Three-Dimensional Optical-Resolution Photoacoustic Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 62

Animal preparation:
9. Anesthetize the animal with 1% isoflurane vaporized by the inhalation gas (the
typical flow rate is 1.0-1.5 l/min, depending on the animal's body weight) and
maintain the anesthesia throughout the experiment. To maintain the animal
under normal physiological status, the recommended inhalation gas is medical-
grade air, unless other type of gas is required for specific functional studies
(e.g., hypoxic challenging).
10. Fix the animal in a stereotactic stage. Set the temperature controller to the
typical body temperature of mice (i.e., 37 ı C).
11. Flatten the mouse ear on a plastic plate and apply a layer of ultrasound gel on
top of the ear. Avoid trapping air bubbles inside the gel.
12. Place the ear under the imaging window of the water tank and slowly raise the
animal holder until the ultrasound gel contacts the bottom of the polyethylene
membrane. Soft contact is preferred. Pressing the ear against the water tank
may affect blood flow.
13. Check again to make sure no air bubble is trapped between the polyethylene
membrane and the mouse ear.
14. Clamp the pulse oximeter to the animal to monitor its physiological status
(recommended).
15. Apply eye ointment to the animal to prevent dryness and accidental laser
damage (recommended).
Image acquisition:
16. Lower down the imaging head (i.e., the acoustic-optical beam splitter) until the
acoustic lens is immersed in deionized water.
17. Remove air bubbles trapped under the acoustic lens.
18. Set the laser to the external-trigger mode and start trial scanning.
19. Adjust the z position of the imaging head until the detected photoacoustic signal
is in the acoustic focal plane (judge from the acoustic delay).
20. Turn on the white-light illumination and check the imaging region under the
integrated transmission-mode optical microscope. Set the scanning origin to
the desired position. This is crucial for chronic monitoring, where the scanning
origin should be the same position throughout the entire monitoring period.
21. Set correct scanning parameters and start image acquisition.
End experiment:
22. Turn off the laser firing once the data acquisition is completed.
23. Lift the imaging head out of water and lower down the animal holder.
24. Clean the mouse ear with deionized water, turn off the anesthesia system and
the temperature controller, and unload the animal from the stereotactic stage.
25. If repetitive imaging is required, put the animal in an incubator with the
temperature controlled at 37 ı C. Return the mouse to the animal facility after
it wakes up naturally. Otherwise, follow the animal protocols to euthanatize
and dispose the animal.
26. Turn off the experimental equipment and clean up the experimental area.