Automated 3D light-sheet screening with high spatiotemporal resolution reveals mitotic phenotypes
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 cel...
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| Hauptverfasser: | , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
1 June 2020
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| In: |
Journal of cell science
Year: 2020, Jahrgang: 133, Heft: 11 |
| ISSN: | 1477-9137 |
| DOI: | 10.1242/jcs.245043 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1242/jcs.245043
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| Verfasserangaben: | Björn Eismann, Teresa G. Krieger, Jürgen Beneke, Ruben Bulkescher, Lukas Adam, Holger Erfle, Carl Herrmann, Roland Eils and Christian Conrad |
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| 520 | |a 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, cell spheroids are imaged for 24 h in toto with a dual-view inverted selective plane illumination microscope (diSPIM) 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 using siRNA knockdown 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. | ||
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