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Macroscopic optical small animal imaging plays an increasingly important role in biomedical research, as it can noninvasively examine structural, physiological, and molecular tissue features in vivo. A novel modality that emerged in the last decade is optoacoustic (photoacoustic) imaging, which combines versatile optical absorption contrast with high ultrasonic resolution and real-time imaging capabilities by capitalizing on the optoacoustic (photoacoustic) effect. Using illumination with multiple wavelengths and spectral unmixing methods, multispectral optoacoustic tomography (MSOT) has the potential to specifically resolve tissue chromophores or administered extrinsic molecular agents noninvasively in deep tissue with unprecedented resolution performance and in realtime. The presented work explores MSOT in the context of small animal imaging. Different instrumentation and detection geometry related effects are analyzed regarding their influence on the imaging performance. Based on the findings, two dedicated MSOT imaging platforms for 2D and 3D real-time imaging of small animals and tissue samples are conceived, implemented and imaging performance is characterized by simulation, on phantoms and ex vivo in mice. Beside instrumentation, the utilized signal processing, image reconstruction and spectral unmixing strategy is of great importance for achieving best imaging results. The research presented shows how reconstruction artifacts can be reduced by compensating for the electrical impulse response of the system and that the calculation of intermediate projections can alleviate artifacts due to sparse angular sampling. Moreover, two regularization approaches for 2D limited view reconstructions are presented and a 3D model-based inversion scheme for improving 3D reconstructions in the developed systems by modeling the shape of the detection elements. Finally, the challenges of multispectral unmixing in deep tissue are discussed; two unmixing schemes for detection of molecular agents are presented and a method to partially compensate for the effect of light attenuation is proposed. Using the unique imaging performance of the developed methods, it is further established that MSOT can actually resolve anatomical, dynamic and molecular information in mice and that it can be used for assessing biodistribution and pharmacokinetic parameters of molecular probes and contrast agents in tissue. A complete whole-body mouse scan is shown, resolving anatomical hemoglobinbased contrast. Functional imaging is presented by tracking contrast enhancement in the kidneys due to perfusion of systemically administered Indocyanine green (ICG). Also the clearance rate of ICG and liposomal ICG from the blood pool is determined by means of MSOT. Molecular imaging performance is shown by detecting optical reporter agents (here a phosphatidylserine targeting fluorescent dye) within mouse xenograft tumors.