Fiji ImageJ 

System requirements Fiji is supported on the following systems: 
Windows XP, Vista, 7, 8, 10, 11, etc. 
Mac OS X 10.8 “Mountain Lion” or later 
Linux on amd64 and x86 architectures
However, Fiji (like ImageJ) should run on any system for which a Java 8 runtime is available (Solaris, Raspbian, etc.).

Installation Caution: “Program Files” not recommended! If you are installing ImageJ2 on Windows, we strongly recommend that you store your ImageJ2.app directory somewhere in your user space (e.g., C:\Users\[your name]\ImageJ2.app) rather than in C:\Program Files or other system-wide directory. If you move ImageJ2.app to such a directory, modern versions of Windows will deny ImageJ2 write permission to its own directory structure, preventing it from being able to update. See also imagej/imagej#72.
Fiji is distributed as a portable application. That means that you do not have to run an installer; just download, unpack and start it.
Source code: https://github.com/imagej/ImageJ. 
Installation time: 15 minutes. 
Tested on Windows 10 Version 21H2 (OS Build 19044.1889)


Analysis
# Code 1 (readme1.txt): Cell Polarity Fixed Confocal (Cell split into two halves and measure fluorescence in each half to calculate cell polarity).
ROI detection: Image stack was opened and split into individual channels. Channels were renamed. Enhance brightness/contrast and subtract background and save these images. For ROI detection, the channel which showed strong signal from the entire cell area was used. ROIs were detected either automatically through thresholding and analysed. In cases where ROIs were not detected or incorrectly detected automatedly, ROIs were manually drawn and added to the ROI Manager. The ROIs were saved as a ROIset.  
Creating binary mask: Binary masks were created by automatic thresholding Ctrl + Shift + T (using appropriate thresholding method). Thresholded images were converted to binary masks (0-255) and saved. 
ROI split and measurement: The images corresponding to specific channels are opened in Fiji and renamed as “A” and “B”. Please ensure that the images are not named as “A.tif” or “B.tif”. Corresponding ROIset was also opened. Binary mask of the image was opened and renamed as “M”. Code1 (.ijm script) was run in Fiji. 
Upon successful running of code, we will get a ‘log’ window, ‘Results’ window and a binary mask image where each ROI has been successfully split into 2 halves. The output ‘log’ file and ‘Results’ was copied in a spreadsheet (‘readme1_log’ and ‘readme1_Results’ sheets of the ‘All_codes_output.xslx’) for further analysis and split ROI image was saved. 
# Code 2 (readme2.txt): Cell polarity Live Confocal Imaging (Cell split into two halves according to direction of movement and fluorescence measured for each half along with displacement of the cell). 
Time-lapse stacks of each channel (Actin-GFP & mCherry) provided. Each channel was renamed as ‘A’ for Actin-GFP and ‘B’ for Piezo1-mCherry. 
Code 2 (.ijm script) was run on Fiji and output log file containing results of analysis (cell displacement, fluorescence signal of front/back half of the each cell, relative polarisation angle of each fluorescent marker, etc.) The output is in ‘readme2_output’ sheet of the ‘Fiji_Codes_Output.xslx’. 






