A newly developed fluorescent calcium sensor delivers brighter, more stable brain imaging while eliminating misleading blue-light signals, expanding possibilities for neuroscience research and diagnostics.
Researchers have developed PinkyCaMP, a new genetically encoded fluorescent calcium sensor, described in a study published in Nature Methods, that improves the accuracy of brain imaging by eliminating false signals caused by blue-light-induced photoswitching. Built using the bright red fluorescent protein mScarlet, the sensor offers enhanced brightness, photostability and compatibility with advanced neuroscience techniques.
Calcium imaging is widely used to monitor neuronal activity because calcium levels rise when neurons fire. While green fluorescent sensors remain the standard, red sensors offer advantages such as deeper tissue penetration and reduced phototoxicity. However, existing red sensors often suffer from dim fluorescence, lower stability and photoswitching, which can produce misleading signals unrelated to actual neural activity.
To overcome these limitations, the researchers engineered PinkyCaMP by modifying mScarlet and combining it with calcium-binding components that alter fluorescence in response to calcium. Thousands of variants were screened before selecting the final design, which was subsequently evaluated in cultured cells, neurons and live mice.
Testing showed PinkyCaMP was substantially brighter and more photostable than three established red calcium sensors. It produced no detectable false fluorescence during repeated blue-light exposure, while comparison sensors generated misleading signal increases. The sensor also reliably tracked neuronal calcium activity during electrical stimulation and enabled simultaneous optical stimulation and recording.
Experiments in freely moving mice demonstrated that PinkyCaMP accurately monitored neuronal activity across multiple imaging techniques, including fibre photometry, two-photon microscopy and miniature head-mounted microscopes. Researchers also showed it could be paired with other fluorescent sensors to monitor multiple biological signals simultaneously with minimal interference.
Although PinkyCaMP responded more slowly than some leading green calcium sensors, researchers believe its improved brightness, stability and resistance to photoswitching make it a valuable tool for neuroscience. They say the sensor could expand studies of brain circuits, behaviour and neurological disease while supporting more reliable long-term imaging experiments.
