Problem with channel bleed-through after splitting channels from RGB image
Posted by FrankyG on Nov 04, 2015; 4:06pm
URL: http://imagej.273.s1.nabble.com/Problem-with-channel-bleed-through-after-splitting-channels-from-RGB-image-tp5014842.html
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
2) After Channel Split
a) Blue
b) Green (I have no clue what is up with the lines, they aren't there when I save the image).
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
b) Green
Now here is what happens when I take the RGB image of this and try to split the channels
c) Original RGB
d) Blue
e) Green
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
b) Green
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!
URL: http://imagej.273.s1.nabble.com/Problem-with-channel-bleed-through-after-splitting-channels-from-RGB-image-tp5014842.html
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
2) After Channel Split
a) Blue
b) Green (I have no clue what is up with the lines, they aren't there when I save the image).
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
b) Green
Now here is what happens when I take the RGB image of this and try to split the channels
c) Original RGB
d) Blue
e) Green
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
b) Green
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!
| Free forum by Nabble | Disable Popup Ads | Edit this page |