Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

The overall goal of this procedure is to demonstrate the integration of three imaging systems in a single device, which enables a simultaneous measurement of the mechanical function, ionic dynamics, and geometrical change of contracting heart tissue. Specifically, we measure the tissue's force production, calcium transients, local displacements, and shape change. Combination of these imaging modalities can help answer key questions in heart disease, with major implications for the development of effective treatment strategies.

The main advantage of this technique is it enables us to study the heterogeneity of muscle activity. The device used in this protocol contains three imaging systems capable of imaging the same sample, a brightfield microscope, fluorescence microscope, and an optical coherence tomograph. A series of dichroic mirrors are used to separate the fluorescence emission and the brightfield transmission image.

The fundamental features of this device are two independently-actuated mounting hooks, a measurement chamber with three optically clear axes and an external trigger line that synchronizes the brightfield and OCT cameras with a stimulator. In this video, we demonstrate how to isolate, prepare, and image cardiac trabeculae and present some representative results. After excising a heart from an anesthetized rat, transfer it to the dissection chamber.

Using two curved forceps, pull the aorta over the perfusion cannula. Hold the aorta in place with one forcep. Meanwhile, open the tubing line to allow dissection solution to flow through the perfusion cannula.

Once the coronary vasculature is cleared of blood and the heart is completely perfused with dissection solution, secure the aorta in place using a suture. Position the heart by rotating the cannula so that the left coronary artery is visible on the superior surface. Pin the apex of the heart to the bottom of the dissection chamber.

Cut off both atria. Cut along the right side of the septum to the apex of the heart. Pin the opened left ventricle to the base of the dissection chamber.

Then, cutting along the left side of the septum, open the right ventricle and pin it to the base of the dissection chamber, too. Identify a free-running trabecula in the right ventricle. Using the spring scissors and a forcep, cut the wall tissue surrounding the trabecula.

Then, cut the wall tissue in half, orthogonal to the direction of the trabecula. Trim the wall tissue until its dimension is appropriate for the mounting configuration used. Leave the excised trabecula in the dissection chamber.

Flush hot water, distilled water, and then superfusate solution through the measurement chamber. Turn on the brightfield microscope light source and press F1 to enable capture. Manually adjust the downstream hook until it is centered in the brightfield image.

Click zero downstream axis, then downstream disable to enable the motor. Move the downstream setpoint slider until the end of the hook aligns with the edge of the default region of interest. Re-zero the downstream axis, then move the downstream setpoint slider to 1, 000.

Repeat the process with the upstream hook, but do not move the upstream setpoint slider after re-zeroing the upstream axis. Click move to mounting. Start the fluorescence illumination system by toggling the lamp switch before quickly turning on the controller subsystems by toggling the main switch.

Switch the operational mode to turbo blanking by pressing the mode button on the front panel, followed by two, then one. Press the online button to enable external control. Pause superfusate flow through the measurement chamber.

Fill the mounting chamber with dissection solution. Using a one-milliliter syringe, transport the trabecula from the dissection chamber to the mounting chamber. Allow the trabecula to descend into the mounting chamber via gravity.

Lower the fluid level in the mounting chamber so that it is level with the midsection of the hooks. Adjust the distance between the hooks to reflect the slack length of the trabecula by moving the downstream setpoint slider. Using a microscope to aid visualization, lightly grip one of the pieces of end tissue with forceps and mount it onto the upstream hook.

Mount the other piece of end tissue onto the downstream hook. Once securely mounted, move the trabecula back into the measurement chamber by pressing move to chamber and resume superfusate flow. Set the stimulus frequency to one, stimulus duration to 10, and stimulus voltage to 10.

Start stimulation by pressing stimulus on. Turn on the brightfield illumination system. Press F1 and select a region of interest that encloses a striated area of the user interface.

Click compute sarcomere length to calculate the average sarcomere length in the highlighted region. Increase the muscle length until the average sarcomere length is approximately 2.32 microns. Move the muscle by adjusting the center setpoint slider on the center and separation control tab so that the edge of the downstream hook is just visible within the brightfield image.

