Single-molecule techniques probe the spectroscopic properties of biomolecules one molecule a time. These approaches allow detecting and quantifying subpopulations which would be masked in ensemble measurements. Single-molecule FRET (smFRET), which indirectly measures the distance between fluorophores, has been extensively used to characterize the conformation of biomolecules or molecular interactions in complex biomolecular assemblies among many applications. FRET efficiency is typically computed ratiometrically from photon counts detected in two spectral channels.