Posted by
Stoyan Pavlov on
Oct 16, 2013; 9:15am
URL: http://imagej.273.s1.nabble.com/correction-for-stray-light-on-epifluorescence-images-tp5005196p5005200.html
Hi Karsten,
As I use a small magnification objective so my sources of positive
fluorescence are below or at least at the resolving power of the microscope
(so they might be considered as point sources).I do not think that in this
case Background subtraction can be useful. I actually do not need to remove
the artifacts completely but to reduce their influence on the brightness of
their neighbourhood. Usually when I am counting objects and measuring areas
I use Top-Hat Opening by Reconstruction to deal with artifacts and
irregularities, but i think in this case this would destroy the intensity
information in the images and render the results incomparable.
As to the shading correction: I do not have a background fluorescence. I
perform the shading correction to correct for the irregularities in the
illumination light - although my lamp is perfectly aligned there is still
unequal distribution of the incident exciting light resulting in difference
in the emission as well. To perform the shading correction I acquired a
background image of an uniformly fluorescing preparation (thin film of my
detector - IgG-Cy3 conjugate) and saved it as a flat-field correction
image in the camera software. It works perfectly - all lamp induced
gradients disappear from my images.
Why is the logarithm prior the division necessary and what will it achieve?
Best regards
Stoyan
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