Edoardo D’Imprima (1), Marta Garcia Montero (2), Sylwia Gawrzak (2), Paolo Ronchi (3), Ievgeniia Zagoriy (1), Yannick Schwab (2) (3), Martin Jechlinger (2), Julia Mahamid (1) (2)
Developmental Cell. (10 April 2023). DOI: https://doi.org/10.1016/j.devcel.2023.03.001

Keywords

FIB-SEM, volume EM, CLEM, patient-derived organoids, cryo-confocal light microscopy, high-pressure freezing, deep-learning image segmentation

Abstract

3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tis- sues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter- scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cul- tures in a single carrier that is amenable to all imaging steps. This allows for following organoids’ growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultra- structure. We demonstrate this workflow on mouse and human 3D cultures and use automated image seg- mentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polar- ized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.

How Our Software Was Used

Dragonfly was used to train neural networks for automatic segmentation of FIB-SEM data. To compute local density maps of the segmentations, the Bone Analysis wizard was used to estimate volume fractions. Nuclear sphericity was subsequently measured in Dragonfly.

Author Affiliation

(1) Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
(2) Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
(3) Electron Microscopy Core Facility, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany