BacStalk: a comprehensive and interactive image analysis software tool for bacterial cell biology

ABSTRACTProkaryotes display a remarkable spatiotemporal organization of processes within individual cells. Investigations of the underlying mechanisms rely extensively on the analysis of microscopy images. Advanced image analysis software has revolutionized the cell-biological studies of established model organisms with largely symmetric rod-like cell shapes. However’ algorithms suitable for analyzing features of morphologically more complex model species are lacking’ although such unusually shaped organisms have emerged as treasure-troves of new molecular mechanisms and diversity in prokaryotic cell biology. To address this problem’ we developed BacStalk’ a simple’ interactive’ and easy-to-use MatLab-based software tool for quantitatively analyzing images of commonly and uncommonly shaped bacteria’ including stalked (budding) bacteria. BacStalk automatically detects the separate parts of the cells (cell body’ stalk’ bud’ or appendage) as well as their connections’ thereby allowing in-depth analyses of the organization of morphologically complex bacteria over time. BacStalk features the generation and visualization of concatenated fluorescence profiles along cells’ stalks’ appendages’ and buds to trace the spatiotemporal dynamics of fluorescent markers. Cells are interactively linked to demographs’ kymographs’ cell lineage analyses’ and scatterplots’ which enables intuitive and fast data exploration and’ thus’ significantly speeds up the image analysis process. Furthermore’ BacStalk introduces a 2D representation of demo- and kymographs’ enabling data representations in which the two spatial dimensions of the cell are preserved. The software was developed to handle large data sets and to generate publication-grade figures that can be easily edited. BacStalk therefore provides an advanced image analysis platform that extends the spectrum of model organisms for prokaryotic cell biology to bacteria with multiple morphologies and life cycles.IMPORTANCEProkaryotic cells show a striking degree of subcellular organization. Studies of the underlying mechanisms and their variation among different species greatly enhance our understanding of prokaryotic cell biology. The image analysis software tool BacStalk extracts an unprecedented amount of information from images of stalked bacteria, by generating interactive demographs, kymographs, cell lineages, and scatter plots that aid fast and thorough data analysis and representation. Notably, BacStalk can preserve the two spatial dimensions of cells when generating demographs and kymographs to accurately and intuitively reflect the intracellular organization. BacStalk also performs well on established, non-stalked model organisms with common or uncommon shapes. BacStalk therefore contributes to the advancement of prokaryotic cell biology, as it widens the spectrum of easily accessible model organisms and enables a more intuitive and interactive data analysis and visualization.