High Throughput Tomography (HiTT) on EMBL beamline P14 on PETRA III

Abstract

Here, high-throughput tomography (HiTT), a fast and versatile phase-contrast imaging platform for life-science samples on the EMBL beamline P14 at DESY in Hamburg, Germany, is presented. A high-photon-flux undulator beamline is used to perform tomographic phase-contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X-ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X-ray imaging data from potentially hundreds of samples during a beam-time shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. The successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin-embedded preparations is demonstrated. Furthermore, the feasibility of HiTT to be used as a targeting tool for volume electron microscopy, as well as using HiTT to study plant morphology, is demonstrated. It is also shown how the high-throughput nature of the work has allowed large numbers of `identical' samples to be imaged to enable statistically relevant sample volumes to be studied.

Keywords: X-ray tomography; beamlines; biology; phase-contrast imaging.

Figure 1

HiTT setup for holotomography on EMBL beamline P14 at PETRA III.

Figure 2

Sample handling at the P14 beamline. Samples are mounted on SPINE-style pins. Mounting schemes for resin-embedded heavy-metal-stained samples, paraffin-embedded as well as wet samples mounted in liquid were established. SPINE pucks hold up to ten samples each. Up to 17 spine pucks (= 170 samples) can be loaded into the MARVIN sample changer. Samples are mounted using a robotic arm with a dedicated sample gripper.

Figure 3

The HiTT pipeline. For each sample four different data collections at increasing propagation distances are performed. Subsequently the data processing is triggered automatically.

Figure 4

HiTT imaging can be successfully performed on various sample types. (a)–(c) Projection image, reconstructed slice and 3D rendering of a paraffin-embedded 1 mm murine kidney punch biopsy. Anatomical structures like glomeruli (red), distal convoluted tubules (cyan) and proximal convoluted tubules (green) can be easily discerned. (d)–(f) Projection image, reconstructed slice and 3D rendering of a Arabidopsis thaliana stem. The zoom-in in (e) shows that individual cells can be easily segmented and labelled. (g)–(i) Projection image, reconstructed slice and 3D rendering of an osmium-stained and resin-embedded mid-gut of an Anopheles stephensi mosquito, which was infected with Plasmodium berghei. Oocysts formed by the parasites are clearly visible in (h) (yellow) and segmented in (i).

Figure 5

HiTT for virtual histology of murine tissues. 1 mm punch biopsies were taken from formalin-fixed and paraffin-embedded mouse organs and imaged at the P14 beamline. (a) Brain (cerebrum), (b) brain (cerebellum), (c) lung, (d) heart, (e) kidney medulla, (f) liver. The mouse sketch was created with BioRender (https://www.biorender.com/).

Figure 6

Stitched acquisition of a mouse kidney punch biopsy. Twenty single acquisitions (2 × 2 × 5 scans) of a mouse kidney punch biopsy were acquired and stitched together. (a) A single reconstructed slice displaying kidney tissue from the kidney cortex (top) to the medulla (bottom). (b) 3D rendering of the same data set showing remarkable resemblance with conventional histology.