Posted by
Daniel James White on
May 30, 2011; 8:17am
URL: http://imagej.273.s1.nabble.com/multi-channel-composite-images-tp3684430p3684431.html
Hi Dave,
On May 28, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:
>
> Date: Fri, 27 May 2011 16:03:46 -0400
> From: David Knecht <
[hidden email]>
> Subject: multi-channel composite images
>
> The sample data has a 5 channel composite image. I am unsure how one creates such an image and how the colors are assigned. If you had 5 grayscale images you wanted to merge to a composite and you want to designate the color of each channel, how would you do that? Thanks- Dave
Are you really sure you want to make a colour merge of 5 channels?
We have only 3 colour receptors , red , green and blue.... the primary colours,
so if you have 4 or 5 or more channels, their colour must consist of a mixture of at least 2 primary colours...
so you must confuse it with either being itself, or 2 of the other channels having signal at that point.
If the structures are very well morphologically/spatially separated, then it might not confuse the brain too much.... but....
in the usual case of diffuse staining in biological samples... its a problem.
To make things worse, a large number of men (approaching 10%) and some women are to some degree colour blind.
That means that 3 colours are already problematic.... So Red / Green is a really bad choice for 2 channel colour merges...
more folks can see Green/Magenta, or red/cyan.... but that still doesnt make it a good idea...
The basic point is that there is usually far too much information in in colour merge images for our brains to get to grips with.
We might get the most obvious features, but even these can be masked by our clever psychovisual system auto adjusting the colour
to get better colour contrast, so the same yellow does not look the same depending on what its next to.
This is super useful for finding fruit in the jungle, but gets in the way of quantitative science.
We miss most of the subtle information that is there and faithfully captured by our digital imaging devices,
but we need to visualise it in a way that our brains can comprehend and not be misled by.
(see the example script spirals in Fiji:
File - open sample - spirals
and
http://www.archimedes-lab.org/color_optical_illusions.html )
If its a question or art: making a pretty cover image, then all bets are off...
To make quantitative assessments... colour merge images could not really be any worse.
ImageJ helps us measure stuff, and so we dont need to be over reliant on our instinctive
tendency to believe what we "see". Much of what we think we see turnes out to be less than true
once you start actually measuring things.... expecially the case in colocalization analysis.
(see
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis )
One might like to ask one's self:
WHY do I want to make a 5 channel colour merge image?
WHAT is it that I am "looking" for to assess?
....just my 2 cents for the benefit of the listers....
(tell me to shut up if I sound like a broken record)
cheers
Dan
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany
+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (Fax MPI-CBG LMF)
http://www.bioimagexd.net BioImageXD
http://pacific.mpi-cbg.de Fiji - is just ImageJ (Batteries Included)
http://www.chalkie.org.uk Dan's Homepages
https://ifn.mpi-cbg.de Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )