WP7: Multispectral Optoacoustic Imaging of the Brain: The project will investigate experimental and processing methods for in vivo MSOT imaging of the brain in mice divided into 4 parts:1) Functional haemoglobin imaging, 2) Cell labelling for sensitive visualization, 3) Stem cell and T-cell imaging and 4) Amyloid-beta imaging. Functional haemoglobin imaging: Functional imaging by means of haemoglobin absorption contrast can provide spatially resolved information in two additional dimensions: spectral and temporal. Thus haemodynamics and oxygenation can be combined to provide a picture of brain function as well as characterization of lesions. While we have demonstrated basic capabilities in this respect, there is a need to further develop imaging protocols and processing methods and validate these on realistic mouse models. In particular, real-time rendering of functional images will be developed to provide researchers with an accurate in vivo picture of function during imaging experiments. Cell labelling: The two most promising approaches demonstrated for optoacoustic genetic reporters are near infrared fluorescent proteins and melanin producing tyrosinase. These two methods differ significantly in terms of absorption spectrum and potential for visualization by other modalities. Within this project, we will investigate the performance of these techniques for the application of cell labelling and assess them in comparison to non-genetic methods for highly sensitive cell imaging in the brain. We will also establish and assess methods for dual- and multichannel channel cell imaging by combining multiple labels at different wavelengths. Stem Cell and T-Cell imaging: the monitoring of multiple events such as stem cell fate (migration, differentiation and function), nanoparticle-release of therapeutic payloads and activity of T-cells will be utilized for WPs 2, 3 & 4. To unravel and understand the relevant processes, simultaneous readouts often need to be made. In WP3, we already have different colored luciferase constructs to detect simultaneously active T-cells, total T-cells and tumour. MSOTTM on the brain for GBM characterization has already been demonstrated by the TUM group. If we can also now detect the stem cells and nanoparticles the mechanisms of therapeutic action can be imaged and revealed. Amyloid-beta imaging: Methods established for conventional fluorescence imaging of amyloid-beta will be adapted in this part for use in mouse models of AD. We will investigate the feasibility and performance of amyloid-beta imaging by means of MSOTTM, utilizing optical agents that have been studied for fluorescence imaging. The objective is to characterize the depth penetration, sensitivity and quantitative accuracy of such imaging in relevant mouse models. Overall, MSOTTM has potential to visualize a range of important biological parameters, from labeled cells through haemoglobin imaging to brain-relevant molecular biomarkers. The ability to monitor these parameters efficiently in vivo could provide new insights to researchers, who must currently rely on a range of methods to obtain such information, often requiring the animals to be sacrificed for analysis. The objectives proposed in this work package will serve to establish MSOTTM as a tool for investigating brain pathophysiology.