The dual raster-scanning photoacoustic small animal imager can be used to visualize various tissue in vivo and is easily adapted to many biomedical research studies with a need for small animal imaging. To align the system, cover an entire 10 x 10 x 4.4 centimeter water tank with a 10 micron thick polyethylene membrane, and fill the tank with ultra pure water. Place the tank on the working stage and turn on the laser switch.
Select the laser control program and reheat the laser for five minutes. Press the on button on the pumping switch and set the laser parameters. Open the laser bevel and select the A-line collected program.
Press start to capture a single point signal and to display the amplitude and spectrum of the current A-line signal. Place a blade at the bottom of the water tank and immerse the bottom part of the ultrasonic transducer in the water tank for acoustic coupling. Adjust the position of the Galva and the X Y translator between the ultrasonic transducer and the confocal objective lens to avoid an oscillation signal.
Then adjust the height of the working stage to maximize the amplitude of the signal and determine the focus position. After confirming a lack of response to pedal reflex, use a trimmer and depilatory cream to remove the hair from the back, ear, scalp, and abdomen of the anesthetized mouse, and place the mouse onto an 8 x 2.8 x 2 centimeter holder in the prone position. Place ultrasound gel onto the area to be imaged and place the holder onto the working stage.
Press start to collect an A-line signal and adjust the position of the Galva XT Translator and working stage to align the region to be imaged with the laser. Then press stop to end the collection. To collect a wide field image, select the wide field imaging mode and name the newly created folder.
Under the scanning speed tab set the scanning speed to 20 millimeters per second. The scanning area to 20 x 20 millimeters and the step to 20. When the scanning parameters have been set, click collect to begin scanning.
At the end of the acquisition, click stop and return to zero to bring the motor to zero. Pose the laser bevel and set the trigger setting to internal trigger. Press off to stop the pumping switch and select the real-time imaging mode trigger.
Connect the real-time imaging mode trigger to the external laser trigger, and turn on the pumping switch. Then set the trigger setting to the external trigger and click exit to exit the wide field imaging mode. To collect a real-time image, initiate the A-line signal collection and open the laser bevel.
Align the laser as demonstrated and click stop to end the collection after the alignment. Select the real-time imaging mode and name the newly created folder, click collect to start scanning. When the acquisition is complete, click stop.
Then click exit to exit the real-time imaging program. For widefield imaging of the brain vascular systems, select a focused ultrasonic transducer with a central frequency of 25 megahertz, a bandwidth of more than 90%and a focal length of 8 millimeters. Use a scalpel to make a small incision on the lateral side of the cranial temporal top of the mouse and use ophthalmic scissors to cut the scalp around the outer edge of the skull.
Compress any bleeding point to achieve homeostasis, and wash the wound with normal saline. Place ultrasound gel onto the exposed skull and initiate A-line signal collection. Click stop to end the collection after the alignment, and select the wide field imaging mode.
Name the newly created folder and set the scanning speed to 10 millimeters per second. The scanning area to 10 x 10 millimeters and the step to 10. Click collect to start scanning.
At the end of the acquisition click stop. Then click return to zero to return the motor to zero and click exit to exit the wide field imaging program. For dynamic real time imagining, place the mouse in the supine position and place ultrasound gel onto the abdomen.
Collect an A-line signal and align the laser as demonstrated. Select real-time imaging mode and name the newly created folder. Then click collect to start scanning.
At the end of the acquisition, click stop and exit to exit the real-time imaging program. Here a maximum amplitude projection image of the mouse back acquired in widefield imaging mode can be observed. In this B scan, the mouse back was imaged using the real time imaging mode.
Images of the vascular networks of the mouse ear and brain can be obtained using a focused ultrasonic transducer. In this analysis, B scan images of the mouse abdomen could be observed, revealing the presence of a vessel within the 100 micron imaging range. In addition, maximum amplitude projection imaging allows comparison of the depth direction of the mouse abdomen versus time.
To obtain optimal quality image, it is essential to achieve a good confocal adjustment of the optical and acoustic focus. The special image processing algorithm can be developed for quantitative analysis and can provide valuable information for early diagnosis and treatment of diseases.
