Quantify the fluorescence intensity over background

Hello everyone. I am a new user of ImageJ. We are interested in measuring fluorescence intensity/background of stained cells. This is how I did the analysis:

  1. Split the picture into green channel.
  2. Select individual cells by drawing a straight line through it, then obtain the plot profile. From there, I get the gray value and calculate the ratio.
    The problem for this is only few cells can be selected and the ratio is cell-dependent as some cells are brighter than others. If we want to get a more accurate measurement based on the whole picture, will anyone be able to help?

Have you read this:


You can automatically select all areas with a particle analysis if necessary to automate your analysis:

On YouTube you will find some examples, e.g.:

See also:



Hello Bio7, thank you very much for your help. I followed the steps from Youtube. After adjusting the threshold and analyzing the particles, I obtained a table summarizing area integrated intensity and mean grey values from all cells I selected. However, the mean, min and max are all 255. Do I need to worry about this or it is totally irrelevant with my analysis?

Yes, you have to worry about that!

When you “Apply” a threshold you create a binary from the original image (values: 0, 255) which you don’t want to measure subsequently.

Just threshold without the use of “Apply” and then measure the values of the original image.

Another way would be to add the particle measurments to the ROI Manager. See the following screenshot:

Then enable the ROI’s on the original image to measure them (Select all ROI’s in the ROI Manager and then measure).

It’s always a good practice to take a sample and compare the pixel values with the measured values (e.g. hover with the mouse over a region and compare if the measured values are in the expected range, etc.)

I tried to use ROI manager, but I can’t transfer my selection from the binary to the original picture. so I can’t do the measurement as “the active image doesn’t have a selection”. How do fix this problem? Thank you again for your help.

Please measure the particles again and select the option “Add to Manager”. In your screenshot the ROI Manager is empty so something went wrong. You can’t activate it after the measurement has been done already.

It worked now. Thanks again for help.

Note that another easy way to measure the properties of the ROIs of one (binary) image with regard to another (original) image is the Redirect to: option in Analyze > Set Measurements…:

From the user guide:

Redirect to The image selected from this popup menu will be used as the target for statistical calculations done by Analyze▷Measure… [m] and Analyze▷Analyze Particles… commands. This feature allows you to outline a structure on one image and measure the intensity of the corresponding region in another image.

This is what I figured out. Thanks.