FRET YFP-CFP recordings start with the donor fluorophore excitation with a 420-445 nm wavelength band. The excited donor fluorophores lose part of that energy to fluorescence in the 460-500 nm band. The rest goes to acceptor fluorophores in a distance-dependent, radiationless manner. The acceptor then emits the fluorescence in the 528-556 nm window.

E is for excitation at the cube's schematic, F1 and F2 are for two spectrally different fluorescences, and S is for the fixed sample port. The numbers in the brackets are for the corresponding wavelength bands.

Compared to Fluorescence Mini Cubes with only FC connectors on all ports, the ones with integrated photodetectors provide a 100% better signal-to-noise ratio (30% signal increase and a 40% noise reduction). Moreover, the versions where LEDs are integrated as well reduce the number of optical connections and the system’s overall footprint.