What are the limitations of FRET?

One limitation is that FRET efficiency is highly dependent on the relative orientation of the donor and acceptor molecules. FRET also exhibits a low signal-to-noise ratio. Additionally, the fluorescence properties of some fluorophores used in FRET experiments can be sensitive to changes in pH. Another con is that free fluorophores in the sample can compete with the labeled molecules for excitation energy, potentially masking the FRET signal. FRET also only provides information about the proximity and interaction between the labeled molecules. Additionally, the size of the probes used in FRET experiments can introduce steric hindrance.