The application of new molecular diagnostics to probe cellular process in vivo is leading to a greater understanding of molecular cytology at a sub-nanoscale level and is opening the way to individualized medicines. We review here three distinct fluorescence-based molecular probes, HyBeacons, split-probe exciplexes and GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) systems. Through this, we highlight the insights into the mechanism and design that a combined computational and experimental approach can yield.