Micro Computed tomography has emerged as a powerful imaging tool to study the micro-structure of cardiac tissue of small mammals. However, conventional techniques in whole organs of large mammals, is compromised due to the use of high molecular weight contrast agents and the relative thickness of samples.

To develop a tissue preparation method to optimize microCT imaging of whole hearts with sizes including those of humans.

Male pig hearts (N=5) were treated by: hydrated samples with conventional contrast agent (iodine, 0.5%) or dehydrated in ethanol and air-dried under hexamethyldisilazane (HMDS). Human hearts (N=3) were treated using air-drying method. Hearts were imaged at 21.7μm isotropic resolution by microCT (SkyScan 1276 Bruker). Images were quantified with Amira software using a 3D cylinder correlation technique.

The X-ray attenuation of air-dried samples was better optimized for the bandwidth of the X-ray detector, indicated by reduced x-ray attenuation (pig 65%, human 55), compared to almost complete signal attenuation from hydrated samples (93%). Voxel intensity histograms showed two populations corresponding to the tissue and the background media and sample support structures. Yet, the separating minima of histogram intensities were always observed at lower intensities for air-dried hearts (Figure 1A) than hydrated hearts, increasing the dynamic range for tissue-specific signals (Figure 1B). Imaging air-dried pig or human hearts permitted a simple threshold-based segmentation to isolate microstructures of the heart, including vessels, myocardial fibers and Purkinje from the background (1C). Fiber orientation quantification could be performed directly on unfiltered images. Fiber orientation was validated against subsequent histology of the same samples.

Air drying with HMDS enables microCT imaging of human-sized hearts while preserving micro-structure and the identification of individual cardiac muscle fibers.