Dual-polarization imaging of a dual-fluorophore ion sensor: a single-molecule study

The fluorescence emission of the dual-fluorophore Ca(2+) ion sensor molecule, calcium-green 2 (CG-2), has been characterized using dual-polarization imaging at the single-molecule level. By comparing the fluorescence intensity of individual CG-2 molecules in two mutually orthogonal polarization image channels, information about the relative orientation of the two constituent fluorophores in the molecule is obtained. Experimental results from polarization measurements are compared with those predicted from a geometric model based on coupled-fluorophores that are randomly distributed in space. The results confirm previous optical spectroscopy-based predictions of the orientation of CG-2's fluorophores, and the general applications of this dual-polarization imaging approach for characterizing the optical properties of molecules containing multiple fluorophores is discussed.