The method involves the following steps:
Step 1 - Define the suffix for the processed files
Step 2 - Define the plugins and parameters that need to be run
Step 3 - Identify the folder that contains the .TIF raw files
Step 4 - Select the channel that needs to processed
Step 5 - Identify the folder where the processed files need to be saved
Step 6 - Repeat for another channel
1. Drag and drop the FIJI_MaxSubtract_metamorph.ijm into ImageJ/FIJI
● Line 26 - This code is for Z-projection- maximum intensity projection on Z-stacks
○ This can be changed to Average intensity or sumslices as desired
● Line 27 - This code is for Subtract Background - Rolling ball 50
○ The rolling ball of 50 pixels is default and can be changed as desired
○One can also perform other tasks such as
■ mathematical subtraction of single pixel value from the entire image using'
> run(“Subtract…”, “value=2300”); This can be determined by taking the mean intensity of an
image in an area without cells
■ Median filter to denoise and help smoothen the edges > run(“Median…”, “radius=0.5”);
● Line 29 - Suffix to be attached to the processed file names is “_max.tif”
● Upon Running the pipeline, select the folder where the .TIF raw data is stored
● Select Channel pop-up - Define the channel on which the plugins need to be applied
○ The channel name is in the image filename usually as _w(channel name)
○ Write “w1405” to select the DAPI channel, “w2488” to select Golgi channel
■ Each channel may have different background intensities / noise and may need different parameters; in the example provided all channels are processed similarly
■ If all channels need same processing, then a unique identifier for all channels can be used - something that is part of all the channels such as “3T3”
● Next select the folder where the output of the processed images need to be saved
○ It is recommended to make a new folder where processed images are saved