TITLE: Nuclei:Cytoplasm EKC Quantification Script (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 ratio fluorescence intensity of EKC in both the nuclei and cytoplasm. This protocol is used in the following eLife paper: </span><a href="https://doi.org/10.7554/eLife.62196" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.7554/eLife.62196</span></a></div><br/><div class = "text-block">Briefly this macro takes directories of images and processes them in an automonous batch-wise maner. Images should be 2-channel with channel one containing your EKC (or other tagged protein of interest) and channel 2 containing your nuclei signal. Nuclei are segmented and two selection regions are created (nuclei, and a surrounding band that is user definable). These two regions are measure for each identified nuclei and the mean fluorescence intensity is output into a single spreadsheet.</div></div>

[STEPS]
?. [Downloading & installing the macro]
Retrieve a copy of the macro file from either the GitHub repo  or from the Software Heritige archive
?. [Downloading & installing the macro]
Download the .ZIP copy of the entire repo to your computer, and extract the compressed file.
?. [Downloading & installing the macro]
Launch FIJI/ImageJ on your computer.
?. [Downloading & installing the macro]
Install the plugin into your instance of FIJI by navigating to Plugins > Macros > InstallChoose to install the file called "Nuclei_cytoplasm-measuring_v1-5.ijm"
?. [Running the macro]
To launch the installed macro, navigate to Plugins > Macros > Nuclei_cytoplasm-measuring_v1-5
?. [Running the macro]
The first window to appear when running the macro describes the Author, Licence and details the tasks the script will run.
?. [Running the macro]
The second window warns the user that they will next be required to navigate to the directory which contains the image files. The user should also note the file extension of these files to enter into the upcoming preferences window.
?. [Running the macro]
The preferences window prompts the user to input the File extension (for example .tif, .czi, .lsm) as well as the banding distance (in pixels). The user can choose to run in batch mode (faster, background running) or not by selecting the relevant checkbox.
?. [Running the macro]
The script will then run through each image within the chosen input directory, identifying nuclei, and measing the EKC 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:_cyto.zip [this contains the ROIs for the cytoplasm measurements]_green-selection.tif [this image is the 32-bit thresholded image of Ch1]_nuclei.zip [this contains the ROIs for the nuclei measurements]_nuclei-mask.tif [this image is the thresholded mask of Ch2]_selectionnumbers.png [this image shows the ROI slection numbers corresponding to the quantification results]For the entire script run the following collated files are create:Log.txt [this is a copy of the Fiji Log window, with reported script version, settings used, directory location, and file completions]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.Nuclei#: This is the nuclei ID (as per the corresponding output file (_selectionnumbers.png)Nuclei Intensity: This is the measured Mean Intensity for the nuclei selection regionCytoplasm Intensity: This is the measured Mean intensity for the cytoplasmic band selection regionNumber of Nuclei: This is the total number of nuclei identified in this image (not it will repeat for each neclei of this imageCyto/Nuclei: This is Cytoplasmic Intensity divided by the Nuclei IntensityNuclei/Cyto: This is the Nuclei Intensity dividied by the Cytoplasmic intensity