Fluorochrome (fluorescein) emission decay when linked to macromolecule I measure the intensity and polarization of light emitted from a blood sample that contains excited fluorescein (experiment known as fluorescence polarization assay). The excitation is done, as usual, with vertically polarized light.
In case the sample has certain macromolecules of interest (an antibody, in this case), the fluorescein links to the much-larger macromolecules, yielding a higher polarization factor, giving a positive- or &quot;sick&quot;- result. Alternatively, if there is no antibody, the excited fluorescein randomly rotates and then emits light, and the polarization factor is smaller giving a negative-or &quot;healthy&quot;- result.
The question:
I find that the light intensity (in both vertical and perpendicular polarizations) decreases for the positive case. Why is that so? Could it be that a non-radiative process like FRET acts stronger for fluorescein linked to macromolecules than for free fluorescein?
EDIT 1: I have used two different intensity detection systems, PMTs and SiPMs, and got similar behaviours.
There should be no FRET for the free fluorescein unless you have specifically added an acceptor and in any case this would reduce the fluorescein emission.  When bound to the protein fluorphores are in a different environment (e.g. pH  change, hydrophobic pocket, tight binding, polar/non-polar nearby residues etc.)  and this can affect their fluorescence yield and lifetime up or down, ANS (anilino naphthalene sulphonate) is a good example.  The same type of thing is probably happening in your case, you know the motion is restricted as the polarisation is greater so there has to be some interaction. Fluorescein emits at a relatively long wavelength so aromatic protein residues cannot act via FRET as they absorb in the UV. You would need a chromophore (such as heam/chlorin/porphyrin) that absorbs in the green to red region to be a FRET acceptor from the fluorescein and so reduce its intensity.