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Hello Susan,
I'm not sure what software you are using to interface with the CoolSnap
camera controls but it is highly likely you are correct in your
assesment that autogain is interfering with your intensity comparisons.
    We use  MCID in conjunction with a CoolSnapfx camera on one of our
microscopes--the MCID software has a camera set up dialogue where one
can adjust the exposure time. On this dialogue window is a button
entitled "Camera Settings".  When this button is pressed, another window
pops up which has a check box for "AutoGain".  Unchecking this box will
prevent the image intensity from artificially scaling due to low signal.
 I would assume that the software you are using has a similar checkbox
somewhere in the camera setup options.
Regards,
Karl

Puhalla, Susan wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>We have been trying to acquire images from a negative control using IP Lab
>and Photometrics CoolSnap camera. We should have a fairly dark image for the
>negative controls with little to no fluorescence. Instead, we get a rather
>noisy image that appears to be either auto-scaled or contrast stretched. It
>almost looks as if the gain has automatically been increaased for this image
>in order to "force" an image to appear. When we take an image of our
>positive control, it's a crisp and clear image with no noise. It almost
>seems as if the camera is doing something to the image before it arrives on
>the monitor in order to force something to appear even if there is nothing
>there. We are using the same exposure times for both, obviously. Is there a
>way around this? How do we shut this feature off (if it's even possible)?
>This problem makes it impossible to compare our negative controls to our
>experimental and show side by side images.
>
>Thanks!
>
>Susan Puhalla
>Microscopy Research Assistant
>MRC Clinical Sciences Centre
>Room 5003
>Hammersmith Hospital
>DuCane Road
>London W12 ONN
>UK
>Telephone: 020 8383 8528
>Email: [log in to unmask]
>Website: http://www.csc.mrc.ac.uk/Microscopy/home.html
>
>

--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu