Photoacoustic, bioluminescent, and fluorescent imaging modalities were compared using the same phantom setup to determine the strengths and limitations of each modality. For photoacoustic imaging, two tubes of melA-expressing bacteria were placed side-by-side using a custom tube-holding apparatus, which positioned the tubes 10 mm from the bottom of the holder. The tubes and holder were placed in a clear plastic container that was filled with 1% Intralipid® solution (scattering coefficient of 1% Intralipid® was similar to tissue at ). The tubes were spaced 2, 5, 10, 15, and 20 mm apart from each other and were imaged with the Vevo LAZR system. Tubes were approximately 10-mm distance from the imaging transducer, and multispectral imaging was performed using 680, 700, 750, 800, 850, and 900 nm light. The imaging system lateral, axial, and sagittal resolutions were approximately 165, 75, and , respectively. A stepper motor was used to translate the transducer with fixed distances, allowing reconstruction of three-dimensional images.