TITLE: Dot Measuring (Co-localisation) (FIJI Macro)
AUTHORS: Condon  ND

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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: </span><a href="https://doi.org/10.1016/j.bbamem.2020.183480" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1016/j.bbamem.2020.183480</span></a></div><br/><div class = "text-block">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.</div></div>

[STEPS]
?. [Downloading and installing the macro]
Retrieve a copy of the macro files from the GitHub repo
?. [Downloading and installing the macro]
Download the .ZIP copy of the entire repo to your computer, and extract the compressed file.
?. [Downloading and installing the macro]
Launch FIJI/ImageJ on your computer.
?. [Downloading and installing the macro]
Install the plugin into your instance of FIJI by navigating to Plugins > Macros > InstallChoose 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
?. [Running the macro]
The first window to appear when running the macro describes the Author, and details the tasks the script will run.
?. [Running the macro]
The next window to appear allows the user to input a working directory location using their system file browser.
?. [Running the macro]
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.
?. [Running the macro]
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"
?. [Reviewing the Results]
For each image completed the following files are created:_RoiSet.zip [this contains the ROIs for the lysosomes]_Green.tif [this image is the cleaned up Green (peptide) image]_Red.tif [this image is the cleaned up Red (lysotracker) image]_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]
?. [Reviewing the Results]
Results Spreadsheet informationNote 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 IDCount: This is the measured Mean Intensity for the nuclei selection regionRed Intensity: This is the measured Mean intensity for the cytoplasmic band selection regionCoLocalisation 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.