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Abstract

The three-dimensional (3D) architecture of organs, tissues and cells together with the spatial distribution of specific
molecules, both enables and drives the close interactions between hosts and microbes. To image the 3D anatomy and
molecular composition of an animal-microbe system at a single cell scale we developed a correlative workflow
combining phase-contrast synchrotron radiation based micro-computed tomography (PC-SRμCT) and mass spectrometry
imaging (MSI). The key challenge in combining both techniques was the synchronization of sample preparation and
imaging conditions.
We used PC-SRμCT for high-resolution 3D imaging, which is the most suitable x-ray tomographic technique to image
soft tissue samples without contrast enhancing agents at a single-cell level. For method development, we used small
invertebrates that live in symbiosis with bacteria. For PC-SRμCT samples were paraformaldehyde-fixed and submersed
in a buffer-filled capillary. We tested sample preparation for Matrix-assisted laser desorption ionization (MALDI)-MSI
after PC-SRμCT and achieved the best results for PFA-fixed samples, processed under cryogenic conditions. The
resulting molecular composition permitted imaging of hundreds of different molecule distributions from single tissue
sections on a micrometer resolution. Image analysis revealed organ- and cell-specific metabolite distributions, which we
could match to corresponding structures in the anatomical 3D PC-SRμCT model.
Our PC-SRμCT-MSI sample preparation- and imaging workflow results in a detailed 3D scenario, which provides an
atlas to guide other microscopy or omics approaches towards specific organs and cells. Particularly, for animal-microbe
systems this approach presents a powerful tool to visualize anatomical and chemical processes at a microscale, linking
structure and function in the symbiosis.