Simultaneous dual-color and dual-polarization imaging of single molecules

We report the observation of single-molecule colocalization and quantitativ e fluorescence resonant energy transfer by simultaneously imaging the emiss ion and polarization characteristics of two colocalized fluorophores using a simple optical design. The methodology was tested using the ligand-recept or system streptavidin, fluorescence labeled with the dye Cy5, and biotin l abeled with tetramethylrhodamine. Discrimination of the two dyes permitted the observation of single-pair fluorescence resonant energy transfer with a n efficiency of 89%. The multidimensional character of our fluorescence mic roscopy combined with the robustness of our design provides a simple method suitable to study biomolecular interactions on the single molecule level. (C) 2000 American Institute of Physics. [S0003-6951(00)01451-0].