To make image of colocalized areas only


Hello. I have image with two channels. From this image I want to make another image (and to save it as a single channel) showing colocalized area only. What is the protocol for it?


Hi @1timur1 -

I am not quite sure what you are asking… do you need to do in-depth colocalizaiton analyses ? or are you just interested in seeing the areas of overlap between to images ? If the latter… then you can just use the built-in Image Calculator AND operator for the two images.

Ideally - if you can post the images here … and describe in a bit more detail what you wish to measure - we can better help you.

eta :slight_smile:


Hi etarena,
Right now my plan was to build mask of colocalized pixels. I want to build it because later I will apply it as a mask to another channel (signal of interest) for quantification. I figured way to do it, but it is painful. Here is the protocol. If you see an easier way, please record protocol and share it with me and others.

  1. Open channel with 3 channels (attached).
  2. Split channel and apply threshold to channel 1 and then to channel 2. Hit “Apply” and have binary images built for both channels.
  3. Go to Process-Math and subtract 254 from these two binary images (it makes signal value 1, background 0).
  4. Go to Process-ImageCalculator and add these two channels together. Then to this new image apply threshold 2-255 (this will threshold colocalized pixels only) and hit “Apply”.
  5. Then go to Process-Math and subtract 254 from this binary image (it will make intensity of pixels in thresholded areas 1 and background 0). Threshold it 1-255 (But Don’t click “Apply”)
  6. Go to Process-Math-ImageCalculator and multiply this image with 8-bit channel (which has signal of interest).
  7. Go to Analyze Particles and create measurements of pixels intensities (final product).




So it seems that what you want to measure is co-occurrence - not colocalization. Co-occurrence is simply saying ‘yes’ or ‘no’ to the ‘presence’ or ‘absence’ of both signals in the same space/area/pixel.

I would think just using the Image Calculater AND operator should do the trick in this case with your individual binary images for each channel. But you would have to test it - would help eliminate some of the ‘extra’ steps in your current protocol.

Also - on a side… your image is very noisy. In fact, for the latter two channels - can you be sure that what you are seeing is true signal as opposed to noise? Have you checked against some control stainings to be sure?

Hope this helps a bit.



You recommend to use Operator. Since I am not aware about this option I would appreciate if you would run entire process and share with me (and others) record of it. Thanks.


when I downloaded this image somehow it did not look the same.
Thus I am sending it again as individual images of three separated channels. Please overlap (co-occur) channel one and two, then calculate intensities of image three in areas masked by the overlap of first and second channels. Again, I would appreciate the record (want to understand what is operator here is ). Thank you.


@1timur1 The command to which @etarena refers is the Image Calculator. Find it easily by pressing L for the Command Finder, and typing image cal then pressing enter to run it. I also suggest to check the “Close window after running command” box so that you can repeatedly launch commands via the Command Finder with ease.


Got it. It works.
Just to clarify: is command finder the same option which etarena called “operator”?



Just click on all the links we posted… you will get the answer to that question.



I did. However didn’t find there word “operator”.


Ok figured it out. Instead of “operator” word “operation” should of be used.