Measuring fluorescence intensity within cell outlines

Hi all!
I am new to imagej. I am trying to measure the intensity of fluorescence within many cells. I have done a cell count for each image and have an outline of all cells that were measured and I would like to now measure the intensity of fluorescence within each of these cells and get averages, SD, etc of all of the cells in one image. What is the best way to do this? Do I need to merge the original image with the image of only the cell outlines to start? Thank you any help you can provide!

Dear @Nicole-Y,

welcome to the forum.
Since you mention that you achieved a counting of cells together with outlines already, I assume that you extractzed your cells e.g. via a threshold in form of a binary image (only black and white). If this is the case the easiest solution is to have a look at the Analyze Particles... function. If you set the checkbox Add to manager active you will get all the ROIs for your cells. by activating the original image and pressing the Measure button in the ROI Manager you can retrieve pixel-base values from the original image. That would be the manual and technical part. Finally it still depends on the type of image, if the measurement can be done this way (e.g. if the original image is a 8- or 16-bit image or saved as RGB etc.)
You might want to have a look into the IJ user manual as well for further information.

Hi thanks a lot for your response. I have extracted my cells via a threshold and I am using an 8 bit image. Does this still work if my image is 8 bit? By now activating the original image I can now see that clicking on one of the cells highlights it on the original image and I can click measure, but which measurement is giving me the intensity of fluorescence (I have a green fluorescence image) and in what kind of units is this in? In addition is it possible to not only get the data for each individual cell but somehow get a summary of all the cells to get averages, SD, etc. I am working with an image that has hundreds of cells so is there also a way to measure each of the cells automatically rather than manually?

Thank you very much for you time and help.


8-bit works.
You do not need to select a specific ROI im the ROI Manager. If you unselect all with the respective button and then press the Measure button it measures all ROIs automatically.
The “units” are gray value. So basically there is no real specific unit except the image is calibrated to any specifi units. But this is normally not done on fluorescent micrographs.

Thanks a lot for your help!