BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.

Department of Biomedical Engineering Faculty of Electrical Engineering and Communication Brno University of Technology Technicka 3058 10 Brno CZ 61600 Czech Republic

Department of Biomedical Engineering Faculty of Electrical Engineering and Communication Brno University of Technology Technicka 3058 10 Brno CZ 61600 Czech Republic Department of Physiology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 62500 Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 62500 Czech Republic International Clinical Research Center St Anne's University Hospital Pekarska 664 53 Brno CZ 65691 Czech Republic

Department of Pathological Physiology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 62500 Czech Republic Central European Institute of Technology Brno University of Technology Purkynova 656 123 Brno CZ 612 00 Czech Republic

Department of Physiology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 62500 Czech Republic Department of Pathological Physiology Faculty of Medicine Masaryk University Kamenice 5 Brno CZ 62500 Czech Republic Central European Institute of Technology Brno University of Technology Purkynova 656 123 Brno CZ 612 00 Czech Republic

Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstr 108 Dresden DE 01307 Germany

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