http://imagej.273.s1.nabble.com/correction-for-stray-light-on-epifluorescence-images-tp5005196p5005201.html
Interesting your shading correction. Perfectly aligned light is promising, did you however tea to Köhler the excitation light? That should be easily possible with your reference specimen! How is your shading correction done? by subtraction or by division? Your next step would mean a second correction which might make things somewhat complicated in understanding the complete system!
If you have such small objects I still think that top hat with or without reconstruction is best choice, of course without theresholding. The resulting peaks reflect IMHO good intensity signals for measuring. However don't forget that differing excitations are not corrected. The fluorescence signal is by no means linearly related to the excitation signal strength!
> 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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