Colour to Grey Split 4th channel not recognized


Hi there,

I’m having trouble with splitting a 4 channel image - the channels being blue, green, red and white (the output from the software). They are .tif images that have been exported from the microscope software. I’ve fed them into a colour to grey pipeline that has channel split with 1:Red, 2:Green, 3:Blue, then 4. I believe that the 4th channel is the issue as it’s not transparent but it is the fourth channel of a .tif. Ideas on how to remedy this?

This is the error CellProfiler (using the version 3.0 - is that a good idea?) gives me when it tries to do it:

Traceback (most recent call last):
File “/Applications/”, line 2846, in do_step
File “/Applications/”, line 1993, in run_module
File “/Applications/”, line 310, in run
File “/Applications/”, line 355, in run_split
IndexError: index 3 is out of bounds for axis 2 with size 3





It’d be great if you give us an example image. In the meantime, can you try this:

  • ColorToGray >> Conversion method: Split >>
    Convert Red to gray: Yes > Name output = OrigRed
    Convert Green to gray: Yes > Name output = OrigGreen
    Convert Blue to gray: No

  • ImageMath >> Operation "Absolute Difference"
    First image: your original image
    Second image: OrigRed
    Third image: OrigGreen
    Name the output : ImageAfterMath

  • Another ColorToGray >> Conversion method: Split >>
    Convert Red to gray: Yes > Name output = OrigRed_2
    Convert Green to gray: Yes > Name output = OrigGreen_2
    Convert Blue to gray: Yes > Name output = OrigBlue_2

You would have now all separated channels.

You can also use ImageJ (Fiji) to process it first, as Beth explained here


Here’s an example image - I’ll try to put your suggestions into effect in the meantime.



Apologies - I’m having some trouble uploading it. Here’s a link to the image


Whatever software you used to generate or export this image (your microscope maybe?) exported it as a (3 channel) RGB tif, not as 4 separate channels- you can see this in ImageJ, for example, the type is listed as ‘RGB’. If you want to cleanly separate all 4 channels, you should see if it’s possible to go back to your original source and export it as individual channels, a tif stack, or something equivalent.


Ok, makes sense. I’ll see if I can export the images differently.