Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience. We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains. It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm3 with respective spatial resolution of 35–200 μm. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically encoded calcium indicator GCaMP5G demonstrate, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in scattering brains. The newly developed platform thus offers unprecedented capabilities for functional whole-brain observations of fast calcium dynamics; in combination with optoacoustics' well-established capacity for resolving vascular hemodynamics, it could open new vistas in the study of neural activity and neurovascular coupling in health and disease.

Conventional optoacoustic microscopy operates in two distinct modes of optical resolution, for visualization of superficial tissue layers, or acoustic resolution, intended for deep imaging in scattering tissues. Here we introduce a new microscope design with hybrid optical and acoustic resolution, which provides a smooth transition from optical resolution in superficial microscopic imaging to ultrasonic resolution when imaging at greater depths within intensely scattering tissue layers. Experimental validation of the new hybrid optoacoustic microscopy method was performed in phantoms and by means of transcranial mouse brain imaging in vivo.

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small‐animal neuroimaging methods, a variety of acoustic and light‐related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high‐resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically‐ and optically‐determined resolution scenarios. It is shown that strong low‐pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.