Determination of surface-bound-fluorophore orientation by goniometric fluorescence polarization: application to quantification of DNA-chip readouts.

We present a polarized goniofluorimeter designed to measure the observation-angle and polarization-dependent intensity emitted by a group of surface-bound fluorescent molecules. We studied two types of surface bonding: In one case, dyes were adsorbed into the surface by spin coating, and in the other, dyes were covalently immobilized to DNA strands. Fluorescent dyes consisted of Cy3 and Alexa546. The substrate was a silicon wafer bearing a silicon dioxide layer. The different samples presented a wide panel of reproducible experimental behavior. By confronting experimental behavior with theory and simulation, we can explain these differences as directly linked to the mean orientation of fluorophores with respect to the surface.