Re: Problem with channel bleed-through after splitting channels from RGB image

Posted by Joel Sheffield on Nov 04, 2015; 6:26pm
URL: http://imagej.273.s1.nabble.com/Problem-with-channel-bleed-through-after-splitting-channels-from-RGB-image-tp5014842p5014844.html

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
e-mail: [hidden email]
URL:  *http://tinyurl.com/khbouft <http://tinyurl.com/khbouft>*

On Wed, Nov 4, 2015 at 11:06 AM, FrankyG <
[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.jpg
> >
>
> 2) After Channel Split
> a) Blue
>
> <http://imagej.1557.x6.nabble.com/file/n5014842/Blue_Channel_Test_NT-3.png
> >
>
> b) 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.png
> >
>
> These 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.png
> >
>
> b) Green
>
> <
> http://imagej.1557.x6.nabble.com/file/n5014842/Green_Channel_Test_NT-3_Correct.png
> >
>
> Now 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.jpg
> >
>
> d) Blue
>
> <
> http://imagej.1557.x6.nabble.com/file/n5014842/RGB_EdU30min_Channel_Split_Blue.png
> >
>
> e) Green
>
> <
> http://imagej.1557.x6.nabble.com/file/n5014842/RGB_EdU30min_Channel_Split_Green.png
> >
>
> As 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.png
> >
>
> b) Green
>
> <
> http://imagej.1557.x6.nabble.com/file/n5014842/Green_Channel_Test_NT-3_Correct_2.png
> >
>
> I 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!
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/Problem-with-channel-bleed-through-after-splitting-channels-from-RGB-image-tp5014842.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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