1. Image setup

1. Turn the ultrasound system on using the switch on the back. Turn the monitor on.
2. Plug in the physiological monitoring unit and turn on heart rate and temperature monitoring. Turn on the gel warmer and ensure the light is on.
3. Check the isoflurane level in the anesthetic vaporizer and refill if necessary.
4. Turn on your O₂ tank or filtered air source and adjust the air flow on the vaporizer to approximately 1 L/min.
5. Attach the mouse or rat stage, and then plug the VGA cord into that stage. Secure the corresponding nosecone into place, and check that the isoflurane (black) and waste gas (blue) tubes are properly connected to nosecone.
6. Choose a transducer to use for your procedure and plug it into the “active” port below the imaging system. Run the transducer cable through plastic holders above the probe mount and secure the transducer into the clamp. Note there is a small, raised line on one side of each transducer to determine image orientation.
7. Anesthetize the animal according to AVMA guidelines and preparing it for imaging. Add ophthalmic ointment to the eyes, secure the paws to the stage electrodes, and remove the hair in the area of interest using a depilatory cream. Cover the area to be imaged with warmed ultrasonic transducing gel.

2. Image Acquisition

1. On the system, start a new study by selecting New and Study, or find a study you previously started and select New and Series. Once in a new series, select a user off of the menu and name your series appropriately.
2. Once your series has been created, select B-mode (brightness mode) from the keyboard. All imaging modality keys are on the bottom row of the black keyboard.
3. Roll the transducer down to the desired location making sure to look at the screen to avoid applying too much pressure to the animal. Check the respiratory rate (RR) that appears on the screen as well, because too much pressure may cause the RR to drop.
4. While looking at the B-mode image, adjust the placement of the transducer by gently turning the x- and y-axis knobs found on the stage until you find the desired location.
5. Once you have the desired location, wait for the white bar at the bottom of the image to fill before pressing Image Label to save the image. When labelling your image, the type of modality will be indicated next to the image label on the study management screen, so you do not need to worry about including this in the image label.
6. For M-mode images, select M-mode (motion mode) from the keyboard. Use SV gate to narrow or widen the yellow bars and the cursor to align the bars over the desired location. Once placed correctly, push M-mode again. The placement of the bars may be adjusted while in M-mode.
7. For EKV imaging, select B-mode and ensure you are in the proper location. Then select EKV, adjust the settings, and push Scan. The EKV modality averages many B-mode images over several cardiac cycles.
8. To use color doppler, select B-mode, check that you are in the proper location, and then select Color. Select Update and move the cursor up, down, left, or right to achieve the desired box size, and select Update to lock it. You may then use the cursor to move the box to the desired location. Turning the Velocity knob up increases the velocity threshold and may decrease background signal.
9. To use pulsed wave doppler for measuring blood flow velocity, it is helpful to first be in color Doppler mode before pressing PW. Two yellow, angled lines will appear. The user must align the shorter, dotted line to be parallel to the anterior and posterior vessel walls by adjusting Beam Angle and turning the PW Angle knob. The dotted, yellow line will turn blue if the angle between the two lines is too large. Once this is aligned, press PW, and then adjust the Baseline, Velocity, and Doppler Gain controls to center and brighten the wave forms.
10. To use 3D mode, begin in B-mode and align the probe in the center of the structure you wish to image. Use respiratory gating and EKG-trigger for cardiovascular applications. Press 3D and set the desired scan distance and step size. Once the scan is complete, click Load into 3D to visualize your 3D data.
11. You may view your acquired images at any time during imaging by pressing Study Management from the top right of the two columns of buttons on the left side.
12. When you are finished acquiring images in a series, select Close Series from the study management screen. You may now open a new series within your study, if needed.

3. Data transfer and clean-up

1. To transfer data for analysis, select the studies or individual series you wish to copy from the study management screen.
2. Click Copy to from the top right corner on the study management screen. Select your desired file location, and press okay.
3. You may now remove the anmial and return it to its housing to recover.
4. To clean the ultrasound set-up, spray a paper towel with T-spray and wipe off the heated stage and rectal probe. Never spray the disinfectant directly on the stage.
5. The transducer should be wiped off with 70% ethanol on a paper towel before being placed back in the holder.
6. To turn off the air, shut off the O2 tank or filtered air source. You should see the air flow bead slowly drop to 0 on the vaporizer.
7. Once you are done with the system, click the power button on the study management screen in the top right corner and allow the monitor to turn off completely.
8. Turn the power button on the back of system off only after the monitor is completely off. You should hear the fan stop once it has been properly shut down.