Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting how they
will eventually respond to treatment. The mouse window chamber model is an excellent tool for cancer research,
because it enables high resolution tumor imaging and cross-validation using multiple modalities. We describe a novel
multimodality imaging system that incorporates three dimensional (3D) photoacoustics with pulse echo ultrasound for
imaging the tumor microenvironment and tracking tissue growth in mice. Three mice were implanted with a dorsal skin
flap window chamber. PC-3 prostate tumor cells, expressing green fluorescent protein (GFP), were injected into the skin.
The ensuing tumor invasion was mapped using photoacoustic and pulse echo imaging, as well as optical and fluorescent
imaging for comparison and cross validation. The photoacoustic imaging and spectroscopy system, consisting of a
tunable (680-1000nm) pulsed laser and 25 MHz ultrasound transducer, revealed near infrared absorbing regions,
primarily blood vessels. Pulse echo images, obtained simultaneously, provided details of the tumor microstructure and
growth with 100-μm3 resolution. The tumor size in all three mice increased between three and five fold during 3+ weeks
of imaging. Results were consistent with the optical and fluorescent images. Photoacoustic imaging revealed detailed
maps of the tumor vasculature, whereas photoacoustic spectroscopy identified regions of oxygenated and deoxygenated
blood vessels. The 3D photoacoustic and pulse echo imaging system provided complementary information to track the
tumor microenvironment, evaluate new cancer therapies, and develop molecular imaging agents in vivo. Finally, these
safe and noninvasive techniques are potentially applicable for human cancer imaging.