Evaluations of Oxygenation and Vascular Perfusion in Pre-Clinical Models of Cancer and Wound Healing Using OS-DCE OAI
April 7th, 2026
Here, we present a protocol for performing Oxygen Sensitive – Dynamic Contrast Enhanced Optoacoustic Imaging (OS-DCE OAI) for evaluating pre-clinical models of cancer and wound healing. OS OAI can image oxy‑ and deoxyhemoglobin, mapping tissue oxygenation. DCE-OAI can image the pharmacokinetics of an exogenous contrast agent, thereby mapping vascular perfusion.
My research focuses on optoacoustic imaging system to measure oxygenation and perfusion of wounds and tumors. Current methods are limited to measuring perfusion and oxygenation simultaneously, but our protocol enable real-time and noninvasive assessment. This protocol is applicable to both preclinical and clinical studies of wounds, tumors, and vascular disease, although it is limited to imaging depth of approximately up to 10 centimeters.
To begin, prepare a 2.14-millimolar solution of the indocyanine green, or ICG contrast agent, in sterile water. Filter the solution with a 0.3-micrometer syringe filter and fill a sterile laboratory syringe with 100 microliters of the filtered solution. Now fill the imaging tank with deionized distilled water and set the tank temperature to 36 degrees Celsius for a mouse with less than 25 grams body weight.
If performing DCE-OAI, flush a 27-gauge tail vein catheter with heparinized saline. Insert the catheter into a lateral tail vein of an anesthetized mouse and secure it to prevent it from being removed from the tail vein. Confirm that no air bubbles are present.
Next, prepare a sterile laboratory syringe with a volume no greater than 500 microliters that can connect to the catheter. Fill the syringe with 100 microliters of 2.14-millimolar ICG. Next, apply a thin layer of clear ultrasound gel to the tumor or wound area.
Then apply ultrasound gel to the cradle to make a seal between the cradle and the tumor or wound area. Place the mouse in the cradle according to the manufacturer's instructions. Position the mouse in the holder with the nose firmly inside the nose cone.
Use cuffs to restrain the mouse's hands and feet. Use the tooth loop to stabilize the head. When imaging a mouse, use a cradle with a rat nose cone to lift the head from the bottom of the cradle and reduce the risk of accidental drowning if water leaks into the cradle.
Now place the mouse and cradle into the OAI instrument according to the manufacturer's instructions. Observe the mouse cradle for any leaks. If performing DCE-OAI, connect the catheter to the syringe containing the contrast agent.
Equilibrate the mouse before imaging by allowing 10 minutes for stabilization of mouse physiology in the warm water tank. To position the mouse relative to the OAI transducer, move the mouse or the OAI transducer so that the tumor or wound is located within the multi-slice image set. Optimize the speed of sound for the image set during the equilibration period.
Maintain consistent animal orientation and slice position for all mice and at all time points to ensure rigorous results. Select the image slice parameters to acquire multiple slices covering the entire tumor or wound area with a one-millimeter slice thickness. Then select the absorption wavelengths for OS-OAI at 700, 730, 760, 800, 850, and 875 nanometers.
Configure signal averaging to acquire 10 averages per wavelength before acquiring the next wavelength and before moving to the next imaging slice. Then set the number of repetitions to maintain a total acquisition time of approximately two minutes. Perform a minimum of three repetitions to ensure measurement stability.
Now acquire the OS-OA images using the previously set parameters. Set the DCE-OAI acquisition parameters for a single-slice imaging protocol with multiple wavelengths. Position up to five imaging slices centered on the tumor or wound area with one-millimeter slice thickness.
Select the optimal absorption wavelength for the contrast agent by setting the wavelength to 800 nanometers for ICG in an in-vivo environment. Set the number of averages and repetitions to maintain a temporal resolution of five seconds or less and a total acquisition time of 11.0 to 11.2 minutes for a 10-hertz imaging repetition rate. To acquire the DCE-OA images, start the acquisition and collect 12 image sets.
Then administer 100 microliters of contrast agent via the catheter while continuing acquisition. Continue imaging to acquire the remaining 128 image sets for 10.2 minutes post-injection to capture wash-in and wash-out kinetics. Monitor the imaging process to ensure images are acquired and observe the mouse breathing rate to confirm stable physiology during scanning.
Then remove the mouse and cradle from the instrument. Remove the mouse from the cradle and allow it to recover from anesthesia. Return the mouse to a warmed cage and monitor until ambulatory.
Finally, analyze OA images and compute quantitative parameters to assess tumor or wound physiology and contrast dynamics. Quantitative maps of oxyhemoglobin, deoxyhemoglobin, total hemoglobin, and blood oxygen saturation were generated from multi-spectral OAI data sets using spectral unmixing. DCE-OAI provided empirical pharmacokinetic parameters, including maximum signal enhancement, time to peak, slope, area under the curve, signal loss, and contrast enhancement at 10 minutes.
Pharmacokinetic modeling enabled estimation of wash-in and wash-out rates, represented by NKtrans and Kep maps. Wound models showed slower wash-out rates immediately after injury that increased during healing, indicating changes in vascular perfusion. This protocol allows a dynamic, non-invasive measurement of tissue oxygenation and perfusion.
The key challenge is minimizing motion and maintaining consistent positioning for reliable measurement.
