Fluorescence arising from dye aggregates
Many quantification approaches (such as the use of RiboGreen to quantify DNA/RNA) rely on the the idea that the dye is fluorescent only when attached to the target molecule. This is also the premise of the Acoerela lipid dyes, and many others.
However, dye aggregates always show some residual fluorescence, which will become by all practical means indistinguishable from fluorescence arising from the target molecule.
This background signal can be quantified (running a blank sample, where no targets are present) but it can't be removed from purely spectroscopic properties.
It is one of the Limitations of Fluorescence Labeling for Quantification.
One can argue that the numbers may be low, but that is only relative to the analytes that are being studied. For example, rare-event detection will be swamped by background signals.
Moving forward to co-localization suffers the same problem. It may lower the chances of getting two different fluorescent molecules at the same spot, but whether it is negligible or not will depend on the context.
Orthogonal methods may shine some light. For example, in super resolution microscopy, one gets not only the spectral signature, but also the geometric distribution of the emission. In TRPS coupled with fluorescence, you get size and label.
This means that there are potential ways of separating dye aggregates from true signal if one introduces a complementary way of measuring. When fluorescence is the source of truth, as in the case of the NanoQNT (and in flow cytometer) properly characterizing the labels and doing proper handling of the data is crucial.
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