AutoWrinkleID: a machine learning pipeline for biofilm wrinkle identification and quantitative analysis

Biofilms are widely distributed in both natural and engineered systems and play a fundamental role in microbial ecology and biotechnological applications. When grown on agar substrates, biofilms often exhibit macroscopic structural features due to mechanical instabilities driven by matrix production. These wrinkles encode relevant information about biofilm growth and structural development and have even been shown to possess some functional roles. Despite their relevance, currently available approaches for wrinkle detection from images rely on manual annotation, which is time-consuming, not scalable, and strongly dependent on the annotator. In the present study, we developed an end-to-end machine learning pipeline, named AutoWrinkleID, that starts from bright field images of biofilms and performs wrinkle identification, characterisation, and quantification across multiple bacterial species and strains. The name reflects the automated identification and subsequent characterisation and quantification of biofilm wrinkles. The annotations used to generate the masks, namely images containing only the wrinkle structures, were obtained both through manual labelling and using constitutive fluorescence and motility fluorescence. Interestingly, we found that models trained using only manual annotations perform worse than those trained using motility fluorescence markers. A comparison between motility and constitutive fluorescence masks further indicates that motility based annotations consistently outperform constitutive fluorescence across all strains, strongly suggesting that motile phenotypes are tightly associated with wrinkle structures. Additional computational analysis is conducted on the same dataset to assess the robustness and limitations of the results as a function of training dataset. We also observed that Sholl analysis, originally developed for the study of neuronal dendrites and later applied to wrinkle analysis, can effectively characterize wrinkle patterns, although its applicability is strongly limited by wrinkle shape and by the quality of the masks.