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

Cognet, Laurent; Harms, Gregory S.; Blab, Gerhard A.; Lommerse, Piet H. M.; Lommerse, Piet H.M.; Schmidt, Thomas
December 2000
Applied Physics Letters;12/11/2000, Vol. 77 Issue 24
Academic Journal
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. © 2000 American Institute of Physics.


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