Fluorescence excitation and imaging of single molecules near dielectric-coated and bare surfaces: a theoretical study

Microscopic fluorescent samples of interest to cell and molecular biology are commonly embedded in an aqueous medium near a solid surface that is coated with a thin film such as a lipid multilayer, collagen, acrylamide, or a cell wall. Both excitation and emission of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index and the fluorophore's orientation. For total internal reflection excitation, multiple reflections in the film can lead to resonance peaks in the evanescent intensity versus incidence angle curve. For emission, multiple reflections arising from the fluorophore's near field emission can create a distinct intensity pattern in both the back focal plane and the image plane of a high aperture objective. This theoretical analysis discusses how these features can be used to report film thickness and refractive index, and fluorophore axial position and orientation.