Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke
June 2nd, 2023
This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.
There are a few experimental challenges. For example, the presence of hair in the scalp can cause artifact in the acquired images. The age of the animal also plays a key role, since the thickness of the skull is currently a limitation for the detection of deeper signals.
Many purely optical imaging techniques require an invasive procedure and can only image about one millimeter deep. With PAUSAT, we combine the advantages of both ultrasound and optical imaging. We can get both structural and functional information surrounding ischemic stroke in a completely safe and non-invasive way.
Being able to evaluate the evolution of an injury with or without treatment from the same animal at different time points allows us to collect very valuable information and also to reduce the number of animals required for different studies. We are constantly working on ways to improve the imaging speed, depth, resolution, and contrast for both photoacoustic and ultrasound imaging. With better imaging systems, we can answer more fundamental biological questions and evaluate new experimental treatments.
To set up the imaging system, start by leaving the 532 nanometer laser on for 15 minutes to warm up. Next, place a customized ramp attached to a manually adjustable stage next to the imaging membrane. Attach a mouse tooth holder with a connected breathing tube to the ramp and secure a heating pad on the ramp surface.
Once the laser has warmed up, place a near IR detector card in front of the fiber bundle input and check the laser alignment by ensuring that the laser light enters the bundle. Begin animal preparation by trimming the hair of the anesthetized mouse's head with an electric shaver from near the eyes to behind the ears. Apply a hair removal cream to the top of the head.
After leaving it on for five minutes, wipe the cream off with a wet cotton swab. Repeat the application until the skin of the head is fully depilated. Just before transferring the animal to the system's platform, use a 27-gauge needle to retro-orbitally inject 100 microliters of microbubbles at stock concentration.
Now put one drop of the eye protection lotion on the eyes. Begin PAUSAT imaging by filling the imaging window with distilled water on the surface for acoustic coupling. Next, secure the mouse's head in the tooth holder and ensure proper anesthesia and air flow.
Using a heating lamp and a rectal probe connected to a temperature controller, keep the animal's body temperature at 37 degrees Celsius. Open the imaging application and navigate to the B-mode ultrasound. Use the live ultrasound window to adjust the mouse's head to the desired position manually and the stage height accordingly.
Adjust the value of the ultrasound transmission frequency in B-mode to 16 megahertz, then input the save directory information in the imaging application. Next, select the desired region of the brain scan by using the floating box. Press the acquire static button and check the scan results once the image acquisition is completed to ensure the desired region imaging.
To perform acoustic angiography imaging, return to image acquisition, then change the mode to acoustic angiography. Next, input the desired scan protocol parameters, setting the frame spacing at 0.2 millimeters and 10 frames per position. Check scan results under image analysis to ensure image quality.
To image with photoacoustic tomography, open the optical parametric oscillator or OPO application and set it to 756 nanometers, then manually translate the linear array transducer to the previously determined coordinates to automatically co-register the wobbler volumes and the linear array volumes. Open the laser application and turn on the 532-nanometer laser. Set the scanning parameters as 0.4 millimeters step size, 20 millimeters scan length, and 10 frames averaged per position.
Now open the ultrasound data acquisition system MATLAB program and press the run button. Next, press start in the scanning application. Once the scan is complete, open the MATLAB saving program, change the save name to the desired file name and press run, then change the OPO wavelength to 798 nanometers and repeat scanning.
Acoustic angiographic or AA imaging showed that, in the uninjured brain, both hemispheres present a similar distribution of blood vessels. Similar results are observed in the photoacoustic images at two different wavelengths. Notable signal reduction in the right lateral cortex was observed in the AA images of electrocauterized mice.
The same region also showed reduced tissue oxygenation suggesting ischemia. PAUSAT imaging can identify stroke areas based on the photothrombotic stroke model by identifying upper cortex region with reduced blood flow supply and decreased oxygen saturation. Mouse age can affect imaging since the skull becomes thicker and results in different acoustic impedance, resulting in decreased imaging depth for older mice.
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Overview
This study investigates a multimodal ultrasound-based imaging platform for the noninvasive imaging of ischemic stroke. The system quantifies tissue oxygenation through photoacoustic imaging and assesses impaired brain perfusion using acoustic angiography.
