Acoustic and photoacoustic high frequency imaging (50-100 MHz) can be used to generate images of cell constructs and spheroids with good spatial resolution and contrast. Here we demonstrate how co-registered acoustic and photoacoustic imaging can be used for imaging spheroids. Spheroids are widely used in cancer research and biology since they emulate a 3-dimensional environment such as that experienced in tumors. Spheroids were made by the hanging-drop method using the MCF-7 cancer cell line. To generate photoacoustic contrast, MCF-7 cells were incubated with optical absorbing nanoparticles (e.g. gold nanorods, 780nm absorption) for 24 hours and mixed with native MCF-7 cells prior to spheroid formation. The spheroids were between 0.5 mm and 1mm in diameter. Imaging was performed with the VisualSonics VEVO 770 (25-55 MHz) and a high-resolution SASAM acoustic/photoacoustic microscope for frequencies over 80 MHz (Kibero GmbH, Germany). The spheroid was imaged first using pulse echo ultrasound, then with pho...