Low-level caspase activity, which would otherwise fall below the threshold of detection in some assays, was measured using a fluorescence resonance energy transfer (FRET)-based probe containing enhanced cyan fluorescence protein (ECFP) as the FRET donor and a variant of enhanced yellow fluorescence protein, Venus, as the FRET acceptor. A caspase cleavage site was designed within the peptide linker that connects ECFP and Venus (Takemoto et al., 2003). In situ confocal imaging of the FRET emission ratio (Venus/ECFP) reveals caspase activity in wing imaginal discs (A), including the specific subset of cells that have the potential to give rise to scutellar sensory organ precursors (B, upper panel). This level of caspase activation is dependent on DmIKKε-activated degradation of DIAP1 and is insufficient to induce apoptosis. Rather, it is required for proper regulation of sensory organ precursor cell number (Kuranaga et al., 2006). Suppression of caspase activation via loss of DmIKKεresults in the eventual formation of an extra sensory bristle, which co-stains with a neuronal marker, anti-senseless (B, lower panels). Images courtesy of Masayuki Miura.