When colors contaminate each other
Spectral bleedthrough creates false FRET signal in intensity-based methods:
The problem is worse for fluorophores with significant spectral overlapLoading...—exactly the pairs needed for efficient FRET.
The Problem: Donor light leaking into the acceptor detector looks like FRET signal, but it isn't. This gives wrong answers.
Worse: the same spectral overlap needed for FRET also causes more bleedthrough.
FLIMLoading...-FRET detects energy transfer through donor lifetime shortening:
This fundamental advantage makes FLIM-FRET particularly suited for clinical applications where robustness and reproducibility are essential.
FLIM Solution: FLIM measures how fast the donor fades (lifetime), not how bright the acceptor is. Donor light leaking into the wrong channel doesn't change how fast the donor fades—so bleedthrough doesn't matter.
