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The preparation of two water-soluble 'through-bond energy transfer cassettes' (TBET-cassettes) which have good extinction coefficients at the donor part and transfer energy to the acceptor parts with good 'overall quantum yields', is presented. Fluorescence resonance energy transfer from one protein functionalized with an appropriate donor could be applied to excite the TBET-cassettes on different proteins to probe protein-protein interactions under conditions that would not be possible for single-dye acceptor systems.

The application of ratiometric probes that absorb ultraviolet (UV) excitation at a fixed wavelength and emit at two different fluorescence wavelengths in a pH dependent manner is demonstrated. The newly designed energy transfer approach to pH probe facilitate observation of processes within the cell and have significant potential for modifications with other pH sensitive donor to give a spectrum of probes with systematically varied pH response transitions.