What is the major limitation of tandem dyes?

The most notable limitation of tandem dyes is that they are highly sensitive and degrade easily. This can result in reduced emission from the acceptor and higher emission by the donor, skewing the results. This is especially problematic when using fluorophores that are excited by the same laser.

Exposure to light, fixatives, permeabilization agents and extreme variations in temperature can accelerate tandem dye decoupling and degradation. Minimizing exposure to these elements is important in order to preserve the stability of tandem dyes.