouse hearts, pinacidil (red trace) shortened APD relative to control (black trace), from
68.23 ± 1.67 ms to 44.04 ± 5.54 ms (p < 0.05) without significantly changing CaTD (Fig.6b). Furthermore,
pinacidil did not signicantly alter other measured parameters in whole mouse hearts (Table1). Supplementary
Table7 displays the mean parameter values obtained from both mice and rat.
Pseudo-ECG traces (Fig.6d) from a representative whole mouse heart were recorded by placing the electrodes
in the custom slots on the sideways bath paddle (orange) and at the conventional location at the edges of the bath
(blue) as illustrated in Fig.6f. e close proximity to the heart and secure placement of the electrodes results in a
higher quality signal with less noise and an approximately 35% increase in amplitude. e temperature of the bath
was controlled and recorded throughout the experiments and maintainedat 37.18 ± 0.16 °C (Fig.6e).
Discussion
Although several advancements in technology have improved optocardiography methodology and its scope over
the last few decades, the cost associated with an optical mapping system is still a signicant nancial burden to
many groups20. e advent of 3D-printing and its application in manufacturing components of experimental
setups has opened a novel open-source avenue to design and produce the optomechanical components of an opti-
cal mapping system. Previous attempts at using 3D-printing in producing optomechanical components include
replacement parts for a commercially available uorescence microscope33,34, an LED brightness controller28, and
Figure 4. Perfusion Components. Sideways imaging components. e sideways bath stage (a) houses the
sideways bath (b) and an adjacent cannula holder. Pseudo-ECG electrodes t into the 3 slots of the electrode
paddle that alsostabilizes the heart against the optical window. (c) Image of printed sideways bath. Upright
imaging components. e upright bath stage (d) houses the upright bath (e). PDMS gel secures insect pins
holding tissue in place. (f) Image of upright bath.
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a kinematic mount/platform to house mirrors35. Here, we describe for the rst time, a fully functional, customiza-
ble 3D-printed system of optomechanical and perfusion components that support dual camera, tandem lens opti-
cal mapping studies. is approach also allows easy customization and rapid, inexpensive prototyping, providing
both optical mappers and other investigators an alternative that mitigates the overall cost of optical mapping sys-
tems and catalyzes new study protocol development. e functioning of the system was demonstrated by optical
mapping of whole mouse hearts and rat cardiac slice preparations, to simultaneously record voltage and calcium
signals. Our custom designed ECG electrode holder improved the quality of the recorded pseudo-ECG traces.
Additionally, we present here an updated optical mapping data analysis soware, RHYTHM 1.2. While a previ-
ously released version of this soware allowed for analysis of action potential duration, generation of activation
and phase maps, RHYTHM 1.2 analyzes several additional parameters of voltage and calcium recording. Overall,
a beginner or experienced cardiac electrophysiologist can easily replicate this open-source optocardiography
system for research or teaching use without the need for manufacturing or soware development competencies.
System Features. e primary mechanisms for assembly of the system components were twist locks and
sliding rails, both of which provided stability to the system. e twist locks permit a 90° rotation for locking.
e only exception arethe twist locks on the camera cages, which permit 360° rotation to provide the user with
freedom in image orientation. e sliding rails allow for linear adjustments of the components in two dimen-
sions. Both lab jacks and the tilting platform feature a grid of the twistlocks and a sliding rail system along their
lengths, to allow linear adjustment of both optical and perfusion components and to align and stabilize them.
Furthermore, the assembly mechanisms of various components of the system are compatible such that each major
component can be included or excluded depending on the application, and many parts can be modied in size
or shape while retaining system compatibility. For example, the lens sleeves can be modied for dierent lenses,
the tilting platform can be excluded in a system that does not require upright imaging orientation, an additional
emission lter cube can be included to measure an additional parameter, and the stationary optics holder can
house a lter for single-camera imaging. Furthermore, the individual components of the system can be scaled or
easily modied to match other applications that vary in species of study and optics.
