Visualization of dynamic functional and molecular events in an unperturbed in vivo environment is essential for understanding the complex biology of living organisms and of disease state and progression. To this end, optoacoustic (photoacoustic) sensing and imaging have demonstrated the exclusive capacity to maintain excellent optical contrast and high resolution in deep-tissue observations, far beyond the penetration limits of modern microscopy. Yet, the time domain is paramount for the observation and study of complex biological interactions that may be invisible in single snapshots of living systems. This review focuses on the recent advances in optoacoustic imaging assisted by smart molecular labeling and dynamic contrast enhancement approaches that enable new types of multiscale dynamic observations not attainable with other bio-imaging modalities. A wealth of investigated new research topics and clinical applications is further discussed, including imaging of large-scale brain activity patterns, volumetric visualization of moving organs and contrast agent kinetics, molecular imaging using targeted and genetically expressed labels, as well as three-dimensional handheld diagnostics of human subjects.

Ongoing efforts to scale neuroimaging towards direct visualization of mammalian brain-wide neural activity face major challenges, and a large gap still exists between localized optical microscopy looking at rapid cellular-resolved neuronal activities and whole-brain observations of slow hemodynamics and metabolism provided by macroscopic imaging modalities. Optoacoustic imaging holds inherent advantages for deep tissue observations, but to date has not been applied towards direct activity observation in the mammalian brain. Here we demonstrate in vitro and in vivo functional optoacoustic neuroimaging from mice expressing the genetically encoded calcium indicators GCaMP6, effectively bridging the gap between functional microscopy and whole-brain macroscopic neuroimaging. We yielded instantaneous high-resolution 3D snapshots of whole-brain activity maps with single optoacoustic excitations and enabled non-invasive detection of fast neural responses to sensory stimuli in the presence of strong hemoglobin background absorption. These results demonstrate a new enabling technique towards scalable direct neuroimaging at unprecedented penetration depths and spatio-temporal resolutions.

In many practical optoacoustic imaging implementations, dimensionality of the tomographic problem is commonly reduced into two dimensions or 1-D scanning geometries in order to simplify technical implementation, improve imaging speed or increase signal-to-noise ratio. However, this usually comes at a cost of significantly reduced quality of the tomographic data, out-of-plane image artifacts, and overall loss of image contrast and spatial resolution. Quantitative optoacoustic image reconstruction implies therefore collection of point 3-D (volumetric) data from as many locations around the object as possible. Here, we propose and validate an accurate model-based inversion algorithm for 3-D optoacoustic image reconstruction. Superior performance versus commonly-used backprojection inversion algorithms is showcased by numerical simulations and phantom experiments.

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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.

Optoacoustics provides a unique set of capabilities for bioimaging, associated with the intrinsic combination of ultrasound-and light-related advantages, such as high spatial and temporal resolution as well as powerful spectrally enriched imaging contrast in biological tissues. We demonstrate here, for the first time, the acquisition, processing and visualization of five-dimensional optoacoustic data, thus offering unparallel imaging capacities among the current bioimaging modalities. The newly discovered performance is enabled by simultaneous volumetric detection and processing of multispectral data and is further showcased here by attaining time-resolved volumetric blood oxygenation maps in deep human vessels and real-time tracking of contrast agent distribution in a murine model in vivo.

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.

In this paper, it is demonstrated that the effects of acoustic attenuation may play a significant role in establishing the quality of tomographic optoacoustic reconstructions. Accordingly, spatially dependent reduction of signal amplitude leads to quantification errors in the reconstructed distribution of the optical absorption coefficient while signal broadening causes loss of image resolution. Here we propose a correction algorithm for accounting for attenuation effects, which is applicable in both the time and frequency domains. It is further investigated which part of the optoacoustic signal spectrum is practically affected by those effects in realistic imaging scenarios. The validity and benefits of the suggested modelling and correction approaches are experimentally validated in phantom measurements.