Contrast Mechanisms of Solution-Dispersed Graphene Compounds in Twilight Fluorescence Microscopy

Twilight fluorescence microscopy is a newly developed technique that is capable of imaging a single-layer graphene compound dispersed in a liquid. A graphene solution mixed with a highly concentrated dye is placed on a glass plate and is irradiated by the excitation beam with an incident angle that has a finite width around the total internal reflection angle. Both the evanescence field and the faint refracted beam decay exponentially as they travel from the glass surface. The dye fluorescence excited by both beams is used as illumination. A simplified theory for dark contrast of graphene compounds is developed based on absorption and Förster resonance energy transfer (FRET), assuming that (1) FRET has a sharp cutoff distance, (2) FRET is independent of the number of layers, and (3) Dexter electron transfer is negligible. The contrast of a reduced graphene oxide multilayer, whose layer heights have been determined by atomic force microscopy, shows good agreement with the simplified theory under various dye concentrations. The FRET cutoff distance is found to be much shorter than one expected for graphene and similar to the distance between two small molecules. This short cutoff distance is the main reason for the assumption to be valid.