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.

Figure 2 Stage Components. (a) Lab jack assembly. (b) Tilting platform for upright imaging mode. Emission and excitation filter cubes are mounted onto the upright plate. (c) Lifts. Two hydraulic camera lifts (left and middle) support the cameras in both system orientations. The upright bath lift (right) permits height adjustment of tissue preparation during upright imaging. (d) Camera cage secures cameras to projection lens sleeves. Full size image

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.

Figure 3 Optical Components. Expanded view of components housing filters, dichroic mirrors, and lenses (transparent gray). The objective lens sleeve (1) secures the objective lens to the excitation filter cube (2) that guides excitation light to the tissue preparation. The excitation light adaptor (3) secures the excitation light guide to the excitation filter cube. The stationary optics holder (4), placed in the excitation filter cube in the orientation shown, houses a dichroic mirror and features a circular emission filter holder for single-camera imaging. The emission filter 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). The projection lens sleeves (7) houses the projection lenses and secures one camera at the end of each. Full size image

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.

Figure 4 Perfusion Components. Sideways imaging components. The sideways bath stage (a) houses the sideways bath (b) and an adjacent cannula holder. Pseudo-ECG electrodes fit 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. The upright bath stage (d) houses the upright bath (e). PDMS gel secures insect pins holding tissue in place. (f) Image of upright bath. Full size image

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.

System Assembly and Cost Comparison

The tandem lens optical mapping system was assembled as shown in Fig. 5. The system was designed to be capable of imaging in the sideways and upright orientation as illustrated in Fig. 5a–d. A manual of parts is provided in Supplementary Tables 1–3. The total cost of a fully 3D-printable optomechanical system was compared to commercially available equivalents and is detailed in Supplementary Tables 4–6. The commercial costs recorded were gathered from quotes provided by companies that sell optical mapping equipment and from other laboratory technology manufacturers. The total cost of 3D-printing customizable optomechanical parts necessary to support recording, illumination, and light filtering components is $1341 (Supplementary Table 5).

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. Full size image

Software

Custom Matlab software RHYTHM 1.2 was developed to analyze simultaneously collected voltage and calcium data. The open-source platform includes the software, a user manual, and sample data sets. The 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 duration (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 files 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 system was performed using intact Langendorff-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 experimental 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 after 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 significantly alter other measured parameters in whole mouse hearts (Table 1). Supplementary Table 7 displays the mean parameter values obtained from both mice and rat.

Figure 6 System Demonstration Data. (a) Activation maps of Langendorff-perfused whole mouse heart stained with voltage sensitive dye RH237 (left) 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). The schematic (f) depicts electrode placement in each case. The graph (e) shows continuous temperature recording of a representative experiment. Full size image

Table 1 Voltage and Calcium Parameters for Whole Mouse Hearts Treated with Pinacidil. Full size table

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. The 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. The temperature of the bath was controlled and recorded throughout the experiments and maintained at 37.18 ± 0.16 °C (Fig. 6e).