We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal. The acetoxymethyl esterified sensor variant was readily taken up by cells without toxic effects and thus allowed us for the first time to perform live imaging of Ca fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain via combined fluorescence and photoacoustic imaging.

Pupillometry, a noninvasive measure of arousal, complements human functional MRI (fMRI) to detect periods of variable cognitive processing and identify networks that relate to particular attentional states. Even under anesthesia, pupil dynamics correlate with brain-state fluctuations, and extended dilations mark the transition to more arousable states. However, cross-scale neuronal activation patterns are seldom linked to brain state-dependent pupil dynamics. Here, we complemented resting-state fMRI in rats with cortical calcium recording (GCaMP-mediated) and pupillometry to tackle the linkage between brain-state changes and neural dynamics across different scales. This multimodal platform allowed us to identify a global brain network that covaried with pupil size, which served to generate an index indicative of the brain-state fluctuation during anesthesia. Besides, a specific correlation pattern was detected in the brainstem, at a location consistent with noradrenergic cell group 5 (A5), which appeared to be dependent on the coupling between different frequencies of cortical activity, possibly further indicating particular brain-state dynamics. The multimodal fMRI combining concurrent calcium recordings and pupillometry enables tracking brain state-dependent pupil dynamics and identifying unique cross-scale neuronal dynamic patterns under anesthesia.