What are the factors that affect FRET measurements?

Factors that affect FRET measurements include: 

• The brightness of fluorophores: The brightness of fluorophores used in FRET experiments should be comparable to avoid one channel being saturated while the other is overwhelmed by noise. 
• Spectral-cross talk: Spectral cross-talk (caused by overlapping excitation or emission spectra between donor and acceptor fluorophores) can affect the accuracy of FRET measurements. Since some level of spectral overlap between donor and acceptor is needed for FRET, cross-talk is unavoidable and needs to be managed during FRET measurements. 
• Fluorophore alignment: Proper alignment of fluorophores is essential to prevent false-negative FRET signals, ensuring that interacting proteins are labeled in a way that allows the fluorophores to be within 10 nm of each other. 
• Donor/acceptor stoichiometry: Maintaining a donor-to-acceptor stoichiometry within the range of 1:10 is crucial for ensuring that the FRET signal is not masked by background fluorescence. Additionally, a long RO can result in a high FRET efficiency.