Key Study Components
Area of Science
- Neuroscience
- Imaging Technology
- Stroke Research
Background
- Existing optical imaging techniques often require invasive procedures and have limited depth capabilities.
- Multimodal approaches combine the strengths of ultrasound and optical imaging.
- Understanding ischemic stroke through imaging can provide insights into treatment efficacy.
- Challenges such as artifacts from hair and age-related skull thickness can affect imaging results.
Purpose of Study
- To develop a safe, non-invasive imaging method for real-time assessment of ischemic stroke.
- To enhance imaging speed, depth, resolution, and contrast.
- To facilitate the evaluation of injury evolution with minimal animal use across time points.
Methods Used
- The platform involves photoacoustic imaging and acoustic angiography, optimizing imaging parameters for stroke detection.
- The key biological model includes anesthetized mice, and imaging assessments are conducted following microbubble injection.
- No multiomics workflow is mentioned in the text.
- A series of steps for animal preparation and imaging procedures are detailed, including setting up the imaging system.
- Image acquisition involves configuring ultrasound and photoacoustic imaging parameters, ensuring quality and alignment.
Main Results
- The imaging system successfully identifies stroke areas, showing reduced blood flow and oxygenation in electrocauterized mice.
- Contrasting images reveal significant changes in blood vessel distribution and tissue oxygenation related to ischemic events.
- Age-related factors influence imaging outcomes, requiring considerations for effective stroke assessment.
Conclusions
- This study demonstrates the potential of PAUSAT imaging as a valuable tool for evaluating ischemic stroke in a non-invasive manner.
- The advancements in imaging capabilities can lead to improved understanding of neuronal injury and treatment responses.
- This approach has significant implications for stroke research and therapeutic intervention evaluation.
Frequently Asked Questions
Executive Industry Relevance
Noninvasive, multimodal imaging platforms like PAUSAT address a critical need for quantitative, longitudinal assessment of cerebral perfusion and oxygenation in preclinical stroke models. This capability enhances predictive confidence in target validation and mechanistic de-risking for neurovascular drug discovery. Integrating structural and functional imaging supports risk-adjusted portfolio decisions at early discovery and preclinical inflection points.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables quantitative interrogation of neurovascular injury and recovery mechanisms in vivo.
- Supports functional target validation by correlating vascular changes with ischemic outcomes.
- Facilitates mechanistic de-risking by distinguishing perfusion and oxygenation deficits across models.
- Improves predictive confidence for translational biomarker selection in stroke research.
Screening & Assay Development
- Provides standardized, reproducible imaging outputs for evaluating candidate interventions in animal models.
- Delivers quantitative readouts of blood flow and oxygenation for assay development.
- Enables high-content screening of therapeutic effects on cerebral vasculature.
- Supports platform reuse across multiple preclinical studies and models.
Translational & Preclinical Research
- Aligns imaging outputs with translational biomarkers of cerebral perfusion and infarct volume.
- Enables longitudinal monitoring of disease progression and therapeutic response in the same animal.
- Reduces animal use by supporting within-subject study designs.
- Improves continuity from discovery through preclinical validation in neurovascular pipelines.
Pipeline & Workflow Integration
PAUSAT integrates into the preclinical discovery continuum, bridging early mechanistic studies and translational validation for neurovascular targets.
- Discovery Biology: Quantifies perfusion and oxygenation changes to test neurovascular hypotheses in vivo.
- Screening: Provides reproducible, quantitative imaging endpoints for candidate evaluation.
- Analytics: Enables statistical comparison of infarct volume, perfusion, and oxygenation across experimental groups.
- Translational Research: Aligns imaging outputs with established histological and perfusion biomarkers.
- Enterprise Reuse: Offers a scalable, noninvasive imaging platform adaptable to diverse preclinical models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neurovascular target validation.
- Operational Value: Standardizes imaging protocols and supports reproducibility across studies.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by providing robust, quantitative endpoints.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of neurovascular assets.
Implementation Considerations
- Requires expertise in multimodal imaging and animal model preparation.
- Needs specialized instrumentation for photoacoustic, ultrasound, and angiographic tomography.
- Demands cross-team standardization of imaging protocols and data analysis workflows.
- Imaging depth may be limited by animal age and skull thickness, affecting model selection.
- Potential for imaging artifacts from hair or improper animal preparation must be managed.
