Multidimensional Analysis of the Adult Human Heart in Health and Disease using Hierarchical Phase-Contrast Tomography (HiP-CT).

Cardiovascular diseases (CVDs) are a leading cause of death worldwide. Current clinical imaging modalities provide resolution adequate for diagnosis but are unable to provide detail of structural changes in the heart, across length-scales, necessary for understanding underlying pathophysiology of disease. Hierarchical Phase-Contrast Tomography (HiP-CT), using new (4th) generation synchrotron sources, potentially overcomes this limitation, allowing micron resolution imaging of intact adult organs with unprecedented detail. In this proof of principle study (n=2), we show the utility of HiP-CT to image whole adult human hearts ex-vivo: one 'control' without known cardiac disease and one with multiple known cardiopulmonary pathologies. The resulting multiscale imaging was able to demonstrate exemplars of anatomy in each cardiac segment along with novel findings in the cardiac conduction system, from gross (20 um/voxel) to cellular scale (2.2 um/voxel), non-destructively, thereby bridging the gap between macroscopic and microscopic investigations. We propose that the technique represents a significant step in virtual autopsy methods for studying structural heart disease, facilitating research into abnormalities across scales and age-groups. It opens up possibilities for understanding and treating disease; and provides a cardiac 'blueprint' with potential for in-silico simulation, device design, virtual surgical training, and bioengineered heart in the future.

Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility.

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 µm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.

Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography.

Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)'s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19).

Clinical, intravascular ultrasound, and quantitative angiographic determinants of the coronary flow reserve before and after percutaneous transluminal coronary angioplasty.

This study evaluated the clinical, intravascular ultrasound (IVUS), and angiographic determinants of the coronary flow reserve (CFR) as measured by guidewire Doppler velocimetry. Using standard methodology, 86 consecutive patients were studied before intervention (n = 73 patients, including the assessment of intermediate stenoses) and/or after intervention (n = 39 patients, including after percutaneous transluminal coronary angioplasty (PTCA) in 27 and post-Palmaz-Schatz stent placement + high-pressure adjunct PTCA in 12). Only 5 patients were studied before intervention, post-PTCA, and poststent. Univariate and multivariate clinical, quantitative coronary angiography (QCA), and IVUS correlates of the CFR were evaluated. There was a linear relation between CFR and IVUS minimum lumen cross-sectional area (CSA): r = 0.771, p <0.0001 for the overall cohort; r = 0.831, p <0.0001 before intervention; r = 0.514, p = 0.0061 post-PTCA; and r = 0.623, p = 0.0306 poststent placement. Overall, an IVUS minimum lumen CSA of > or = 4.0 mm2 had a diagnostic accuracy of 89% in identifying a CFR of > or = 2.0. This diagnostic accuracy increased slightly to 92% when only the preintervention observations were considered. Using multivariate linear regression analysis, the independent determinants of the CFR in the overall cohort of 112 observations were IVUS minimum lumen CSA (p <0.0001), angiographic lesion length (p = 0.0101), and diabetes mellitus (p = 0.0371): r2 = 0.6224. When the subset of preintervention observations were analyzed separately, the independent determinants of the CFR were minimum lumen CSA (p <0.0001) and angiographic lesion length (p = 0.0095); r2 = 0.7176. Thus, the major determinants of the CFR in patients with coronary artery disease are lumen compromise (which is best assessed by the IVUS measurement of the minimum lumen CSA) and lesion length. A minimum lumen CSA > or = 4.0 mm2 has a high diagnostic accuracy in predicting a CFR > or = 2.0, especially before intervention.

Effect of varying the ultrasonic power setting on canal preparation.

The purpose of this study was to evaluate the effect of instrumenting root canals using an ultrasonic device (Enac) at different power levels on the prepared canal shape and instrumentation time, and to compare these results with those obtained using a standardized hand instrumentation technique. The mesial canals (120) of 60 human mandibular first and second molars were randomly assigned to one of six groups. In groups A through D, the canals were instrumented using the Enac ultrasonic unit at different power settings (1, 2, 3, and 5, respectively). Group E was hand instrumented. Group F served as uninstrumented controls. The roots were then sectioned horizontally and the canal shapes examined, as was the mesiodistal canal diameter as it relates to the external root surface. Instrumentation time for each group was recorded. Ultrasonic instrumentation at the different power settings was significantly faster than hand instrumentation (p less than 0.001). However, the difference between the different power levels was not significant. There were no significant differences between the different groups as to the effect on the prepared canal shape. The only problem arising from using the Enac at a power level more than the recommended (5) was the greater tendency to break size #15 files during instrumentation. Using the Enac at power level 3 provided satisfactory instrumentation capability with minimal risk of file breakage.