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As one of the principal cytoplasmic second messengers, the calcium ion (Ca(2+)) 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 Ca(2+) in live cells using noninvasive methods such as genetically encoded biosensors based on Förster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca(2+) biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca(2+) 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 Ca(2+) 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.

analytical chemistry

Förster Resonance Energy Transfer-Based Biosensors for Multiparameter Ratiometric Imaging of Ca<sup>2+</sup> Dynamics and Caspase-3 Activity in Single Cells

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

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