Noninvasive Tracking of Embryonic Cardiac Dynamics and Development with Volumetric Optoacoustic Spectroscopy

Abstract Noninvasive monitoring of cardiac development can potentially prevent cardiac anomalies in adulthood. Mouse models provide unique opportunities to study cardiac development and disease in mammals. However, high‐resolution noninvasive functional analyses of murine embryonic cardiac models are challenging because of the small size and fast volumetric motion of the embryonic heart, which is deeply embedded inside the uterus. In this study, a real time volumetric optoacoustic spectroscopy (VOS) platform for whole‐heart visualization with high spatial (100 µm) and temporal (10 ms) resolutions is developed. Embryonic heart development on gestational days (GDs) 14.5–17.5 and quantify cardiac dynamics using time‐lapse‐4D image data of the heart is followed. Additionally, spectroscopic recordings enable the quantification of the blood oxygenation status in heart chambers in a label‐free and noninvasive manner. This technology introduces new possibilities for high‐resolution quantification of embryonic heart function at different gestational stages in mammalian models, offering an invaluable noninvasive method for developmental biology.


Figures S1 to S5 Legends for movies S1 to S5
Other Supplementary Material for this manuscript includes the following:

Supplementary Text
All experimental procedures, including animal handling, were performed in compliance with the guidelines of the University of Houston Institutional Animal Care and Use Committee, protocol No. 15037.

Note S1: Evaluation of embryonic heart cycle
To evaluate the embryonic cardiac cycle, we analyzed optoacoustic (OA) images of the embryonic heart across various stages at gestational day (GD) 16.5 (Figure S2).Captured at a frequency of 25 Hz, these images sequentially depict the heart's motion throughout the cycle, showcasing distinct morphological changes and phases of systole and diastole.The initial frame captures the ventricular wall at the end of systole, while the subsequent frame illustrates the onset of ventricular expansion and the isovolumetric relaxation (ventricular diastole).This expansion continues for 40 ms, with a later frame displaying the complete relaxation of all heart chambers.As the ventricles further expand, the pressure in the ventricles starts to decline.Concurrently, the initiation of atrial contraction propels blood towards the relaxed ventricles, a process vividly depicted in frame 5.The sequence advances to the next frame, depicting the heart in diastole.After 40 ms, by frame 7, atrial expansion starts with no change in ventricular volume.Frame 8 then illustrates the ventricular walls beginning to contract, making the start of isovolumetric contraction.This phase precedes the ejection of blood during systole captured in the concluding frames 9 to 12.

Note S2: Embryonic oxygen saturation state
The OA image captured at an 850 nm illumination wavelength reveals higher signal intensity than those obtained at 760 nm and 800 nm, as depicted in Figure S3 A. This enhancement in signal strength from organs with oxygen-rich blood is attributed to the increasing optical absorption (extinction coefficient, ) of oxygenated blood with wavelength (), as shown in Figure S3 B. The OA image at 800 nm, where oxygenated (HbO2) and deoxygenated (Hb) hemoglobin absorptions converge (isosbestic point), displays total hemoglobin absorption contrast.Spectrally unmixed biodistribution maps of the HbO2 and Hb, presented in Figure S4 A and B, highlight a pronounced HbO2 signal from the embryonic cardiovascular system and the umbilical vessels.Molecular-specific volumetric optoacoustic spectroscopy (VOS) enables the identification of embryonic vasculature.Notably, the arrow in Figure S4 A points out the vessel with deoxygenated blood, which is visible on the Hb map but not on the HbO2 map.Conversely, arrows in Figure S4 B indicate vessels with oxygenated blood, absent from the Hb map.The overlay of these images, as illustrated in Figure S4 C, features both oxygenated and deoxygenated blood vessels and organs, providing a comprehensive view of the embryonic vasculature.

Note S3: Spatial resolution characterization
An optically transparent agar phantom embedded with black paramagnetic polyethylene microspheres (Cospheric LLC Santa Barbara, CA, size 38-45 μm) was utilized to characterize the VOS system capability.The laser wavelength was set to 680 nm with a peak energy output of 13 mJ.The agar phantom was made using 1.3% agar powder (05038; Sigma-Aldrich, St. Louis, MO) dissolved in distilled water.Next, black microspheres were sparsely embedded in this tissue-mimicking agar matrix.To evaluate the resolution of the system, the agar phantom was moved against the ultrasound transducer array in various directions from the center of the spherical transducer array.
The spatial resolution at each position was evaluated by determining the full width at half maximum (FWHM) of the reconstructed image profiles of the microspheres near the center of the ultrasound detection array.First, the point spread function (PSF) was calculated using the three-dimensional (3D) back-projection algorithm.Then, the Gaussian functions representing the signal profile of the absorbing microsphere were convolved with the PSF.The FWHM of the resulting signal was quantified, and the average squared difference between the measured FWHM and the diameter of the microsphere was considered the resolution.The field of view was defined as the volume where the amplitude of the reconstructed microspheres at a certain position relative to the transducer is greater than 50% of the maximum amplitude achieved at the center of the field of view.An approximately isotropic volume of 15×15×15 mm 3 was measured as the effective field of view.A 3D reconstructed image of a microsphere at the geometrical center of the transducer array is displayed in Figure S5 A.
Figure S5 C. Figure S5 D illustrates the reconstructed size of the microsphere at various

Figure S2 .
Figure S2.Embryonic cardiac dynamics (GD 16.5) visualized at an 800 nm illumination wavelength.OA images of the heart are shown for twelve timeframes (1-12).Arrows indicate the directions of expansion and contractions of the heart chambers.Scalebar = 2 mm.