Cyan colour instead of Blue - how do I convert it into blue??

[apo_99]

Dearest members,

I take pictures using a Leica Confocal, staining my samples with blue (DAPI) and green (Bodipy) fluorochromes. After applying the different excitation and emission channels for the two fluorochromes (in sequential scan mode), I obtain the pictures. However this time, instead of using "Blue" colour to depict my blue-emission channel, I chose Cyan colour (thus the nucleus becomes better visible to the naked eye) for the same (blue) emission range.

Now, normally in the imageJ I split the 3 colour channels of my obtained pictures and then I count particles. In this way, I can measure the size of the nucleus (blue fluorochrome, blue channel) and my lipids (green fluorochrome, green channel).

BUT in the pictures I obtained choosing the cyan colour for the blue fluorochrome, the ImageJ splits my channels in the blue one (showing only the DAPI-blue nucleus) and in the green channel which shows BOTH the green stained lipids AND the blue stained nucleus!

Is there any way to get rid of the blue stained nucleus in my green channel pictures? Any answer would be highly appreciated... Thanks!!!

/Apo_99
Karolinska Institutet,
Stockholm Sweden

[Glen MacDonald-2]

Dear Apo_99,
It sounds as though you converted your confocal images to RGB.  The RGB color scheme is just that: red, green and blue
Cyan is the result of adding blue and green.  Overlapping pixels in the blue channel were assigned a similar intensity in the green channel.  You might be able to accomplish removal by channel subtraction but the result may be artifactual.  The best option is to start over and set the source image to blue and green.  If you want separate channels for analysis, it is usually best to remain in the higher bit-depth of the original images rather than immediately converting to RGB.  Visualize the original files in ImageJ as ‘Composite’ images, where the Channels Tools will allow viewing in any color combination you wish without losing bit depth or mixing channels.  Then you can convert to a desired RGB combination.  BTW, cyan is much easier to visualize by the human eye and is often used in publications.  

Regards,
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Oct 21, 2015, at 8:07 AM, apo_99 <[hidden email]> wrote:

> Dearest members,
>
> I take pictures using a Leica Confocal, staining my samples with blue (DAPI)
> and green (Bodipy) fluorochromes. After applying the different excitation
> and emission channels for the two fluorochromes (in sequential scan mode), I
> obtain the pictures. However this time, instead of using "Blue" colour to
> depict my blue-emission channel, I chose Cyan colour (thus the nucleus
> becomes better visible to the naked eye) for the same (blue) emission range.
>
> Now, normally in the imageJ I split the 3 colour channels of my obtained
> pictures and then I count particles. In this way, I can measure the size of
> the nucleus (blue fluorochrome, blue channel) and my lipids (green
> fluorochrome, green channel).
>
> BUT in the pictures I obtained choosing the cyan colour for the blue
> fluorochrome, the ImageJ splits my channels in the blue one (showing only
> the DAPI-blue nucleus) and in the green channel which shows BOTH the green
> stained lipids AND the blue stained nucleus!
>
> Is there any way to get rid of the blue stained nucleus in my green channel
> pictures? Any answer would be highly appreciated... Thanks!!!
>
> /Apo_99
> Karolinska Institutet,
> Stockholm Sweden
>
>
>
> --
> View this message in context: http://imagej.1557.x6.nabble.com/Cyan-colour-instead-of-Blue-how-do-I-convert-it-into-blue-tp5014698.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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ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[gankaku]

Hi Apo_99

Not knowing the microscopic software you are using, I assume you saved the
image by exporting it in a specific format like tiff. Problem with saving
RGB images a single layr images is that splitting the channels only allows
you to retrieve red, green and blue. All oter colors are mixed using those
three. Therefore splitting the image results in the phenomenon you see.
Knowing Leica I assume you can also save the file as .lif or .lsm or any
similar original format od Leica. This actually should solve the problem
because theae formats normally saves the image a composites giving you
individual access to insividual channels. Anorher option (but not as
convenient) would stull be to export each channel as a separate image. The
original file format also allows you to save higher bit depths like 12, 14,
or 16 bit. Retrieving a channel from an RGB image alows you only having the
three channels as 8-bit images which imposes a limitation as soon as you
adyitionally want to measure intensities.

Regards,
Jan
Am 21.10.2015 17:26 schrieb "apo_99" <
[hidden email]>:

> Dearest members,
>
> I take pictures using a Leica Confocal, staining my samples with blue
> (DAPI)
> and green (Bodipy) fluorochromes. After applying the different excitation
> and emission channels for the two fluorochromes (in sequential scan mode),
> I
> obtain the pictures. However this time, instead of using "Blue" colour to
> depict my blue-emission channel, I chose Cyan colour (thus the nucleus
> becomes better visible to the naked eye) for the same (blue) emission
> range.
>
> Now, normally in the imageJ I split the 3 colour channels of my obtained
> pictures and then I count particles. In this way, I can measure the size of
> the nucleus (blue fluorochrome, blue channel) and my lipids (green
> fluorochrome, green channel).
>
> BUT in the pictures I obtained choosing the cyan colour for the blue
> fluorochrome, the ImageJ splits my channels in the blue one (showing only
> the DAPI-blue nucleus) and in the green channel which shows BOTH the green
> stained lipids AND the blue stained nucleus!
>
> Is there any way to get rid of the blue stained nucleus in my green channel
> pictures? Any answer would be highly appreciated... Thanks!!!
>
> /Apo_99
> Karolinska Institutet,
> Stockholm Sweden
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/Cyan-colour-instead-of-Blue-how-do-I-convert-it-into-blue-tp5014698.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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