Re: background removal in fluorescence

Posted by Bevic, Jordan on Sep 20, 2005; 7:33pm
URL: http://imagej.273.s1.nabble.com/background-removal-in-fluorescence-tp3704817p3704820.html

Have you tried running the Background Subtractor on each separate image
before making the montage?  Although I think there are some minor bugs
in it (the bottom few rows show artifacts on contrast stretching) it
works quite well at flattening out illumination differences.

Jordan Bevic
QuadTech Advanced Vision Systems

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Martin Wessendorf
Sent: Tuesday, September 20, 2005 1:12 PM
To: [hidden email]
Subject: Re: background removal in fluorescence

Dear ImageJ-ers--

I realize that I didn't articulate the problem particularly well.  --We
want to make some montages of images.  Our illumination is not perfectly

even and so parts of the field are illuminated more brightly than
others.  Thus when we try to make the montage, there are abrupt
inconsistencies in the lighting across it.

I had thought that the following *should* work:

1) Take an image of a uniform fluorescent field (image A)

2) Take an image of the specimen (image B)

3) Divide B by A; multiply by the mean intensity of A.

(--I think that this is what Wayne's plugin does.)

However, when we use that method, the inconsistencies of the
illumination are greatly accentuated rather than being reduced.  My
guess is that this is due to non-linearity of response in our CCD
camera.

I'd be delighted if anyone has a work-around for this problem!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                E-mail: [hidden email]