Spectral fluorescence measurements on reflecting surfaces shedding light onto conformation and orientation of macromolecules

The functionality of macromolecules depends strongly on their structural arrangements that form the functional sites and on the accessibility of these sites. These molecules are often immobilized on surfaces to study their interactions with other molecules, in biological and medical sciences. When macromolecules are in contact with a surface, a new structural arrangement, or conformation, may be adopted due to free energy minimization mechanisms, and the functional sites may be rendered inactive. Additionally, the molecular orientation acquired for surface attachment may hinder the accessibility of the functional sites. Therefore, conformation and orientation of macromolecules are of utmost importance to sustain their functional behavior on a surface and require systematic studies. However, the length scale necessary to directly measure macromolecular conformation is far beyond the capability of conventional optical microscopy.;We have developed a spectroscopy technique to study the surface conformation and orientation of fluorescently tagged macromolecules. The technique, spectral self-interference fluorescence microscopy (SSFM), utilizes the spectral oscillations in the radiated intensity due to the interference of direct and reflected fluorescence from emitters near a dielectric surface. Using the sub-nanometer emitter axial localization capability of this technique, we have determined the conformational change of surface adsorbed polymer chains upon hydration. The results revealed the importance of this conformational change in the efficiency of the polymeric structure for surface binding of probes in microarray applications to study ligand-analyte interactions.;SSFM in the traditional configuration lacked high lateral resolution due to the low numerical aperture (NA) requirement to achieve sub-nanometer axial localization. However, for applications that involve resolving finer structures such as topographical mapping of cell surfaces or study of sub-cellular components, high lateral resolution is required. Using high-NA objectives results in a diminished fringe contrast of spectral oscillations, and thus the localization capability is reduced. Through angular tailoring of the radiated intensity, we have achieved sub-micron lateral resolution while maintaining sub-nanometer axial localization capability. The elimination of the low-NA requirement with this angular tailoring approach also opened up the possibility of determining the dipole orientation information from the spectral signature in addition to the axial position information. Simulations demonstrate that the dipole orientation of the emitters can be detected with a high precision.