Posted by
Joel Sheffield on
URL: http://imagej.273.s1.nabble.com/Problem-with-channel-bleed-through-after-splitting-channels-from-RGB-image-tp5014842p5014859.html
Hi,
It is not a matter of excitation so much as emission, and the way that
images are recorded. Even if you illuminate DAPI with a relatively narrow
exciting wavelength, the emission is over a broad range. Look, for
instance, at this spectrum (not by me), www.botanik1.uni-karlsruhe.de
<
http://r.search.yahoo.com/_ylt=A0LEVinodztWAngA1scnnIlQ;_ylu=X3oDMTEzZjZpNzAxBGNvbG8DYmYxBHBvcwMxBHZ0aWQDRkZVSUMwXzEEc2VjA3Nj/RV=2/RE=1446766696/RO=10/RU=http%3a%2f%2fwww.botanik1.uni-karlsruhe.de%2fNick-Lab%2f377.php/RK=0/RS=LN2eASsJgEHyENeKaG9oKRpfZ0w->
. You will see significant emission in the range from 500 almost to 600
nm. This is in the green end of the spectrum, and if the filter cube that
you use allows these wavelengths to be transmitted, they will be detected
by the green channel of your camera. I am assuming that you are using some
form of rgb camera, which separates the light into three channels by using
an internal filter mask.
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 Thu, Nov 5, 2015 at 10:18 AM, FrankyG <
[hidden email]> wrote:
> Dr. Sheffield,
>
> Our scope is a bit different in that for each channel a different light
> source is used. It still filters the light a bit but the DAPI light source
> is different than the GFP light source. Unless I understood you wrong and
> you were not saying that the cubes all filter from one main light source.
>
> I will try your method of saving and then merging the files again. Though
> it
> requires more macros I'm all for efficient workflow so it will get done
> somehow.
>
> Dr. Swayne,
>
> I will check with the EVOS people if it is possible to change the LUT used.
> If it is possible and that solves the problem than you have many thanks
> from
> me for saving me from a lot of extra programming.
>
>
> Thanks for the answers, it's time to get back to work! I will post if I
> have
> further issues.
>
>
>
> --
> View this message in context:
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