The formation of dual-component organic nanoparticles by a modified emulsion-templated freeze-drying approach leads to aqueous nanosuspensions showing fluorescence (Förster) resonance energy transfer (FRET) from within a distribution of single nanoparticles. The combination of both FRET dyes within dual-component nanoparticles (<200 nm) allows the spatial and physical monitoring of the particles, as the FRET signal is lost on dissolution and breakdown of the nanoparticles. The monitoring of accumulation by Caco-2 cells and macrophages shows very limited internalization within the non-phagocytic cells. Conservation of FRET within the macrophages confirms extensive whole-particle internalization. The cellular permeability through Caco-2 monolayers is also assessed and movement of intact dual-component particles is observed, suggesting a mechanism for enhanced pharmacokinetics in vivo.