Forster Resonance Energy Transfer-Based Biosensors for Multiparameter Ratiometric Imaging of Ca2+ Dynamics and Caspase-3 Activity in Single Cells

As one of the principal cytoplasmic second messengers, the calcium ion (Ca2+) is central to a variety of intracellular signal transduction pathways. Accordingly, there is a sustained interest in methods for spatially- and temporally resolved imaging of the concentration of Ca2+ in live cells using noninvasive methods such as genetically encoded biosensors based on Forster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca2+ biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca2+ affinity for various applications. We now report the development and systematic optimization of a pair of spectrally distinct FRET-based biosensors that enable the simultaneous imaging of Ca2+ in two compartments of a single cell without substantial spectral crosstalk between emission channels. Furthermore, we demonstrate that these new biosensors can be used in conjunction with previously reported caspase-3 substrates based on the same set of FRET pairs.