Multi-parametric optocardiography is an important tool in studies of the mechanisms of cardiac physiology.
For multi-parametric imaging, spatial accuracy when splitting signals to multiple photodetectors using a dichroic
mirror is essential. e design of the emission lter cube achieves this by allowing the dichroic mirror angle to
be nely adjusted and then secured in place using 3D-printed clips. e hydraulic lis also contributed to spatial
alignment by allowing ne adjustment of the camera height.
Figure 5. Full Optical System Assembly. Sideways imaging mode rendering (a) and photo (b). Upright
imaging mode rendering (c) and photo (d). In the renderings, stage components are shown in light gray, optical
components in dark gray, and perfusion components in gold.
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For whole-heart studies, a high quality pseudo-ECG was recorded using the customized electrode paddle,
which secured the electrodes in place to maintain polarity and close proximity to the tissue for increased signal
quality. e curved surface of the paddle stabilized the heart against the optical window to create a at imaging
Figure 6. System Demonstration Data. (a) Activation maps of Langendor-perfused whole mouse heart
stained with voltage sensitive dye RH237 (le) and calcium sensitive dye Rhod2AM (right). Representative
action potential and calcium transient recordings of whole mouse heart (b) and rat cardiac slice (c) during
control Tyrode or 300 µM pinacidil treatment. (d) Comparison of pseudo-ECG traces using electrodes (+, −,
gnd) placed in customized electrode paddle (orange) vs. traditional electrode placement (blue). e schematic
(f) depicts electrode placement in each case.e graph (e) showscontinuous temperature recording of a
representative experiment.
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surface. A similar design goal has previously been achieved for a Langendor-perfused mouse heart chamber,
however this design featured a tissue restrainer device with a separate electrode holder attached to the chamber
using screws25. While both of these chamber designs optimize placement of the heart and the electrodes, our new
design achieves heart position stabilization and electrode placement by the paddle alone, which requires far less
plastic and is more compact, a necessity for smaller tissue baths such as those for mouse hearts.
e waterproong method used for the tissue chambers is eective for sealing o pores in ABS plastic, how-
ever if a dierent model material is desired, waterproong measures may need to be modied according to mate-
rial properties.
O-the-shelf (OTS) Components. One previous study in macroscopic imaging applications demonstrates
an optical mapping system comprised entirely of OTS components30. Another study reported that the cost and
complexity of uorescent microscopy was eectively reduced by the assembly of a microscope that includes OTS
components, including a dichroic mirror, an excitation lter, a barrier lter, a microscope, and a ashlight34. Yet
another study presents an electrochemical-scanning probe microscope (EC-SPM) that uses 3D-printed stage
platform and electrode holder, metal rods and an electronic stepper motor to produce motor-controlled linear
stages for ne adjustment28. In these systems, 3D-printed parts are essential in two ways: they have a primary
function within the system and secondarily, their customizability permits optimal incorporation of any OTS
equipment. In all studies, simple OTS parts increase the range of application of low-cost 3D-printed components
to make an integrated, customized system.
e system presented here incorporated several cost-ecient OTS components which improved functional-
ity and usability of the system. e plastic syringes featured in the hydraulic lis easily and precisely adjust the
camera height and takes up little space on the setup. ree-prong extension clamps t into the cannula holder
and directly held the cannula and pacing electrodes in the sideways orientation. An optical window was used in
the sideways bath to allow imaging of hanging heart preparations. Finally, PDMS gel was used to layer the inner
bottom surface of the upright bath to secure tissue and ECG electrodes in place.
Rhythm 1.2. e custom Matlab soware, RHYTHM 1.2, expands upon a previous version of optical map-
ping data analysis soware that we developed and published, RHYTHM 1.031. e updates in this version of the
soware include analysis of voltage and calcium signals obtained using our dual camera system. In addition to
the signal conditioning functions of RHYTHM 1.0, RHYTHM 1.2 is equipped to analyze and measure action
potential durations, rise times and conduction velocities from voltage recordings, as well as calcium transient
durations, rise time, and decay rate constants from calcium recordings. Soware use was demonstrated by meas-
uring each of the parameters listed above from both mouse hearts and rat cardiac slices. A detailed user manual
and sample data sets are provided with the soware on the open-source platform for other researchers to utilize
or modify the soware.
