Abstract High grade glioblastomas are aggressive and highly invasive tumors that rarely are curable. To investigate novel treatments, accurate orthotopic tumor-models are crucial to evaluate efficacy. Because caliper measurements are not possible in this setting, accurate imaging strategies are needed for temporal tumor-tracking. Here we present a novel imaging strategy that leverages the recent development of fluorescent proteins absorbing light in the near infrared region, enabling their application in visualizing biological processes in deep seated tissues such as orthotopic glioblastomas. While purely optical in vivo imaging methods can only provide insufficient resolution limited by scattering, multispectral optoacoustic tomography (MSOT) is a very well suited hybrid imaging modality that can provide noninvasive imaging of optical absorbers at ultrasound resolution. MSOT is based on the photoacoustic effect; the generation of ultrasound waves as a result of the transient heating and expansion that takes place after the absorption of nanosecond laser pulses. We have developed a preclinical MSOT whole animal scanner that makes use of a tomographic ultrasound transducer array to achieve fast image rates of 10Hz, while high penetration depths can be achieved using light in the near infrared (NIR) optical window. Optical absorption of intrinsic tissue chromophores enable rich anatomical contrast, while multispectral imaging permits the separation of distinct optical absorbers and promotes the applicability of MSOT for functional and molecular imaging. The utilized imaging setup consists of a tunable NIR laser (680-950nm) and a 64 element ultrasound transducer array covering 172° around the sample (center frequency 5Mhz) to achieve an in-plane resolution of ∼150µm; further detailed in A. Buehler et al, 2011, OpticsLetters. For the presented study 8-10 weeks old CD1 nude mice were stereotactically implanted with 105 U87MG cells stably expressing iRFP (Filonov, G.S. et al., NatBiotechnol, 2011) in depths of 1.5mm and 3.5mm. MSOT imaging was performed in vivo 12 and 25 days after implantation. Excellent accordance was observed between in vivo MSOT imaging and subsequent post-mortem epi-fluorescent cryoslicing, demonstrating the unique performance of optoacoustic imaging in the presented scenario. MSOT was able to discriminate the iRFP signal over strong background absorbers such as oxygenated and deoxygenated hemoglobin as early as 12 days post implantation of the tumor cells. MSOT offers considerable advantages over MRI and bioluminescent imaging, such as high spatial resolution, ease of use and cost-efficiency. The presented study illustrates the capabilities of this emerging imaging modality for the tracking of deep seated tumors with a multitude of settings and applications being feasible. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2445. doi:1538-7445.AM2012-2445