Automated 3D light-sheet screening with high spatiotemporal resolution reveals mitotic phenotypes.
J Cell Sci
; 133(11)2020 06 01.
Article
em En
| MEDLINE
| ID: mdl-32295847
ABSTRACT
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 24â
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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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Processamento de Imagem Assistida por Computador
/
Esferoides Celulares
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article