System Demonstration. Dual imaging of voltage and calcium signals from whole mouse hearts and rat
slices demonstrate eective functioning of the 3D-printed optical mapping system. Optical recordings obtained
before and aer administration of pinacidil, an adenosine triphosphate-sensitive potassium-channel opener,
revealed APD shortening in whole mouse hearts, as previously reported36–38. Additionally, dierences in other
analyzed parameters in whole mouse hearts were not measurable between control and pinacidil treatment. In
using a single rat heart slice preparation, we only demonstrate that the system can be used for physiological stud-
ies of dierent types of preparations. All values reported in Table1 are comparable to previously published mouse
data (references indicated in Table1)39–44. Dierences between these previously reported values are attributable
to dierences in protocol, including dyes, data acquisition method, and data analysis methods. Due to lack of pre-
viously published data from optical mapping of rat cardiac slices using similar optical mapping methods, there is
limited ability to compare the parameters we report. More studies must be conducted to determine precise values
Parameter Control Pinacidil (300 μM) p-value
Vol tag e
APD80 (ms) 68.23 ± 1.6739 44.04 ± 5.54 0.01
RT (ms) 5.05 ± 0.0340 6.92 ± 0.72 0.19
CVT (m/s) 0.30 ± 0.0241 0.30 ± 0.04 0.96
CVL (m/s) 0.67 ± 0.0341 0.54 ± 0.09 0.32
AR 2.24 ± 0.1642 1.79 ± 0.09 0.08
Calcium
CaTD80 (ms) 70.82 ± 5.6639 64.72 ± 5.03 0.51
RT (ms) 13.98 ± 2.7243 18.054 ± 5.40 0.58
Tau (s−1) 32.78 ± 12.5944 34.72 ± 15.15 0.93
Table 1. Voltage and Calcium Parameters for Whole Mouse Hearts Treated with Pinacidil. Values are reported
as mean ± standard error (n = 4). Voltage parameters include Action potential duration at 80% repolarization
(APD80), rise time (RT), transverse conduction velocity (CVT), longitudinal conduction velocity (CVL),
and anisotropic ratio (AR). Rise time was calculated as the time from 20% to 90% of maximum amplitudeof
upstroke of action potential. Anisotropic ratio was calculated as the ratio of CVL to CVT. Calcium parameters
include calcium transient duration at 80% repolarization (CaTD80), rise time (RT), and the calcium transient
decay constant (Tau). 300 μM of pinacidil signicantly reduced action potential duration (p = 0.01).
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for physiological parameters in rat cardiac slices, however we demonstrate that this optocardiography system can
be used to do so.
License. Hardware design was completed using a free academic license of AutoCAD. e Matlab code was
written using a free academic license of Matlab 2017. e hardware designs and Matlab soware are released with
this publication under the MIT open-source soware license.
Limitations
While the 3D-printed system decreases cost and increases system functionality through customization, the
production of the system requires access to an industrial-grade printer with a minimum resolution of 0.01in
(0.254 mm). Most universities have in-house 3D-printing facilities, however prints can be outsourced to one of
several commercial vendors including e UPS Store and FedEX. At this resolution, there is some variability
present between replicates, which can alter the ease of t between pieces. In this case, sand paper can be used to
achieve the desired tightness of t. e system also requires longer assembly time due to the number of parts and
the use of OTS components. However, the additional assembly time is less than the time one is likely to wait for
commercially ordered parts to be delivered. e modication of the open-source designs also requires 3D-design
skills.
For eective dual mapping, the dyes chosen should have non-overlapping spectra. In this study we chose
RH237 for membrane potential imaging and Rhod2-AM for calcium imaging45. RH237, though ideal for this
application, yields weaker uorescence intensity. is results in noisier signals especially in the thinner rat slice
preparations. is can be further developed using better uorescent markers with suitable spectra.
