Single-cell analysis of bacteria and subcellular protein localization dynamics has shown that bacteria have elaborate life cycles, cytoskeletal protein networks and complex signal transduction pathways driven by localized proteins. The volume of multidimensional images generated in such experiments and the computation time required to detect, associate and track cells and subcellular features pose considerable challenges, especially for high-throughput experiments. There is therefore a need for a versatile, computationally efficient image analysis tool capable of extracting the desired relationships from images in a meaningful and unbiased way. Here, we present MicrobeJ, a plug-in for the open-source platform ImageJ1. MicrobeJ provides a comprehensive framework to process images derived from a wide variety of microscopy experiments with special emphasis on large image sets. It performs the most common intensity and morphology measurements as well as customized detection of poles, septa, fluorescent foci and organelles, determines their subcellular localization with subpixel resolution, and tracks them over time. Because a dynamic link is maintained between the images, measurements and all data representations derived from them, the editor and suite of advanced data presentation tools facilitates the image analysis process and provides a robust way to verify the accuracy and veracity of the data.
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The authors thank all members of the Brun laboratory for many discussions. In particular, the authors thank B. LaSarre and C. Ellison for useful feedback about MicrobeJ and D. Kysela and L. Espinosa for discussions and reading the manuscript. The authors thank S. Schlimpert, N. Feirer, C. Grangeasse, T. Doan, P. Brown and B. LaSarre for providing images from S. venezuelae cells, A. tumefaciens, S. pneumoniae, B. subtilis, P. hirshii and R. palustris, respectively. The authors thank M. Thanbichler for the pvan-ftsZ-yfp carrying the Caulobacter strain. This research was supported by National Institutes of Health grants GM51986 and GM113172, and by seed funding from the Indiana University Office of the Vice-President for Research. This project was supported, in part, by the Indiana Clinical and Translational Sciences Institute funded, in part, by grant UL1TR001108 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Presentation of the MicrobeJ workflow.
FtsZ-YFP localization in C. crescentus.
Example of an advanced image analysis with MicrobeJ in real-time.
FtsZ-YFP localization in a filamentous C. crescentus cell.
Example of automated and manual segmentation with MicrobeJ.