We report the observation of single-molecule colocalization and quantitative fluorescence resonant energy transfer by simultaneously imaging the emission and polarization characteristics of two colocalized fluorophores using a simple optical design. The methodology was tested using the ligand-receptor system streptavidin, fluorescence labeled with the dye Cy5, and biotin labeled with tetramethylrhodamine. Discrimination of the two dyes permitted the observation of single-pair fluorescence resonant energy transfer with an efficiency of 89%. The multidimensional character of our fluorescence microscopy combined with the robustness of our design provides a simple method suitable to study biomolecular interactions on the single molecule level.

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