Limitations of fluorescence labeling for quantification

The crucial limitation of fluorescence labeling for quantification purposes is that it is almost impossible to have a 100% efficient process.

If we want to quantify the number of a given antibody on the surface of a membrane, we could attempt to do single-molecule fluorescence detection, and assuming each antibody is uniquely labelled.

This is the challenge which is pervasive in the use of Flow Cytometry to determine loading ratio of nanoparticles (see: 202111301443 determining drug loading and release kinetics in nanoparticles) or to properly identify particles with low numbers of labeling sites.

Another problem is the separating the signal from the background generated by the fluorescence arising from dye aggregates.