The Intensity Macro for Background Subtraction is customized for the image processing and analysis done by the Ogle Lab. It prompts the user to choose a background region of interest, a percentage of background to retain and a region of interest to which the subtraction should be applied. The intensity level to subtract from the fluorescent regions of interest is calculated by computing the level at which the background intensity histogram corresponds to the amount of background the user desires to retain.
The command is: Plugins > LOCI > Intensity Macro for Background Subtraction.
The macro will prompt the user to open a brightfield image and select a cell region of interest. The macro then prompts the user to open a fluorescent image and select a fluorescent region of interest. The next prompt allows the user to choose the percentage of background the user wants to retain - high amounts (~100%) will result in very little background subtraction, while lower amounts (~0%) will result in significant background subtraction. After determining the level of background to retain, another prompt allows the user to choose a background region to measure the background of the image. After background subtraction, the results window will display statistics of Intensity / BF area, Intensity / Fluorescent area, % background retained, and others.