Mouse Inguinal Lymph Node Volume Analysis: An Ultra-high-frequency Ultrasound Imaging Technique to Determine the Volume of Inguinal Lymph Node in Mouse Model

After confirming anesthesia, transfer the animal to a heated board of the ultra-high frequency ultrasound imaging station, holding the animal in a supine position. Adjust the board temperature using a rectal probe lubricated with petroleum jelly to maintain the mouse's body temperature in the physiological range.

Coat the fore and hind paws with conductive paste, and tape them to the ECG plate electrodes embedded in the board. Check that the physiological parameters are correctly acquired and displayed.

Remove hair from both inguinal areas by applying a depilatory agent, and coat them with an acoustic coupling medium. Then, clamp the ultra-high frequency ultrasound linear probe into a specialized 3D motor embedded in the imaging station, allowing automated and stepwise movement of the probe.

Properly orient and adjust the position of the ultrasound probe to obtain short-axis images of the inguinal lymph node, and place the region of interest in the focal zone.

Set the scan distance between two and five millimeters, step size to 44 micrometers, with an outcome of 46 to 114 scan steps per lymph node slice, and an acquisition time of 1 to 3 minutes per animal.

Scan the entire volume of the inguinal lymph node as a sequence of 2D B-mode images, acquiring images at multiple levels by linear movement of the transducer with step sizes on a micrometer scale, to generate 3D data in terms of respiration and cardiac-gated gated cine loops.

After imaging, digitally store the acquired images in raw format for further offline analyses.