PT  - JOURNAL ARTICLE
AU  - Daniel Zyss
AU  - Susana A. Ribeiro
AU  - Mary J.C. Ludlam
AU  - Thomas Walter
AU  - Amin Fehri
TI  - Cell Segmentation in Images Without Structural Fluorescent Labels
AID  - 10.1101/2023.01.12.523757
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.01.12.523757
4099  - http://biorxiv.org/content/early/2023/01/16/2023.01.12.523757.short
4100  - http://biorxiv.org/content/early/2023/01/16/2023.01.12.523757.full
AB  - High-content screening (HCS) provides an excellent tool to understand the mechanism of action of drugs on disease-relevant model systems. Careful selection of fluorescent labels (FLs) is crucial for successful HCS assay development. HCS assays typically comprise (1) FLs containing biological information of interest, and (2) additional structural FLs enabling instance segmentation for downstream analysis. However, the limited number of available fluorescence microscopy imaging channels restricts the degree to which these FLs can be experimentally multiplexed. In this paper, we present a segmentation workflow that overcomes the dependency on structural FLs for image segmentation, typically freeing 2 fluorescence microscopy channels for biologically relevant FLs. It consists in extracting structural information encoded within readouts that are primarily biological, by fine-tuning pre-trained state-of-the-art generalist cell segmentation models for different combinations of individual FLs, and aggregating the respective segmentation results together. Using annotated datasets that we provide, we confirm our methodology offers improvements in performance and robustness across several segmentation aggregation strategies and image acquisition methods, over different cell lines and various FLs. It thus enables the biological information content of HCS assays to be maximized without compromising the robustness and accuracy of computational single-cell profiling.Impact Statement This methodological paper describes a framework enabling cell segmentation for datasets without structural fluorescent labels to highlight cell organelles. Such capabilities favorably impact costs and possible discoveries in single-cell downstream analysis by improving our ability to incorporate more biological read-outs into a single assay. The perspective of computational and experimental biologist coauthors ensures a multidisciplinary viewpoint and accessibility for a wide readership.Competing Interest StatementThe authors have declared no competing interest.