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Overview
Oxygen-sensitive optoacoustic imaging (OS OAI) and dynamic contrast-enhanced optoacoustic imaging (DCE OAI) provide complementary, noninvasive readouts of tissue physiology. OS OAI uses multispectral detection of endogenous oxyhemoglobin and deoxyhemoglobin to generate blood oxygen saturation (%sO₂) maps, while DCE OAI tracks the in vivo pharmacokinetics of a near-infrared absorber to quantify vascular perfusion and permeability. This protocol presents a unified OS-DCE OAI workflow for simultaneous assessment of oxygenation and perfusion in orthotopic breast cancer tumors and full-thickness cutaneous wounds in mouse models.
Key Study Components
Area of Science
- Biomedical imaging
- Optoacoustic imaging
- Tumor physiology
- Wound healing
Background
- OS OAI enables noninvasive measurement of tissue oxygenation via spectral unmixing of hemoglobin signals.
- DCE OAI assesses vascular function by tracking contrast agent kinetics after intravenous injection.
- Combining OS and DCE OAI allows correlated evaluation of hypoxia and perfusion in disease models.
- Preclinical models such as orthotopic tumors and cutaneous wounds exhibit dynamic changes in oxygenation and vascular transport.
Purpose of Study
- To establish a unified protocol for simultaneous OS and DCE OAI acquisition.
- To enable early detection of physiological changes in tumors and wounds before anatomical alterations are visible.
- To provide a flexible imaging approach adaptable to various preclinical disease models.
Methods Used
- Orthotopic breast cancer tumor and full-thickness laceration wound mouse models were prepared.
- For OS OAI, multispectral scans were acquired between 700–875 nm for spectral unmixing and %sO₂ estimation.
- For DCE OAI, repeated imaging at ≤5-second resolution was performed before and after ICG bolus injection, using either multispectral (700–875 nm) or single-wavelength (800 nm) sampling.
- Time–OA signal amplitude curves were generated to analyze vascular perfusion and permeability.
Main Results
- The OS-DCE OAI workflow yielded integrated functional maps of hypoxia and vascular transport in tumors and wounds.
- Physiological changes were detectable earlier than with gross anatomical imaging.
- The protocol demonstrated adaptability across different disease models.
- Key troubleshooting addressed motion artifacts, superficial absorbers, and failed injections.
Conclusions
- OS-DCE OAI provides a powerful, noninvasive tool for assessing tissue oxygenation and perfusion in vivo.
- The unified workflow enables early detection of pathophysiological changes in preclinical models.
- The method is readily transferable to other disease models beyond breast cancer and cutaneous wounds.
Frequently Asked Questions
Executive Industry Relevance
OS-DCE OAI enables simultaneous, noninvasive assessment of tissue oxygenation and vascular perfusion in preclinical models, providing early functional readouts that precede anatomical changes. This integrated approach supports mechanistic de-risking in oncology and wound healing programs by correlating hypoxia and perfusion dynamics. The method enhances predictive confidence in target validation and lead identification stages by delivering quantitative, reproducible physiological metrics.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by mapping hypoxia and perfusion changes in response to perturbations.
- Operational Value: Provides noninvasive, longitudinal monitoring of target engagement effects on tumor and wound microenvironments.
- Strategic Value: Supports target de-risking through functional validation of oxygenation and vascular pathways.
Screening & Assay Development
- Scientific Value: Generates quantitative %sO₂ and perfusion metrics suitable for assay standardization across compound screens.
- Operational Value: Delivers reproducible, high-temporal-resolution readouts (≤5 s) for consistent pharmacokinetic and pharmacodynamic profiling.
- Strategic Value: Enables platform reuse across disease models, reducing assay development overhead.
Translational & Preclinical Research
- Scientific Value: Bridges discovery and preclinical stages by providing translatable functional biomarkers of hypoxia and vascular transport.
- Operational Value: Facilitates cross-functional collaboration through standardized, noninvasive imaging outputs.
- Strategic Value: Informs risk-adjusted advancement decisions by detecting physiological changes earlier than gross anatomical endpoints.
Pipeline & Workflow Integration
OS-DCE OAI fits within the discovery continuum from target validation through preclinical assessment, enabling continuous monitoring of tissue physiology without terminal endpoints.
- Discovery Biology: Supports hypothesis testing and pathway clarification by correlating oxygenation and perfusion responses to genetic or pharmacological modulation.
- Screening: Enhances assay readiness through standardized, quantitative oxygenation and perfusion readouts compatible with compound library evaluation.
- Analytics: Delivers time–OA signal amplitude curves and %sO₂ maps that enable intergroup comparison and effect size quantification.
- Translational Research: Connects to preclinical continuity via functional biomarkers that align with clinical hypoxia and perfusion imaging modalities.
- Enterprise Reuse: Positions the workflow as a reusable imaging capability applicable across oncology, wound healing, and ischemic disease models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in tumor and wound pathophysiology.
- Operational Value: Ensures standardization and reproducibility through defined acquisition protocols and troubleshooting guidelines.
- Strategic Value: Improves go/no-go decisions by providing early, functional efficacy signals that reduce late-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization through objective, longitudinal physiological monitoring.
Implementation Considerations
- Requires expertise in optoacoustic imaging, spectral unmixing, and contrast agent pharmacokinetics.
- Dependent on multispectral optoacoustic systems capable of 700–875 nm imaging and ≤5-second temporal resolution.
- Necessitates standardization across sites for consistent ICG dosing, injection timing, and post-processing pipelines.
- Involves adaptation considerations for varying tissue optical properties in different disease models.
- Limited by motion artifacts, superficial absorbers, and injection success rates, as noted in troubleshooting guidance.
