Single-molecule fluorescence resonant energy transfer (FRET) is a widely accepted method for determining the spatial separation between molecules. In combination with pulsed interleaved excitation (PIE), additional information about the stoichiometry of molecular interactions is obtained. PIE-FRET, however, as implemented with standard confocal optics, requires the dilution of the sample to biologically low concentrations. Here, we show that PIE-FRET measurements inside nanometer-sized apertures yield meaningful biochemical data at 1000 x higher concentrations.