Posted by
Mario Faretta on
Mar 24, 2010; 5:34am
URL: http://imagej.273.s1.nabble.com/Normalizing-background-and-fluorescence-intensity-tp3688820p3688821.html
Dear Dawn,
there are a lot of factors to be considered in your problem. Variations in
intensity can be related to the way you acquire images (depending on the
setting of your microscope, filters, lamp alignment...) or to the preparation
of the samples (efficiency of the staining, batch of reagents,...).
It could be a tricky stuff to compare "quantitatively" different coverslips
and preparations.
To correct the first factors you can try to apply a "flatfield" correction to
make illumination homogeneous (in the plugin section on the ImageJ website you
can find some solutions with good explanations and you can also search archive
of the list for the topic). You can use empty fields of view (no cells) to
acquire images for background subtraction using the image calculator or to
apply one of the background functions.
For the second story, i.e. normalize signals for comparative measurements, the
best thing is probably to have an internal "biological" standard to refer as
unit of measure of your experiments...
Hope it helps
Mario
Dawn Koh (
[hidden email]) wrote:
>
> Hi all,
>
> Im currently working on labeling DNA with Alexa Fluor Dye 488. In
my images
> I can see nuclei
> with maximum fluorescence at S-phase and slight fluorescence when
undergoing
> DNA repair.
> This is actually another method to assay for unscheduled DNA
synthesis (UDS)
> instead of using radioisotopes.
>
> My problem begins when I try to compare images from different
views or cover
> slips.
> As I try to compare nuclei at S phase from different cover slips,
> I notice that the fluorescence intensity slightly stronger for one
cover
> slip and weaker for the other.
> I need both cover slips to have the same intensity in order to
compare the
> nuclei undergoing DNA repair.
> Does anyone know how I can normalize the images on Image J so that
they can
> be compared fairly?
>
> Thanks!
>
> Best Regards
> Dawn
>
--
Mario Faretta
Department of Experimental Oncology
European Institute of Oncology
c/o IFOM-IEO Campus for Oncogenomics
via Adamello 16
20139 Milan
Italy
Phone: ++39-02574303054
email:
[hidden email]
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