INFORMATION
Software for background subtraction and kymograph fitting for fluorescence microscopy image stacks of vertically immobilized bacteria using VerCINI (Vertical Cell Imaging by Nanostructured Immobilization). First published in Whitley et al, Nature Communications 2021. This software is provided as is and without warranty.
OVERVIEW
Performs circular kymograph analysis for VerCINI microscopy data of vertically immobilized bacteria. Two analysis methods are provided:
- verciniAnalysis.m: Automated kymograph analysis where septa/ cell circumference is localized to sub-pixel precision by fitting an explicit model. The fitted background contribution is subtracted from each image, and a kymograph calculated along the circular line profile of the cell circumference. This should work for most datasets
- manualVerciniAnalysis.m: Manual analysis where the kymograph is calculated along a circular line profile manually selected for each image stack, based on the maximum intensity projection of the stack. This is mostly used for sparse single molecule datasets if automated VerCINI analysis has failed
SYSTEM REQUIREMENTS
This package does not require any special hardware. It can be performed on a standard computer.
This package development system has been tested on Windows 10, but should be compatible with other operating systems.
The package requires the following software:
- MATLAB (>=R2018b)
- MATLAB Image processing toolbox
- MATLAB Optimization toolbox
INSTALLATION
Add the 'ring-fitting2' directory to your MATLAB path, and save the updated path so that it will remain installed next time you start MATLAB. Typical install time should be a couple of minutes.
USAGE INSTRUCTIONS
To run the software, please see detailed help documentation in the main analysis functions verciniAnalysis.m, manualVerciniAnalysis.m, which can be accessed by typing 'help verciniAnalysis' on the MATLAB command line. Each function should take between 10 s and 10 min to run on a 'standard' computer, depending on the size of the dataset.
DEMO
Test data and example analyses are included in ring-fitting2/testing. Three scripts are provided:
- testCytoplasmicGFP_Analysis.m
- testVerciniAnalysis.m
- testManualVerciniAnalysis.m
Each script can be run either from the command line (i.e. by typing the function name and pressing Enter) or by running them from the Editor. They will automatically analyse example data provided in the ring-fitting2/testing, and produce a new directory ring-fitting2/testing/analysed with the output data. Each should take between 10 s and 1 min to run on a 'standard' computer. Note that for testManualVerciniAnalysis you must manually draw a circle where you see a ring in the image, then press Enter.
The output files will be
- file_bgsub.tif (video with cytoplasmic background subtracted)
- file_diamInfo.txt (text file showing fitted diameter of ring for frame 1)
- file_fitData.mat (MATLAB file containing information about the fitting)
- file_fitResult.png (image showing first frame of video with fit to model)
- file_kymo.tif (radial kymograph from 0 to 360 degrees)
- file_kymoRaw.tif (radial kymograph without background subtraction from 0 to 360 degrees)
- file_kymoRawWrap.tif (radial kymograph without background subtraction from 0 to 720 degrees)
- file_kymoWrap.tif (radial kymograph from 0 to 720 degrees)
LICENSING INFORMATION
All files are distributed under the GPLv3 and (c) 2020 Seamus Holden, Newcastle University unless otherwise stated. See LICENSE.txt for full terms.
CITATION
If you use this software in work leading to a scientifc publication, please cite:
FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division Kevin D. Whitley, Calum Jukes, Nicholas Tregidgo, Eleni Karinou, Pedro Almada, Yann Cesbron, Ricardo Henriques, Cees Dekker, Séamus Holden Nature Communications 12, 2448 (2021). https://doi.org/10.1038/s41467-021-22526-0
