Posted by
Swayne, Theresa C. on
Nov 04, 2015; 7:08pm
URL: http://imagej.273.s1.nabble.com/Problem-with-channel-bleed-through-after-splitting-channels-from-RGB-image-tp5014842p5014846.html
I agree with Joel that DAPI bleeds through like crazy, but I don’t think it’s the problem in this case. A filter issue wouldn't explain why the results are different when the images are saved separately (assuming they were all captured in the same way through the same filters).
Instead, I think the problem comes from the default lookup table (LUT) the camera software uses for DAPI. It is not pure blue, but a mix of blue with a little bit of green. I have several software packages that default to some intermediate color like this.
Software vendors do this because it makes your “blue” channel easier to see on screen — our eyes are not very sensitive to pure blue. But it causes a problem like you observed when colors are merged in the RGB format. When you split the channels, the “Green” channel now contains your actual green acquisition, plus some of the DAPI signal.
For this reason I always recommend to my users to use the Composite format, especially because more of us are using 4+ colors.
However, I sympathize with the desire to stick with a successful workflow!
So I think you could make it work if you could change the default colorization of the DAPI channel to a pure blue.
Hope this helps,
Theresa
On Nov 4, 2015, at 1:33 PM, JOEL B. SHEFFIELD <
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We have also seen this, and it certainly presents a problem. The DAPI
filter cubes often have a very wide bandpass on the emission side, and so
some longer wavelength DAPI signal gets through and is picked up by the
camera. One solution is to take images of the three channels separately,
separate each of the three images into RGB components, and then reassemble
them using the color>merge option. Alternatively, you could look into a
narrow bandpass DAPI cube.
Joel
Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
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On Wed, Nov 4, 2015 at 11:06 AM, FrankyG <
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[hidden email]>> wrote:
I have been trying to conduct IF analysis with ImageJ and it seems to be
giving me a lot of trouble. I always save my images as .tif and they each
have a GFP and DAPI channel to them.
When I split them there is no red channel, as expected, but something funky
goes on with the GFP channel. It's almost as if ImageJ bleeds over a lot of
the DAPI(blue) channel onto the GFP(green) channel after splitting.
Here are some images to showcase what I am talking about:
1) Original RGB
<
http://imagej.1557.x6.nabble.com/file/n5014842/HUminus_EDUminus_NT3_RGB_0001.jpg2) After Channel Split
a) Blue
<
http://imagej.1557.x6.nabble.com/file/n5014842/Blue_Channel_Test_NT-3.pngb) Green (I have no clue what is up with the lines, they aren't there when
I
save the image).
<
http://imagej.1557.x6.nabble.com/file/n5014842/Green_Channel_Test_NT-3.pngThese cells are a negative control and were not treated with EdU. When
treated with AlexaFlour (488nm) the EdU present will show up in the GFP
channel on the microscope. When imaging these cells there is clearly no
signal in the GFP channel but when splitting the channels in ImageJ it
creates signal.
The only way I have gotten around this is by saving the channels separately
but for my negative control it runs into another issue.
First, for reference, here are images of non-target cells that have had EdU
treatment. As you can imagine there is signal in the GFP channel.
3) Channels saved separately, NOT split through ImageJ
a) Blue
<
http://imagej.1557.x6.nabble.com/file/n5014842/Blue_Channel_Test_NT-3_Correct.pngb) Green
<
http://imagej.1557.x6.nabble.com/file/n5014842/Green_Channel_Test_NT-3_Correct.pngNow here is what happens when I take the RGB image of this and try to split
the channels
c) Original RGB
<
http://imagej.1557.x6.nabble.com/file/n5014842/HUminus_EDU30min_NT3_RGB_0001_2.jpgd) Blue
<
http://imagej.1557.x6.nabble.com/file/n5014842/RGB_EdU30min_Channel_Split_Blue.pnge) Green
<
http://imagej.1557.x6.nabble.com/file/n5014842/RGB_EdU30min_Channel_Split_Green.pngAs you can see the green image is simply lower in intensity but ImageJ
seems
to be unable to separate the DAPI from the image and it completely masks
the
intensity from the GFP channel.
Now here is what happens when I save the cells that have no EdU treatment.
4) Channels saved separately, not split through ImageJ, No EdU
a) Blue
<
http://imagej.1557.x6.nabble.com/file/n5014842/Blue_Channel_Test_NT-3_Correct_2.pngb) Green
<
http://imagej.1557.x6.nabble.com/file/n5014842/Green_Channel_Test_NT-3_Correct_2.pngI would much rather be able to save all images as just RGB because my macro
works perfectly and allows my workflow to be efficient.
If I have to switch to the separate channel method I have to re-write the
macro and find some way to make it open the GFP image that corresponds to
the DAPI one that I choose. I would also need to solve the background issue
seen in the negative control (#4). I am willing to learn how to do that but
I'd rather find a way that allows me to use what I have now.
If anyone has any idea why splitting channels in ImageJ produces
bleed-through from other channels that would be great!
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Theresa Swayne, Ph.D.
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Herbert Irving Comprehensive Cancer Center
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