BactoMate: an integrated platform for reproducible bacterial microscopy analysis

Quantitative microscopy of microorganisms increasingly produces large, multidimensional datasets, yet their analysis often depends on fragmented workflows spanning file conversion, segmentation, quality control, fluorescence quantification, tracking, and visualization. Here, we present BactoMate, an open-source, cross-platform graphical user interface that integrates these steps into a unified workflow for microbial image analysis. BactoMate incorporates established segmentation methods and supports both single-file and batch processing. Its modules enable image preprocessing, cell segmentation, morphology-based quality control, fluorescence and foci quantification, single-cell tracking, lineage reconstruction, structured data export, and generation of quality-control and visualization outputs. We demonstrate the applicability of BactoMate across multichannel fluorescence imaging, bacterial swimming assays, microcolony lineage analysis, phage infection assay and a microfluidic time series. All user-configurable parameters are exposed through the interface, are recorded alongside structured outputs and can be loaded for reproducible image analyses across experiments to reduce introduction of bias. By reducing workflow handoffs while preserving parameter control and exportable results, BactoMate enables accessible, reproducible, and scalable quantitative analysis of microbial microscopy data.