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Date: | Thu, 22 Oct 2009 07:47:38 +1300 |
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Hi John
Obviously, subtracting a single number is not correct. If you know what
the background is due to you can subtract a model for its behavior (e.g.
if it is reflection it should follow the z-response of the microscope) .
If you have no knowledge of the backgound behavior you can directly
measure the background in a region where you _know_ there is no signal
-this could be another z-stack in a region without stained cells. If you
have a source of "non specific" signal in the specimen (e.g. cell
autofluorescence), you have a bigger problem as that can't be simply
subtracted -it's actually data. This will increase the number of
measurements needed to detect a difference between control and treated
cells for example.
If it's a reflection problem can't you fix it (e.g better filter
combinations and/or a polarizer)?
My 2c
Cheers Mark Cannell
John Oreopoulos wrote:
> I have some confocal z-stacks of cells expressing a fluorescent
> protein and I've encountered something I haven't had to deal with
> before. I'm wondering what is the best way to subtract the background
> signal from a z-stack like this. My problem is that the background is
> different for each z-step, peaking a the z-height corresponding to the
> bottom of the cells that are in contact with the substrate -
> supposedly since this is the height where you have the most reflection
> of the excitation light from the substrate itself backwards to the
> detector.
>
> Which is better? Should I subtract the maximum background level of the
> substrate slice from all slices or subtract the background of each
> individual slice from themselves only? Or I could subtract the average
> background over all slices from each slice instead of the maximum
> value at the substrate slice. Alternatively, should I create the
> z-projection and subtract the background of the z-projection image?
>
> Thanks for anyone who can clear this up for me.
>
>
> John Oreopoulos, BSc,
>
> PhD Candidate
>
> University of Toronto
>
> Institute For Biomaterials and Biomedical Engineering
>
> Centre For Studies in Molecular Imaging
>
>
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