Three-dimensional mapping of organ geometry Laura Blackiea*, Pedro Gaspara, Lisa Gartnera, Hannah Beckwithb, Salem Moslehc, Lingjin Kongb, Yuhong Jina, Agata P. Zielinskaa, Todd Schoborgd, L. Mahadevane, John Thomasb, Ben Stattonb, Uday Patelf, Philip Lungf, Bernhard Kainzg, Irene Miguel-Aliagaa a Francis Crick Institute, London, UK; b Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK; c Department of Natural Sciences, University of Maryland Eastern Shore, MD, USA; d Department of Molecular Biology, University of Wyoming, WY, USA; e School of Engineering and Applied Sciences, Harvard University, MA, USA; f London North West University Health Care NHS Trust; g Department of Computing, Imperial College London, London, UK *laura.blackie@crick.ac.uk References (1) Blackie, Gaspar et al (2024) The sex of organ genometry. Nature. (2) Masselink et al (2019) Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development. (3) Schoborg et al (2019) Micro-computed tomography as a platform for exploring Drosophila development. Development. (4) Arshadi et al (2021) SNT: a unifying toolbox for quantification of neuronal anatomy. Nature Methods (5) Yushkevich et al (2006) User-guided 3D active contour segmentation of anatomical structures: Significantly improved efficiency and reliability. Neuroimage. Loss of 3D information Opaque cuticle and toxic, long protocols Hard to label multiple organs at once Low throughput Loss of organ relationships Aim: map 3D organ shape and relationships Imaging method Confocal Light sheet Requires dissection Dissected Drosophila gut stained with DAPI imaged by confocal microscopy MicroCT Analysis methods Proximity to organ (μm) 590 295 442.5 147.5 0 767 0.00 0.25 0.50 0.75 1.00 75 25 50 100 0 Position along whole gut length (%) Distance to ovaries (mm) Left ovary n=44 Right ovary n=44 Extending to the human gut Segmentations - organ proximity analysis The gut tube of flies and humans is longer than the body so must fold and coil in order to fit into the abdomen. Does the gut fold into a stereotypical defined shape or is it random? Gut length differs between male and female flies - does gut shape also differ? Is gut shape important for gut function? To answer these questions we developed 3D imaging methods and 3D image analysis techniques(1). We scaled up micro-Computed Tomography (microCT) scanning(3), an x-ray based technique, by improving mounting methods. We mount 10 flies in a p10 pipette tip and stack four tips to scan ~40 flies in one run. Coupled with an automated sample changer, this allows us to scan 100s of flies automatically. Fly with GFP labelled gut cleared by 2Eci(2) and scanned by lightsheet microscopy. (Left) autofluorescence gives overview of fly body, (right) coiled gut can be seen in situ. 3D info remains intact - no dissection Most variable Least variable Overall mean Female mean Male mean 0.00 0.05 0.10 0.15 0.20 0.25 R L Ovary volume (mm3) *** n=60 90O 81.8% 6.8% 11.4% n=44 We used the ITKsnap software(5) to segment visible organs including the gut, ovaries and central nervous system. We then developed a method to measure the proximity between these organs and along the length of the gut. We identified several interesting asymmetries in organ arrangement for example the stomach more often contacts the fly’s right ovary. The fly’s left ovary is on average bigger than the right (left). Secondly we noted the fly’s stomach most often joins the gut from the left-hand side but a minority of individuals demonstrated an inverted arrangement (right). Left, example heatmap displayed on segmentation of gut showing gut proximity to ovaries (red = near, blue = far). Right, graph showing distance between gut and ovaries plotted along the length of the gut. We extracted parameters from the centreline including curvature, torsion and radius and found that females have higher curvature of their central midgut loops than males. Centrelines