Imaging of three-dimensional (3D) tumor scaffolds, engineered or naturally-derived tissue architectures, provides spatial, molecular, and phenotypic information for the extracellular environment and cells. Traditional optical techniques used to image two-dimensional cell cultures rely on light transmission through the sample. However, absorption and scattering by 3D tumor scaffolds impede light transmission. Appropriate sample preparation such as tissue clearing can reduce scattering and improve imaging depth. Epi-illumination, an imaging technique in which light is collected in the backward direction, combined with microscopy techniques with optical sectioning, such as multiphoton fluorescence, allow imaging of scaffolds with high 3D spatial resolution. Optical microscopy can evaluate fluorescent probes targeted to a specific area or molecule of interest, autofluorescent properties of cells and the extracellular matrix, and additional tissue properties such as light scattering or absorption. In addition to optical imaging, MRI can be used to image 3D tumor scaffolds for applications requiring imaging depths beyond optical limits. MRI of implanted tumor scaffolds provide assessment of microenvironment factors including tumor vascularization, pH, and hypoxia. Quantitative analysis of images provides spatial and heterogeneity information of both the extracellular matrix and cellular components of 3D tumor scaffolds to reveal insights into the tumor microenvironment.
