This repository contains scripts and templates used for image preprocessing and analysis in the publication specified below.
Krister Vikedal, Synnøve Brandt Ræder, Ida Mathilde Riisnæs, Magnar Bjørås, James Booth, Kirsten Skarstad, and Emily helgesen
RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin exposure in Escherichia coli
bioRxiv (2024)
doi: 10.1101/2024.11.15.623168
Please see the Methods and Materials section of the paper for details on preprocessing and analysis.
All scripts for preprocessing of images from different microscopes before analysis are located in the folder Preprocessing. These scripts are intended for use in the Fiji distribution of ImageJ, using Jython, an implementation of Python that runs on the Java platform. Different scripts were written for each type of microscope, due to differences in image formats and imaging methods. Each script is described under their relevant heading below.
The Z-stacks were first processed using the script FocusedZSlicePrompter.py to select the best-focused slice from each frame, and save them as a new .tif hyperstack. The input hyperstack should ideally also be a .tif file. The images in the new in-focus hyperstack were then manually preprocessed in Fiji (ImageJ) using the procedure described in Preprocessing_ZeissZ1_images.txt.
Images in the .nd2 format were preprocessed using the script Preprocessing_NikonTi2_images.py.
A separate flat-field correction image is required for preprocessing of the brightfield channel and is optional for fluorescence channels. To create a flat-field correction image, we averaged 20 images acquired from different locations on an empty agar pad using the same microscope settings as for the images to be processed.
Images in the .tif format with filenames describing well number, imaging site, and channel were preprocessed using the script Preprocessing_ImageXpress_images.py. The time_map variable needs to be modified to ensure correct time_stamp annotations on output image filenames.
Images in the .lif format were first saved as individual .tif images and then preprocessed using the script Preprocessing_DM6000B_images.py.
See License Details
All scripts and templates for analyzing images with the ObjectJ project Coli-Inspector and the MicrobeJ plugin in Fiji (ImageJ) are located in the folder Analysis.
We have customized the embedded macros of the ObjectJ project Coli-Inspector (version 03f) to allow grouping of cells by image number and, consequently, by time frame. To use the customized embedded macros, open the file Coli-Inspector-03f-KVmod.ojj in Fiji (ImageJ). The changes made to the original embedded macros can be reviewed in the corresponding file Coli-Inspector-03f-KVmod.txt, where code modifications are annotated with the comment Added by KV.
Please refer to the project's documentation for detailed instructions on its use.
In our study, we used two different versions of MicrobeJ for image analysis:
- Main version 5.13p (1): For creating kymographs and analyzing DNA distribution.
- Beta version 5.13p (13): For tracking GFP-RecN and RecA-mCherry foci and analyzing their distribution and localization within cells.
Templates for analyzing all cells from each time frame:
MJtemplate_AvgKymograph.xml: To create kymographs.MJtemplate_RecNinDNA_tracking.xml: To track RecN and assess its colocalization with DNA.MJtemplate_RecAcolRecN_tracking.xml: To track RecA and assess its colocalization with RecN.
Templates for single-cell analysis:
MJtemplate_SingleCell_Kymograph_Profile.xml: To create kymographs and analyze DNA distribution profiles for strains harboring only HU-mCherry.MJtemplate_SingleCell_Kymograph_RecNinDNA_tracking.xml: To create kymgographs and track GFP-RecN for strains harboring GFP-RecN and HU-mCherry.MJtemplate_SingleCell_Kymograph_RecNonly_tracking.xml: To create kymographs and track GFP-RecN for strains harboring GFP-RecN and RecA-mCherry.MJtemplate_SingleCell_RecAcolRecN_tracking.xml: To track RecA-mCherry for strains harboring GFP-RecN and RecA-mCherry.
Please refer to the plugin's documentation for detailed instructions on its use.
Scripts for preprocessing and the modifications to the Coli-Inspector macro script in this repository are licensed under the MIT license, included in the LICENSE file. Templates for MicrobeJ are also licensed under the MIT license, but note that the license applies exclusively to the contributions made by the author of this repository.
Permissions to distribute the MicrobeJ templates have been requested from the original developer, but have not yet been granted or denied. Only the modifications are currently under the MIT license, and the original software remains under its respective rights held by the original developers. Users of the templates should respect the original licensing terms of the MicrobeJ software until full permissions are obtained.
Krister Vikedal
The customized embedded macros Coli-Inspector-03f-KVmod.ojj is based on the ObjectJ project Coli-Inspector (version 03f), developed by Norbert Vischer (University of Amsterdam). The original embedded macros can be downloaded from the documentation page. MicrobeJ was developed by Adrien Ducret (Université de Lyon), Yves Brun (Indiana University), and Christophe Grangeasse (Université de Lyon). The plugin can be downloaded from its download website. We thank Adrien Ducret (Université de Lyon) for assistance with MicrobeJ analysis and code development for tracking of foci, and Thierry Oms (Université Libre de Bruxelles) for assistance with kymograph plots. ChatGPT by OpenAI was utilized for suggesting code for scripts included in this repository.