Abstract In designing new biomarker imaging approaches for cancer research, the heterogeneity and dynamics of the tumor microenvironment cannot be disregarded. The interplay between the different components of the tumor stroma and parenchyma play a significant role in tumor progression, invasion and treatment response. In order to study such dynamic processes by (molecular) imaging, volumetric imaging modalities with a high temporal and spatial resolution need to be combined with contrast agents that possess appropriate pharmacokinetic parameters. In this work, we assessed tumor heterogeneity and specific cell-populations within the tumor microenvironment by combining Multispectral Optoacoustic Tomography (MSOT) with three different fast-clearing contrast agents. MSOT is a biomedical imaging modality based on the photoacoustic effect. In the used setup, mice are illuminated at multiple wavelengths in the NIR range (680 - 980nm) and by detecting the acoustic waves with a 5MHz tomographic ultrasound array, cross-sectional images can be obtained at a resolution of 150µm in less than a second. Imaging multiple cross-sections allows for volumetric reconstruction of the data and multispectral data analysis enables the specific identification of endogenous absorbers (hemoglobin and melanin) and/or multiple injected contrast agents. The pharmacokinetic parameters of the imaging agents were optimized to allow for relatively fast imaging regimens (<1 hour) that can be employed daily. Firstly, we assessed inter- and intra-tumoral heterogeneity in tumor perfusion and vascular permeability by systemically administering novel formulations of Indocyanine Green (ICG). Pixel-by-pixel analysis was then performed to create parametric maps, thereby revealing a high degree of heterogeneity in dynamic contrast enhancement between and within tumors. Secondly, apoptotic regions within orthotopic tumors were visualized using a caspase-targeted probe and compared to the hypoxia status of each tumor region, before and after treatment. Before treatment, maximal apoptosis-signal was co-localized with more hypoxic regions in the tumor. However, overall signal intensity was significantly increased after systemic treatment with doxorubicin. Lastly, the tumor associated macrophage (TAM) cell population within the tumor stroma was visualized by a novel NIR marker specific for macrophage. Again, intra-tumoral distribution of TAM-specific signal was compared to images of Hb and HbO2, revealing higher TAM densities in areas of relative hypoxia. In summary, MSOT offers an imaging modality that can provide anatomical, functional, molecular and kinetic information at high temporal and spatial resolution. When combined with (molecular) imaging agents that possess appropriate pharmacokinetic properties, this modality can be leveraged for new biomarker imaging approaches in cancer research. Citation Format: Stefan Morscher, Neal C. Burton, Thomas Sardella, Daniel Razansky, Vasilis Ntziachristos, Wouter H. P. Driessen. Novel approaches for dynamic tumor microenvironment imaging by Multispectral Optoacoustic Tomography (MSOT). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2014-2050