Calcium sensors
Calcium imaging is one of the primary methods neuroscientists use to monitor brain activity in real time, since calcium levels inside neurons typically rise when they fire.
Thus far, green fluorescent sensors have been the standard tools for calcium imaging because of their sensitivity and speed. However, red fluorescent sensors offer multiple advantages, including deeper tissue penetration and reduced phototoxicity. They can also be combined with blue-light-based tools, such as optogenetics, with better spectral separation, although some sensitive light-activated proteins can still respond to the illumination used for red-sensor imaging.
Despite these benefits, red calcium sensors have generally been dimmer and less photostable than their green counterparts. Many red fluorescent calcium sensors also exhibit photoswitching, in which blue light artificially boosts their fluorescence, creating a signal unrelated to actual calcium changes. Several mApple-based red calcium sensors can accumulate in lysosomes, potentially interfering with long-term recordings. These limitations have restricted the widespread use of red sensors in combined imaging and optogenetic studies.
About the study
In the present study, the research team used mScarlet, a red fluorescent protein known for its brightness, to build PinkyCaMP. They created a circularly permuted version of the protein and attached it to calmodulin (CaM) on one end and a CaM-binding peptide on the other, two elements that allow calcium binding to alter fluorescence.
Candidate constructs were tested in bacterial colonies, and the two most promising were subjected to repeated rounds of directed evolution, during which specific amino acid positions were mutated, and thousands of variants were screened for brightness and calcium responsiveness. This process produced three refined candidates, from which one was selected as the final sensor and named PinkyCaMP.
The researchers then measured the basic biophysical properties, including light absorption and fluorescence efficiency. They expressed PinkyCaMP in human embryonic kidney 293T cells to compare its brightness with that of three established red sensors, and exposed the cells to brief blue-light pulses to assess for false fluorescence signals.
The team next introduced PinkyCaMP into cultured mouse neurons, paired the fluorescence recordings with direct electrical measurements, and combined the sensor with a light-activated ion channel in a single genetic construct to enable simultaneous stimulation and imaging.
For the in vivo experiments, the researchers delivered viral vectors encoding PinkyCaMP into specific brain regions in live mice. They used fiber photometry in freely moving mice, conventional two-photon imaging in awake head-fixed mice, and head-mounted miniature microscopes, including a two-photon system for freely moving animals. They also compared it with a green fluorescent sensor.
Key findings
The study found that PinkyCaMP was substantially brighter and more photostable than the three red calcium sensors tested, while showing no detectable false signals from blue light exposure. In the same cells, PinkyCaMP produced baseline fluorescence more than twice as strong as two widely used red sensors and considerably stronger than a third.
Under repeated blue light pulses, PinkyCaMP fluorescence stayed flat, whereas a comparison sensor showed a marked, misleading rise in signal unrelated to any actual change in calcium.
In cultured neurons, PinkyCaMP tracked gabazine-induced spontaneous network activity and reliably reported calcium responses to controlled electrical stimulation, with signal strength proportional to the number of delivered stimuli. When paired with a light-activated channel in the same cells, the sensor allowed researchers to trigger neuronal firing with blue light while reading out activity with orange light, although the imaging light weakly activated the sensitive channel and could itself generate a calcium response.
In live mice, PinkyCaMP detected population-level calcium responses in the prelimbic medial prefrontal cortex during an aversive air puff and captured shifts in activity as animals moved between the enclosed and open sections of an elevated zero maze, a test of approach-avoidance conflict. When PinkyCaMP was combined with a separate serotonin sensor, it allowed neuronal activity and serotonin release to be tracked simultaneously.
Across the tested imaging setups, the sensor performed well, including two-photon microscopy in awake, head-fixed, and freely moving mice, and could be paired with a green sensor to record two neuron populations simultaneously with limited crosstalk.
One limitation observed in the study was that PinkyCaMP responded and decayed more slowly than state-of-the-art green sensors, such as GCaMP8. The researchers attributed these slow kinetics to its strong calcium-binding affinity and noted that they could limit its usefulness for studies requiring precise detection of rapid, high-frequency firing. They may also merge closely spaced signals, while overexpression could buffer calcium, although the high affinity may aid detection in sparsely active neurons.
Conclusions
In summary, the study established PinkyCaMP as a red fluorescent tool that combines high brightness, improved photostability, and no detectable blue light-induced photoswitching, addressing several long-standing weaknesses of red calcium sensors. It showed minimal lysosomal accumulation and no detectable toxicity or aggregation in the tested systems. Its compatibility with optogenetics and green fluorescent tools broadens the range of experiments scientists can design to study brain circuits, though residual activation of sensitive opsins, slower kinetics, and potential calcium buffering at high expression levels leave room for future refinement.
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