Experimental Design and Imaging Setup

To image both electrical and mechanical activity in the heart, high-speed fluorescence imaging was used in combination with high-speed ultrasound imaging. Both measurement techniques were used simultaneously with their clocks synchronized to acquire movies of the electrical as well as accord-ing mechanical activity of the cardiac muscle, at the same time on its surface as well as within cross-sections inside the muscle. The experiments were conducted with isolated rabbit hearts, which were kept in retrograde Langendorff-perfusion, see figures 6.1-6.4. This allowed to conduct exper-iments with intact hearts ex-vivo under physiological conditions, being able to film coupled elec-tromechanical wave activity during normal sinus rhythm as well as during induced and controlled cardiac tachyarrhythmias, such as ventricular tachycardia and fibrillation.

Figure 6.1 shows the principle design of the experiment. The setup allows to image (a) the fluores-cence on the surface, (b) the deformation of the surface and (c) the deformation several millimeters underneath the surface, within the cardiac muscle, at high speeds. The two images on the right show the left ventricular surface of the heart as imaged with fluorescence imaging, with the left image showing voltage-sensitive (V_m) fluorescence emission and the right image showing calcium-sensitive ([Ca²⁺]i) fluorescence emission, see section 6.4. The image on the left shows a cross-section of the left ventricular wall as imaged with ultrasound. The images are still frames from high-speed videos acquired at acquisition speeds ranging in between 250−500frames per second to image the fast electrophysiological processes and mechanical contractions appropriately. All three videos show the contracting and deforming heart and therefore can be used to track and capture the time-varying

Chapter 6. Intramural Scroll Wave Imaging during Ventricular Tachycardia and Fibrillation

λ_Exc1 = 532nm

λ_Exc2= 650nm

2kHz Tyrode ECG

Electrode

Defibrillation Paddle

Ultrasound

Aquarium

Acquisition & Synchronization

37^◦

Camera Filter

Figure 6.2:Schematic drawing of experimental setup for electromechanical wave imaging in intact, isolated rabbit hearts. Rabbit heart inside perfusion bath connected to retrograde Langendorff-perfusion.

Synchronized acquisition of fluorescence and ultrasound videos and electrocardiogram. Simulta-neous high-speed fluorescence and ultrasound imaging in left ventricular wall to measure electro-physiological activity and mechanical deformation.

elasto-mechanical activity. However, in addition to the elasto-mechanical activity, the two videos on the right contain fluorescence, which carries information about the electrophysiological activity.

In total, the imaging configuration allows to acquire five spatial-temporal patterns, three related to mechanical activity and two related to electrical activity.

Figure 6.2 shows a schematic drawing of the setup and figures 6.3 and 6.5 show the corresponding photographs. The heart is placed inside an eight-sided aquarium filled with heated Tyrode solution at 37^◦Cand connected to retrograde Langendorff-perfusion, see section 6.3. The tubing that connects the perfusion to the aorta holds the heart in place. The camera capturing the fluorescence emission films the heart’s ventricular surface through one of the glas windows of the aquarium. The ultrasound transducer head is immersed from the top into the bath, filming the ventricular wall from the top di-rected towards the apex, see figure 6.4.

The idea of the imaging configuration was to image the same part of the heart using both imaging modalities and to align the two imaged areas in a way that the captured two-dimensional patterns of the respective electrical and mechanical activities would show two congruent areas. Therefore, the ultrasound imaging plane was positioned and aligned within the ventricular wall with its plane normal facing the camera. Accordingly, the in-plane horizontal orientation was aligned along the cir-cumferential direction of the heart, with the plane being located approximately at midwall in parallel with the epi- and endocardial walls, see figures 6.3, 6.4 and 6.18. It was assumed that the muscle fiber orientation would also lie within or approximately parallel to the imaging cross-section. Ac-cordingly, the camera filming the surface showed a projection of the surface that was parallel to the internal cross-section.

Next to filming, the setup also allowed to record electrocardiograms and to induce and terminate

Perfusion

37^◦C

λExc1= 532nm Ultrasound LED

Camera

Ultrasound Transducer

Figure 6.3:Experimental setup with simultaneous optical fluorescence and ultrasound imaging: (a) Imaging configuration with scanning position of ultrasound transducer for cross-sectional imaging of left ventricular wall: (a) perfusion tubing holding heart in position with left ventricular wall facing glass wall of aquarium (b) ultrasound transducer intersecting lect ventricular wall from the top imaging cross-section of wall at midwall as indicated in figure 6.5, epicardial surface visible to camera (c) camera and diode positioning in front of bath, camera filming left ventricular surface

λExc1= 532nm

LV LV

3D-CT

Figure 6.4:Imaging configuration with scanning position of ultrasound: side view of ultrasound transducer scanning head and rabbit heart during imaging with ultrasound imaging plane intersecting left ventricular wall with the plane located approximately at midwall in parallel to epi- and endocardial walls to image a circumferential cross-section. Wall thickness of rabbit heart about2−5mm.

Illumination from the right atλ_Exc1= 532nm.

Chapter 6. Intramural Scroll Wave Imaging during Ventricular Tachycardia and Fibrillation

λ_Exc1= 532nm λ_Exc2= 650nm

Camera

from LED Driver

(a)

(b)

Electrode

Defibrillation Paddle

(c)

Figure 6.5:Experimental setup with multi-parametric fluorescence imaging to measure transmembrane volt-ageVmand intracellular calcium concentration[Ca]²⁺ on the epicardial surface as well as me-chanical deformation on surface, as well as intramural meme-chanical deformation inside the ven-tricular wall using ultrasound imaging. Two excitation bandwidths atλExc1 = 532nmto excite calcium-sensitive dye andλExc2= 650nmto excite voltage-sensitive dye and rapid switching of illumination at500fps (250fps per parameter, see below).

500fps

250fps

250fps

Figure 6.6:Switching scheme of LED illumination for multi-parametric imaging: custom-made driver switch-ing illumination every 2ms from red to green light for interleaved excitation of two dyes for multi-parametric imaging, see section 6.4, resulting in an effective acquisition speed of250fps per acquired parameter.

arrhythmias, see section , by applying pacing pulses, cardioversion shocks or low energy antifibrilla-tion pacing (LEAP). To be able to asociate the according frames of the fluorescence and ultrasound recordings with each other and with the electrocardiogram recordings, a commercial data acquisi-tion was used, see secacquisi-tion , recording the trigger signals of each device. Recordings were acquired and analyzed using custom-made acquisition and post-processing software, see following sections, to visualize spatial-temporal patterns of electrical and mechanical activity respectively. Experiments were conducted together with M. Chebbok. In total, 20 experiments were conducted in between June 2012 and July 2014.