Measuring Intensity Using ImageJ

Question

we have to measure the intensity of the fluorescence in certain regions of images using imagej. we came up with the below steps to measure the intensity. while it does seem correct, my question is --> are we actually measuring intensity correctly using the following steps or are we erroneously measuring something else and believing that that value is the intensity?

1. Make the image a 8-bit
2. Threshold the image (Image > Adjust > Threshold) to outline all the regions and click Apply
3. Open Analyze > Analyze particles. Make sure “add to manager” is clicked
4. Analyze > Analyze particles > Show > Bare Outlines. This will open a new image.
5. Open the color microscopy image. Then, Image > Overlay > From ROI Manager.
6. Image > Overlay > To ROI Manager.
7. In ROI Manager: press “measure.” (a Results window with individual data points will pop up)
8. Right click in the Results window and click Summarize.
9. Record mean intensity data

are we correctly measuring mean intensity data using the above steps?

Answer 1

The are some things you need to be aware of when measuring intensity in imageJ:

1. ImageJ automatically converts images to 8-bit
2. You should ALWAYS use RAW if available
3. If your microscope cannot save files as RAW format you must use .tif, other formats create artifacts
4. Save channels separately not as an RGB stack if you are using .tif format

Before threshold you must split the channels, especially if you are looking for florescence from a certain wavelength.

You also need to use the "Watershed" function after the threshold so it will outline individual cells allowing you to avoid outlining a group of cells globed together. This way you can measure their individual intensities. However it is not perfect so once ROI pops up and the cells have been outlined by the particle analysis you must go through and ensure it is measuring singular cells. Any outlines containing more than one cell should be deleted. Also look for odd shaped cells or micro-nuclei, which should also be deleted.

Now I assume you are measuring human cancer cells. You should use these settings:

After all of this then you apply the overlay. Whatever image you are overlaying on you must also split the channels of that as well and use the channel that contains the wavelength you are measuring (ie: Alexaflour 488 would be in the GFP channel).

If you have been collecting data without doing this I would trash all of it as the procedure done to collect it hasn't controlled for anything.

Also as for where to go to ask this stuff.