Optoacoustic Tomography (OT) is now widely used in preclinical imaging, however, precision (repeatability and reproducibility) of OT has yet to be determined.

METHODS: We used a commercial small animal OT system. Measurements in stable phantoms were used to independently assess the impact of system variables on precision (using coefficient of variation, COV), including acquisition wavelength, rotational position, frame averaging. Variables due to animal handling and physiology, such as anatomical placement and anesthesia conditions were then assessed in healthy nude mice using the left kidney and spleen as reference organs. Temporal variation was assessed by repeated measurements over hours and days both in phantoms and $in\; vivo$. Sensitivity to small molecule dyes was determined in phantoms and $in\; vivo$; precision was assessed $in\; vivo$ using IRDye800CW.

RESULTS: OT COV in a stable phantom was less than 2% across all wavelengths over 30 days. The factors with greatest impact on the signal repeatability in phantoms were rotational position and user experience, both of which still resulted in a COV of less than 4%. Anatomical ROI size showed the highest variation at 12% and 18% COV in the kidney and spleen respectively, however, functional SO₂ measurements based on a standard operating procedure showed exceptional reproducibility of <4% COV. COV for repeated injections of IRDye800CW was 6.6%. Sources of variability for $in\; vivo$ data included respiration rate, user experience and animal placement.

CONCLUSION: Data acquired with our small animal OT system was highly repeatable and reproducible across subjects and over time. Therefore, longitudinal OT studies may be performed with high confidence when our standard operating procedure is followed.