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

Abstract

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.

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

“…During evisceration, the pericardium and lungs were removed. The hearts were prepared following the protocol of Brunet et al (23). Briefly, hearts were post-fixed in 4% neutral buffered formalin for 4 days at room temperature, they were then partially dehydrated to 70% ethanol using multiple baths of increasing ethanol concentration (4 days per bath).…”

“…Further details on the reconstruction pipeline can be found at Xian et al(27). Data ranged from 150-915 GB per volume and required ultra-high-performance computing to process and visualise as described by Brunet et al(23). Data was made publicly accessible through the web interface Neuroglancer, as detailed in the supplementary methods, and illustrated in Supplementary Fig.…”

“…We show proof of principle for mapping cardiac components in 3D at histologic resolution and for demonstrating interconnectivity between and within structures without the use of a destructive approach. This data could be linked to clinical data and or functional modelling as well as to advanced techniques such as targeted spatial transcriptomics (23).…”

Cardiovascular Brain Circuits

“…This work is predicted to come from postmortem studies of human neuronal tissues using single nuclear RNAseq analyses, 199,200 conventional immunohistochemical analyses, and multiple brain imaging approaches. 201,202 We highlight here 6 major core methods that are all required to make progress to investigate the CBC (Figure 8).…”

Unsupervised segmentation of 3D microvascular photoacoustic images using deep generative learning

“…The past decade has brought about a revolutionary change in our ability to visualise vascular networks in 3D at a spatial resolution never before possible. Advances in photoacoustic mesoscopy [1][2][3] and ultrafast ultrasound 4,5 provide in vivo images of vessel form and function at the micron scale in living subjects, while tools such as X-ray histology 6,7 and light sheet fluorescence microscopy 8,9 yield unprecedented insight into vascular architectures at the sub-micron scale ex vivo in intact specimens. The segmentation of microvascular networks from these 3D image volumes and interpretation of their meaning in the context of physiological and pathological processes is now a crucial task in biological research, yet currently lags behind the phenomenal pace of development in imaging hardware and scale of data output.…”