Evaluations of Oxygenation and Vascular Perfusion in Pre-Clinical Models of Cancer and Wound Healing Using OS-DCE OAI.

Oxygen-sensitive optoacoustic imaging (OS OAI) and dynamic contrast-enhanced optoacoustic imaging (DCE OAI) provide complementary, noninvasive readouts of tissue physiology. OS OAI leverages multispectral detection of endogenous oxyhemoglobin and deoxyhemoglobin to generate maps of blood oxygen saturation (%sO₂), while DCE OAI tracks the in vivo pharmacokinetics of a near-infrared absorber to quantify vascular perfusion and permeability. The goal of this protocol is to present a unified OS-DCE OAI workflow that enables simultaneous assessment of oxygenation and perfusion in two widely used pre-clinical settings: orthotopic breast cancer tumors and full-thickness cutaneous wounds. In this protocol, we demonstrated OAI of mouse models of breast cancer or a laceration wound. For OS OAI, multi-slice multispectral scans are acquired at 700-875 nm to allow spectral unmixing and estimation of %sO₂. For DCE OAI, repeated acquisitions at ≤5-s temporal resolution are performed before and after intravenous bolus injection of indocyanine green (ICG), using either multispectral sampling at 700-875 nm or single-wavelength sampling at 800 nm to generate time versus OA signal amplitude curves. The development of animal models, preparation for OA imaging, OS OAI and DCE OAI acquisitions, and analyses to measure characteristics of oxygenation and vascular perfusion are detailed, along with key troubleshooting guidance for motion artifacts, superficial absorbers, and failed injections. Together, this OS-DCE OAI protocol yields an integrated functional portrait of hypoxia and vascular transport that can detect physiological changes in tumors and wounds earlier than gross anatomical measures. OS-DCE OAI is readily adaptable to other disease models.