Methods
All experimental protocols were approved by the Institutional Animal Care and Use Committee at e George
Washington University and conform to the NIH Guide for the Care and Usage of Laboratory animals.
Printing and Design of Hardware. Academic version of AutoCAD 2017 was used for the 3D design of all
system components. To achieve optimal ts between attached pieces, distances ranging from 0.1–0.5 mm were le
between adjacent surfaces depending on total surface area of contact and desired tightness of t. Each AutoCAD
design (.dwg format) was exported in .stl (stereolithography) format, which describes the surface geometry of a
3D object. To print, the .stl les were imported to Insight soware for pre-processing and then relayed to Control
Center soware for printer communication.
A Stratasys Fortus 250mc was used to print all system components in acrylonitrile butadiene styrene (ABS)
Plus 430 ($130/60in3 with educational discount). We strongly discourage the use of polylactic acid (PLA) mate-
rial for 3D-printing any components of the system due to its low quality: it deforms with time and has a low
melting point compared to ABS or ABSP. All parts were printed at high density at a layer thickness of 0.01in
(0.254 mm). For parts with complex geometry, such as hollow regions, printers ejected a soluble support material
(SR-30, $250/60in3) during the printing process. Aer printing, the support material was dissolved o of the
plastic part in a heated, sonicated solution of concentrated sodium hydroxide (Soluble Concentrate P400-SC,
WaterWorks). e part was then soaked in DI water for approximately 5 hours to drain any uids remaining in the
pores of the plastic. Each part was allowed ample drying time (~24 hours) prior to assembly. e printing soware
provided the volume (in3) of model and support material used for each print job, which was used to calculate the
cost of each component (Supplementary Table5).
Additional Preparation of 3D-Printed Components. To prevent leakage, the porous surface of each
tissue chamber was treated with a 100% acetone vapor bath. e chamber was fully submerged in acetone vapor
(~1 inch above boiling surface) for 3–6 second bursts until the surface appeared shiny and smooth. Aer a drying
time of 24 hours, the chamber was tested for leakage using DI water. If leakage was present, the waterproong
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, rst, 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. e mixture was then slowly
poured into the upright bath and le at room temperature for 24 hours to dry.
e 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 aer the adjustable paddle was attached. To t
the slot on the chamber and leave room for glue, the glass was cut 1/32” shorter than the slot on each side. Aer 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 le formats. e open-source
platform includes the code and user manual to analyze voltage and calcium data simultaneouslycollected using
the 3D-printed system.
Tissue Preparations. e system was tested to optically map Langendor-perfused mouse hearts and
rat cardiac organotypic slice preparations. Briey, mice were anesthetized using isourane vapors, hearts were
excised following thoracotomy, and the aorta was cannulated as previously described46. Mouse heart studies uti-
lized the sideways system orientation. For the rat heart slices, approximately 1.5 × 1.5 cm section from the LV was
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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. Briey, 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 car-
bogen bubbling) e tissue was stained with RH237, a voltage sensitive dye, and Rhod2-AM, a calcium indica-
tor46. e 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 rst passed through an excita-
tion lter (ET500/40×, Chroma) before reecting o of a dichroic mirror (T550LPXR-UF1, Chroma) towards
the tissue. e 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 emis-
sion lter (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 ow 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 lis to ensure the spatially accurate alignment of the signals (see Supplementary text and
Supplementary Fig.6). Aer 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 signicant dierence between groups. P < 0.05 was reported as signicant.
Code Availability. e 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
e optical mapping and ECG data used for system demonstration are available from the corresponding author
upon request. e 3D-printed hardware designs will be available in both drawing (.dwg) and printing (.stl) format
at: (https://github.com/optocardiography).
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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 Contributions
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 soware 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 gures.
B.C. and S.G. wrote the manuscript. All authors reviewed the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36809-y.
Competing Interests: e authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
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