Multiscale cardiac imaging to capture the whole heart and its internal cellular architecture, with applications to congenital heart disease

Abstract

9Efficient cardiac pumping depends on the morphological structure of the heart, but also on its 10 sub-cellular (ultrastructural) architecture, which enables cardiac contraction. In cases of 11 congenital heart defects, localized sub-cellular disruptions in architecture that increase the risk 12 of heart failure are only starting to be discovered. This is in part due to a lack of technologies 13 that can image the three dimensional (3D) heart structure, assessing malformations; and its 14 ultrastructure, assessing disruptions. We present here a multiscale, correlative imaging 15 procedure that achieves high-resolution images of the whole heart, using 3D micro-computed 16 tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). 17This combination of technologies has not been possible before in imaging the same cardiac 18 sample due to the heart large size, even when studying small fetal and neonatal animal models 19 (~5x5x5mm 3 ). Here, we achieved uniform fixation and staining of the whole heart, without losing 20 ultrastructural preservation (at the nm resolution range). Our approach enables multiscale 21 studies of cardiac architecture in models of congenital heart disease and beyond. 22 23 51 whole heart morphology and its sub-cellular organization (ultrastructural architecture). Our 52 multiscale procedure uses micro computed tomography (micro-CT) imaging to capture heart 53 morphology at micrometer resolution, and scanning electron microscopy (SEM) to capture 54 cardiac tissue ultrastructure at nanometer resolution. Current SEM technologies allow for three-55 dimensional (3D) imaging of sub-cellular architecture, enabling reconstruction and quantification 56 of ultrastructural features within a tissue volume 14-16 . Among 3D SEM methods, we have 57 selected serial block-face SEM (SBF-SEM) for ultrastructural imaging, as it allows 3D imaging of 58 relatively large volumes (sample size 40x60x40 µm 3 ). The methodology we present herein 59 improves upon previous protocols by achieving uniform staining of a relatively large heart 60 sample (3-4 mm wide, 5-6 mm long), circumventing micro-CT x-ray penetration issues, and 61 allowing sample screening and selection prior to full sample preparation. Our multiscale imaging, 62 further, enables mapping of structural and ultrastructural heart features. 63 129 thickening and enlargement of the RV wall. RV hypertrophy in TOF, however, develops over 130 time as the stenosis of the pulmonary artery increases pressure in the RV after birth 17 and was 131 not present in the heart examined in this study (see Figure 3 for a comparison of the selected 132 normal and TOF hearts). The TOF heart analyzed here featured supravalvular pulmonary 133 stenosis, a ventricular septal defect, and an overriding aorta. The right ventricle was enlarged 134and thin-walled compared to the control heart (Figure 3). Further, the TOF heart was missing 135 the right branch of the pulmonary artery. In humans, this rare condition, called unilateral 136