Preparation of large biological samples for high-resolution, hierarchical, multi-modal imaging

Abstract

Imaging the different scales of biological tissue is essential for understanding healthy organ behavior and pathophysiological changes. X-ray micro-tomography using both laboratory (μCT) and synchrotron sources (sCT) is a promising tool to image the 3D morphology at the macro- and micro-scale of large samples, including intact human organs. Preparation of large samples for high resolution imaging techniques remains a challenge due to limitations with current methods, such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe a protocol to prepare, stabilize, and image large soft-tissue samples with X-ray microtomography. We demonstrate the protocol using intact human organs and Hierarchical Phase-Contrast Tomography (HiP-CT) imaging at the European Synchrotron Radiation Facility, but the protocol is equally applicable to a range of biological samples, including complete organisms, for both laboratory and synchrotron source tomography. Our protocol enhances the contrast of the sample, while preventing sample motion during the scan, even in case of different sample orientations. Bubbles trapped during mounting and those formed during scanning (in case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging (MRI), CT, and histological observation. We describe a protocol for sample preparation and mounting which requires 25 to 34 days for a large organ such as an intact human brain or heart. This 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 pixel size of one micron using HiP-CT.

A closer look at high-energy X-ray-induced bubble formation during soft tissue imaging

“…The postmortem study was conducted according to QUACS (Quality Appraisal for Cadaveric Studies) scale recommendations [68]. The postmortem processing of organs (human brain, lung, and liver) follows the previously discussed procedure involving formalin fixation and ethanol dehydration [6], [25], [69]. The human liver sample was imaged twice with HiP-CT, an X-ray phase-contrast imaging technique compatible with large soft tissue samples [6].…”

“…The slices fit laterally into the cylindrical plastic containers such that the section plane is parallel with the lid to simulate the imaging geometry in tomography [6] and to facilitate gas escape as they are formed on the tissue interior and surfaces. The tissue slices were immersed in 70% ethanol solution in water and immobilized with crushed agar [6], [25], [69]. The non-tissue controls contain only the agar-ethanol mixture.…”

“…Compared with b , the imaging outcome of the same sample without bubbles (produced by imaging after tissue degassing), the bubbles create huge streaking artifacts in the reconstructed image. Image modified form Brunet et al[69] c , Bubbles revealed (black arrows) by differential intensities between a and b within the region specified in a . The contrast change, such as the negative differential signal within the liver vasculature is primarily caused by bubble formation.…”

Mapping the blood vasculature in an intact human kidney using hierarchical phase-contrast tomography

“…The kidney was then dehydrated through an ethanol gradient over 9 days to a final equilibrium of 70%. 14 The volume of each dehydration solution was four-fold greater than the volume of the organ and during dehydration, the solution was degassed using a diaphragm vacuum pump (Vacuubrand, MV2, 1.9m3/h) to remove excess dissolved gas. The dehydrated kidney was transferred to a polyethylene terephthalate jar where it was physically stabilised using a crushed agar-agar ethanol mixture, and then imaged.…”

“…Imaging was performed on the BM05 beamline at the ESRF following the HiP-CT protocol. 12,14 Initially the whole kidney was imaged at 25 µm/voxel (edge length). Volumes of interest within the same kidney were also imaged at 6.5 and 2.6 µm/voxel.…”