Fluorescence Excitation and Imaging of Single Molecules Near Coated Surfaces: A Theoretical Study

Abstract

Microscopic fluorescent samples of interest to cell and molecular biology are almost always in aqueous medium near a solid surface. Frequently, that surface is coated with a thin film such as: a lipid monolayer, bilayer, or multilayer; a collagen or agarose layer deposited before cell plating or deposited by the cells themselves; acrylamide gel to immobilize beads or single molecules (such as in commercial preparations for nucleic acid sequencing); or a cell wall interposed between the substrate and cellular organelle. 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 TIR excitation, multiple reflections in the film lead to unique resonance peaks in the evanescent intensity vs. incidence angle curve. For emission, multiple reflections coupled to the fluorophore's near field emission create a distinct intensity pattern in the back focal plane (BFP) of a high aperture objective. In principle, these features should allow retrieval of information about local film thickness and refractive index, and about fluorophore axial position and 3D molecular orientation. This entirely theoretical analysis with computer-generated images explores these possibilities. Supported by NIH grants 2R56NS038129-11 and 1R21NS073686-01 to DA and Ronald V. Holz.