Sep 30, 2021
Dot Measuring (Co-localisation) (FIJI Macro)
This protocol is a draft, published without a DOI.
- Condon ND1
- 1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia
Protocol Citation: Condon ND 2021. Dot Measuring (Co-localisation) (FIJI Macro). protocols.io https://protocols.io/view/dot-measuring-co-localisation-fiji-macro-byp6pvre
Manuscript citation:
https://doi.org/10.1016/j.bbamem.2020.183480
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 30, 2021
Last Modified: September 30, 2021
Protocol Integer ID: 53726
Keywords: Microscopy, Image Analysis, ImageJ, FIJI, Macro, Quantification, Colocalisation, mean fluorescence intensity of region, fluorescence intensity, mean fluorescence intensity, labelled peptide, fluorescence, lysosome, channel images of cell, peptide, lysome, dot measuring, channel image, labeleld with lyostracker,
Funders Acknowledgements:
CZI
Grant ID: 2020-225648
Abstract
This protocol describes how to download, install, and run the ImageJ/Fiji macro for quantifying the mean fluorescence intensity of regions determined in one channel, based upon the intensity of the second channel. This protocol is used in the following manuscript: https://doi.org/10.1016/j.bbamem.2020.183480
Briefly, this script takes 2 channel images of cells which have taken up fluorescently labelled peptides, and have their lysomes fluorescently labeleld with Lyostracker. Lysosomes are identified using a Find Maxima command, and selections generated. These selections are then measured individually for the fluorescence intensity of the labelled peptide. Results are output into a spreadsheet for further analysis.
Troubleshooting
Before start
Having an up-to-date version of FIJI/ImageJ is critical for this macro. A fresh installation can be downloaded from Fiji.sc
Downloading and installing the macro
Download the .ZIP copy of the entire repo to your computer, and extract the compressed file.
Launch FIJI/ImageJ on your computer.
Install the plugin into your instance of FIJI by navigating to Plugins > Macros > Install
Choose to install the file called "Aurelie - Reversed Green of RedArea.ijm"
Running the macro
To launch the installed macro, navigate to Plugins > Macros > Aurelie - Reversed Green of RedArea
The first window to appear when running the macro describes the Author, and details the tasks the script will run.
The next window to appear allows the user to input a working directory location using their system file browser.
The script will then run through each image within the chosen input directory, identifying lysosomes, and measing the fluorescent peptide signal automonously with no user input required.
Upon completion the script will prompt the user with a dialog box stating "Put down that coffee! Your job is finished"
Reviewing the Results
Navigate to the input directory location using your computers filesystem browser.
Select the newly created results directory called "Results"
For each image completed the following files are created:
<Filename>_RoiSet.zip [this contains the ROIs for the lysosomes]
<Filename>_Green.tif [this image is the cleaned up Green (peptide) image]
<Filename>_Red.tif [this image is the cleaned up Red (lysotracker) image]
<Filename>_Points.tif [this image shows the ROI slections resulting from the Find Maxima]
For the entire script run the following collated files are create:
Results.xls [results spreadsheet for quantification, see below]
Results Spreadsheet information
Note if opening with Microsoft Excel you will see the following warning, this is because the file Results.xls was created outside of Microsft Excel, click Yes to continue opening the document.
The following columns are provided in the Results.xls output file:
Filename: This is the filename for the input image.
Cell: This is the cell ID
Count: This is the measured Mean Intensity for the nuclei selection region
Red Intensity: This is the measured Mean intensity for the cytoplasmic band selection region
CoLocalisation Boolean: This column determines if the intensity of Peptide (green) is above a certain cut off (500) within the lysosome region, it is considered colocalised and will have a value of 1, if below this cut off the value 0 will be displayed. This cut off can be modified in line 69 of the code.