PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities.

Genetically encoded calcium (Ca ) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches.

A chemigenetic indicator based on a synthetic chelator and a green fluorescent protein for imaging of intracellular sodium ions.

A chemigenetic indicator with an affinity suitable for imaging of intracellular sodium ions (Na) in mammalian cells was developed. The indicator, based on a chimera of green fluorescent protein (GFP) and HaloTag labeled with a synthetic crown ether chelator, was produced by a combination of rational design and directed evolution. In mammalian cells the indicator exhibited an approximately 100% increase in excitation ratio when the cells were treated with 20 mM Na and an ionophore.

The best of both worlds: Chemigenetic fluorescent sensors for biological imaging.

Synthetic-based fluorescent chemosensors and protein-based fluorescent biosensors are two well-established classes of tools for visualizing and monitoring biological processes in living tissues. Chemigenetic sensors, created using a combination of both synthetic parts and protein parts, are an emerging class of tools that aims to combine the strengths, and overcome the drawbacks, of traditional chemosensors and biosensors. This review will survey the landscape of strategies used for fluorescent chemigenetic sensor design.

Chemigenetic indicators based on synthetic chelators and green fluorescent protein.

Molecular fluorescent indicators are versatile tools for dynamic imaging of biological systems. We now report a class of indicators that are based on the chemigenetic combination of a synthetic ion-recognition motif and a protein-based fluorophore. Specifically, we have developed a calcium ion (Ca) indicator that is based on genetic insertion of circularly permuted green fluorescent protein into HaloTag protein self-labeled with a ligand containing the Ca chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.