A tomographic microscopy-compatible Langendorff system for the dynamic structural characterization of the cardiac cycle

Figures

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  Figure 1. (A) X-ray tomographic microscopy-compatible Langendorff system at the TOMCAT beamline. (1) PowerLab data acquisition hardware. (2) Circulating water bath to keep the circuit at ~37°C. (3) 95% O2/5% CO2 gas bottle for perfusate oxygenation. (4) Perfusate reservoir. (5) Peristaltic pump circulating the perfusate up to the compliance chamber. (6) Compliance chamber. The height difference with the aorta determines the perfusion pressure (60 mmHg/~90 cm water column). (B) Close-up of the custom stage tower. (7) Rotary union system to ensure that the heart can be rotated without entangling tubes and wires. (8) Motor stage. (9) Modular sample holder, where the heart is attached at the ends of metallic aortic and left atrial cannulae. (10) Aquarium to keep the heart warm and collect the perfusate. (C) TOMCAT ultra-fast end-station. (11) High-numerical aperture 4x macroscope (53). (12) GigaFRoST detector (54). (D) Simplified sketch of the system components shown in (A, B).

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  Figure 2. Sketch of the retrospective gating procedure. In dynamic scans, 160,000 projections (1 ms exposure) were acquired over 720o of continuous rotation. The ECG of the beating heart was recorded by the PowerLab through an Animal BioAmp. Simultaneously, the exposure signal from the detector was sent through a pulse shaper to the PowerLab. For the retrospective gating, (1) the feature corresponding to the start of the contraction was detected in the ECG. (2) Then, all the corresponding projections acquired at that specific time-point were identified. To identify the projections in the rest of time-points, 1 ms offsets were applied until the time-series were completed. (3) With all projections assigned to each time-point, the data was gated and all individual datasets for every time-point were created. (4) All datasets were corrected using dynamic flat-field correction and (5) reconstructed to obtain the 3D volumes for every time-point in the heartbeat.

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  Figure 3. Time-series of an illustrative slice showing the region where left ventricular wall (LV), septum (S) and right ventricular wall (RV) join. Time is indicated using the start of the contraction as reference (0 ms). An ECG signal corresponding to a full heartbeat is included in every slice. The green dot on the ECG indicates the start of the contraction (reference time-point), while the advancing red dot corresponds to the exact reconstruction location. A right ventricular trabeculation has been marked (*) to clearly follow myocardial contraction and relaxation over time. Yellow arrows point at coronary vasculature, while red arrows point at cardiomyocyte aggregates. The bottom right image shows a sketch of the presented heart region. Scale bar is 1 mm.

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  Figure 4. Time-series of an illustrative reslice (see bottom right, yellow line) from the scanned region including part of the left ventricular chamber (LV), septum (S) and right ventricular wall (RV). Time is indicated using the start of the contraction as reference (0 ms). An ECG signal corresponding to a full heartbeat is included in every slice. The green dot on the ECG indicates the start of the contraction (reference time-point), while the advancing red dot corresponds to the exact reconstruction location. Scale bar is 1 mm.

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  Figure 5. Series of representative window-reconstructed slices from 0–180o to 180–360o in 20o steps. Black and white arrows indicate example structures progressively changing over time, thus indicating function and/or tissue degradation. The star marks a large variation on the heart surface, probably due to sliding perfusate. Scale bar is 1 mm.