Segmentation of dense and multi-species bacterial colonies using models trained on synthetic microscopy images

The spread of microbial infections is governed by the self-organization of bacteria on surfaces. Limitations of live imaging techniques make collective behaviors in clinically relevant systems challenging to quantify. Here, novel experimental and image analysis techniques for high-fidelity single-cell segmentation of bacterial colonies are developed. Machine learning-based segmentation models are trained solely using synthetic microscopy images that are processed to look realistic using state-of-the-art image-to-image translation methods, requiring no biophysical modeling. Accurate single-cell segmentation is achieved for densely packed single-species colonies and multi-species colonies of common pathogenic bacteria, even under suboptimal imaging conditions and for both brightfield and confocal laser scanning microscopy. The resulting data provide quantitative insights into the self-organization of bacteria on soft surfaces. Thanks to their high adaptability and relatively simple implementation, these methods promise to greatly facilitate quantitative descriptions of bacterial infections in varied environments.