Collect the fluorescence information for 10 twitches. Increase the center setpoint value by 200 and collect another 10 twitches worth of fluorescence information. Repeat this process until the brightfield image contains the upstream hook.

Collect the final window's worth of fluorescence information. Return the trabecula to a central position by setting the center setpoint value to zero. Reduce the stimulus frequency to 0.2 Hertz and switch from the K-H superfusate to the Fura-2 loading solution.

Measure the fluorescence signal every 10 minutes. Visualize the signal on the PMT signal tab. After the 360 nanometer signal has increased by a factor of 10, return the stimulus frequency to one Hertz and switch back to the K-H superfusate solution.

Check the ratio measurement every 10 minutes until the signal stabilizes, at which point, data collection can begin. Return the muscle to the position where the edge of the downstream hook is just present within the brightfield image. Capture fluorescence information by clicking enable fluorescence source.

Start streaming hardware data. On the brightfield imaging user interface, set the capture mode to external trigger, increase the frame rate to 100 Hertz, and set the number of images to capture to 100. Press control-shift-S, followed by F1, to record the brightfield imaging data for this window.

Increase the center setpoint value by 200 and repeat the brightfield capturing process. Continue with the scanning protocol until the imaging data has been collected for the final window. Return the trabecula to a central position by setting the center setpoint value to zero.

Turn on the OCT illumination source by turning the master key, pressing the power button, followed by the SLDs button. Cover the galvanometer head and click get background to measure the background interference pattern and subtract it from the measurement. Set the image capture mode to live view.

Center the trabecula in the X and Y axes by adjusting the X and Y offset values. With the trabecula centered, scan along the y-axis by adjusting the Y position to find the positions corresponding to the upstream and downstream hooks. Note these positions down.

Set range Y to the absolute difference between these values divided by 10. Set the image capture mode to stimulus triggered, range X to 100, and click the set active parameters button. Click stream data, then acquire.

In order to capture the regional calcium and brightfield information for the entire length of the trabecula presented here, seven muscle positions were required. This figure of the average force from each of the positions overlaid suggest that twitch force was undisturbed by this motion, revealing that there was no position dependence of the act of force production. These scans collected using optical coherence tomography at a rate of 100 Hertz was segmented using the ImageJ plugin, Weka.

Each cross-section appears distorted due to the difference between the lateral and depth resolutions. This was corrected by scaling each axis independently with its respective resolution. After scaling the raw C-scan of the trabecula, the muscle is approximately cylindrical.

The reflection of the measurement chamber wall can sometimes overlap with the muscle data, but the segmentation software can be trained to account for this. Once segmented, the cross-sectional area along the length of the muscle can be calculated throughout the twitch. Note that this particular trabecula has a small branch.

The motion of the branch is evident approximately halfway along the trabecula. Finally, the segmented images can be converted into meshes to aid the construction of geometrical models. Imaging data captured at each of the seven positions at a rate of 100 frames per second was stitched together to create a single complete image of the trabecula.

The ratio of the omitted fluorescence associated with the 340 and 380 nanometer excitation light correlates to the intracellular calcium after the trabecula has been loaded with Fura-2. The average of 10 intracellular calcium transients from each window are aligned with the region they were imaged. While the peak of the transients for this trabecula appear reasonably consistent, the diastolic calcium reduced along its length.

Similarly, the results of the displacement tracking and sarcomere length calculations also indicate the presence of regional variability. The markerless tracking technique used is able to calculate the displacement of each pixel. The suitability of the sarcomere length estimates were tested based on the width and amplitude of the Gaussian fit to the FFT signal.

These conditions were not met in the muscle region between zero and 500 microns, so no sarcomere length information could be computed there. Given the associated displacements, it is likely that the sarcomeres in this region elongated during the contractile phase of the twitch. After watching this video, you should have a good understanding of how to isolate cardiac trabeculae and prepare them for multimodal imaging reliably.

This process establishes a pathway for collecting a dataset required for the construction of physiologically-informed finite element models of cardiac tissue contraction.