Automated screening by 3D light-sheet microscopy with high spatial and temporal resolution reveals mitotic phenotypes

• Published in: bioRxiv
• Main Authors: Eismann, Björn, Krieger, Teresa G, Beneke, Jürgen, Bulkescher, Ruben, Lukas, Adam, Erfle, Holger, Herrmann, Carl, Eils, Roland, Conrad, Christian
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 20.01.2020
• Subjects: Automation; Cell culture; Cell cycle; Epigenetics; High-throughput screening; Image processing; Information processing; Microscopy; Mitosis; Neural networks; Phenotypes; siRNA; Spheroids; Tissue culture
• DOI: 10.1101/2020.01.20.912659

3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, three-dimensional cell spheroids are imaged for 24 hours in toto with a dual inverted selective plane illumination (diSPIM) microscope with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network based phenotype classification. We illustrate the potential of our approach by siRNA knock-down and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.