Photoacoustic tomography of calcium activity in the mouse brain could potentially provide whole-brain coverage of neural activity and therefore offer new insights into brain function. Here we report the development and characterization of a novel photoacoustic calcium-sensitive probe based on the HaloTag protein which is suitable for in vivo imaging in mice. The photoacoustic brightness and signal enhancement upon calcium binding were measured using a custom-built spectroscopy setup and compared to the available far-red calcium indicator NIR-GECO1. Additionally, we conducted validation experiments on tissue-mimicking phantoms using a Fabry-Perot-based photoacoustic tomography setup to determine the depth limit and concentration detection threshold of the imaged probes. Furthermore, we tested various in vivo delivery methods, by analyzing brain slices from mice labeled with the photoacoustic probe. These experiments demonstrated that we are able to specifically label neurons targeted brain regions, confirming the suitability of these photoacoustic probes for in vivo calcium imaging applications.
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