Stephen T. Haley, II wrote:

> The trouble arose when I tried to check the
> validity of my measurements with some InSpeck beads from Molecular
> Probes.  The beads are calibrated to several relative intensities. To
> make a long story short I found out that the system is not linear.

Hi Stephen,
        First make sure that your detectors are operating in manual control
mode: you will have all sorts of problems if they are trying to cleverly
adjust ("optimize") your image intensity while you are not looking. A
more likely site of problem is in your setting of the background in your
image. If you take an image where there is no fluorescence (air, water,
whatever), and then make a histogram of the results, all your values in
the image must STILL be above zero. Otherwise when you go to take
fluorescence images, you will not be linear.

In essense I am encouraging you to make sure your background subtraction
is correct prior to quantifying your fluorescence.

Good luck. Chip
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M.H. Montrose, Ph.D.
   Johns Hopkins University             tel: 410-955-9681
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