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 photo thrombotic 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.
