This article discusses the use of ultra-high-frequency ultrasound (UHFUS) for high-resolution imaging of superficial structures in live animals, specifically focusing on inguinal lymph nodes in mice. The methodology involves careful preparation of the animal and the imaging setup to ensure accurate and reliable results.
Ultra-high-frequency ultrasound, or UHFUS, facilitates high spatial resolution imaging of superficial structures in a live animal.
To begin, place an anesthetized mouse in the supine position on the heated platform of an imaging station to regulate its body temperature. Insert a probe to monitor the body temperature.
Next, coat the limbs with conductive paste and secure them to the electrocardiogram electrodes embedded in the platform to monitor the physiological parameters. Remove the hair from the mouse inguinal region. Apply the acoustic coupling medium to provide an efficient path for ultrasound propagation. Position the UHFUS transducer over the inguinal region.
During the scan, the transducer emits ultra-high-frequency ultrasound pulses with reduced penetration depth into the superficial inguinal lymph node. Based on the lymph node density and size, a fraction of the ultrasound pulses with varying amplitudes is reflected and received by the transducer, converting these signals into electrical signals.
Further, the computer reconstructs them to obtain a two-dimensional high-resolution ultrasound image, wherein the brightness of each dot is determined by the amplitude of the reflected signals. Scan the entire region of the lymph node by linear movement of the transducer to obtain a sequence of short-axis images.
Using suitable software, post process the acquired ultrasound images to create a 3D ultrasound image of the inguinal lymph node and measure its volume.
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.
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