Focal Molography: Coherent Microscopic Detection of Biomolecular Interaction

We introduce and theoretically investigate here a novel analytical method that we have called focal molography, in which molecular interactions are made visible through scattering of coherent light by a coherent pattern of molecules. The scattered light quantifies the presence of molecules at molecular interaction sites. It is separated from noncoherent background scatter by a combination of local dark-field illumination, interference enhancement, and spatial filtering. The latter is achieved by holographic focusing of the wave field generated by the coherently assembled molecules onto an Airy disk and by subtraction of the noncoherent irradiance in the focal plane outside the disk from the irradiance in the disk. This new microscopic method allows distinct detection of low-refractive-index contrast in the nanoenvironment of biomolecules from which information on the interaction of the coherently assembled molecules with molecules in a liquid or gaseous sample may be deduced. The noncoherent surroundings of the coherently assembled molecules consist of freely diffusing solvent and solute molecules. The surroundings, as well as changes in temperature, do not contribute to the coherent signal in the diffraction focus. Interference lithography or high-resolution-imaging lithography can be used to synthesize the coherent pattern of molecules on a monolithic substrate. The coherent pattern of molecules constitutes a synthetic phase hologram that creates a diffraction-limited light wave. We suggest the term “mologram” for the coherent assembly of functional nanostructures and the term “focal molography” for label-free or labeled analysis of molecular interactions through the measurement of the properties of light in the focus of the mologram. We derive analytical formulas that express the detection signal and the sensitivity of focal molography on the surface of a high-refractive-index thin-film optical waveguide in terms of known parameters. We discuss the implementation of a readout system for molograms on a thin-film optical waveguide by adapting a confocal laser-scanning microscope to a bifocal laser-scanning microscope.