The overall goal of this procedure is to demonstrate non-invasive handheld photoacoustic imaging at the sentinel lymph node using a clinical ultrasound system and to illustrate real-time photoacoustic needle guidance. Photoacoustic imaging, hybrid imaging modality combining light and ultrasound provides high optical contrast, high resolution, deep tissue imaging in vivo. However, translating for the crystal imaging into clinical application is challenging.

This work presents a hand-held, real time, photoacoustic imaging system by integrating light illumination with the clinical ultrasound platform. Demonstrating the procedure will be Kathyayini Sivasubramanian a PHD student and Vijitha Periyasamy a researcher from our laboratory. Turn on the laser by switching on the AC power and turning the key on the laser controller to the left.

To set the Q-switch delay, press the select key until you see the delay value and increase it to 170 microseconds. Then, set the repetition rate to 10 hertz to ensure low laser energy and start the laser. Open the laser wavelength tuning software.

Select the Go To menu, enter the wavelength at 675 nano meters and press the start button to set the wavelength to 675 nano meters. Then press the shutter button and turn the laser on using the switch. Use a one inch diameter planoconvex lens with a focal length of 15 millimeters to focus the laser beam to the fiber bundle so that all the light falls on the fiber input end.

Once aligned, switch off the laser. Choose the appropriately angled probe holder based on the desired depth of imaging, the size of the object, the shape of the object, and the object's location. For this experiment, fit the bifurcated optical fiber into the side slot of the probe holder at a light illumination angle of 15 degrees.

Loosen the screws on the side and adjust the distance between the transducer and the fiber's end to one centimeter. After adjusting it to the exact distance tighten the screws. Then, switch the laser back on and make sure to obtain a rectangular laser beam spot in front of the transducer.

Once the rectangular beam can be seen in front of the transducer, increase the delay value to 210 microseconds. Next, turn on the clinical ultrasound system and open the imaging software provided by the manufacturer. From the touch screen, select the research button to Operate and Research mode, then click on the combined ultrasound and photoacoustic imaging script and click the run button for imaging in the combined mode.

To begin, place a 23 gauge needle on top of a piece of raw chicken breast tissue. Apply ultrasound gel and then add a slice of chicken tissue on top of it. With the laser on, take photoacoustic images of the needle.

Then, apply ultrasound gel on top of the breast tissue and add a second piece of tissue. Take another photoacoustic image and continue to apply the ultrasound gel and stack the breast tissue one-by-one up to three centimeters thick imaging every time chicken tissue layer is added. Ensure that the animal is anesthetized by checking for reflex following a toe pinch.

Then, remove hair from the region of interest using a hair removal cream and apply an artificial tear ointment to prevent the eyes from dryness and accidental laser damage. Next, place a blue under pad on the table and position the animal sideways on it. Affix the nose cone to administer inhalation anesthesia and clip a pulse oximeter to the hind paw to maintain and monitor the animal during the experiments.

Prior to imaging, apply one to two milliliters of ultrasound gel on the skin, using a syringe, and spread it well using an applicator. Then, place a 0.5 centimeter thick slice of chicken breast tissue on the imaging area and apply more u.s. gel on the chicken tissue to improve coupling.

At this point, switch on the laser and place the hand held probe holder on top of the chicken tissue. Move the holder right to left in the combined ultrasound and photoacoustic modes. Press the freeze button on the ultrasound system and then press the export selected frames button to export the control images prior to injecting the contrast agent.

Then, inject 0.2 milliliters of the methylene blue contrast agent into the fore paw of the animal and massage it well for two minutes to facilitate the move of the contrast agent to the lymph nodes through the lymph vessels. After, five minutes, scan the area again with the handheld probe along the chicken tissue to locate the sentinel lymph nodes with the help of the photoacoustic signal. Once identified, save the frames.

Add two more layers of 0.5 centimeter thick chicken tissue slices on top of the animal one by one and locate and save the sentinel lymph node images up to 1.5 centimeters into the tissue. After imaging, switch off the laser, remove all of the chicken tissue slices and prepare the rat for photoacoustic spectroscopy. Place a 0.5 centimeter thick slab of chicken breast tissue onto the rat.

Then, change the wavelength of the laser to 670 nanometers and switch on the laser. Scan with the probe along the chicken breast tissue to locate the sentinel lymph node with the help of the photoacoustic signal. Hold the probe stable after identifying the sentinel lymph node.

In the laser tuning software, enter the wavelength as 800 nanometers, set the speed to 10 nanometers per second and click the start button. This will vary the wavelength from 670 nanometers to 800 nanometers at a speed of 10 nanometers per second. Observe the change in the photoacoustic signal from the sentinel lymph node on the monitor of the ultrasound system.

To track needles in real time, set the wavelength to 675 nanometers then move the probe to locate and identify a sentinel lymph node on the screen. Inject a 23 gauge needle into the rat parallel to the ultrasound transducer. Guide it in real time as it passes through the chicken tissue and into the animal to reach the sentinel lymph node.

Once finished, turn off the laser. Remove the chicken tissue and pulse oximeter from the animal and move the animal to the work bench. Then, clean the ultrasound gel from the rat with cotton wipes.

Once clean, place the animal on its bedding. Monitor it until it regains consciousness and return it to it's cage after it's behavior is normal. Using the method shown in this video, the full width at half maximum was calculated from the needle images at various depths.

The axial resolution was calculated to be 207 micrometers and the lateral resolution, limited by the ultrasound transducer, was 351 micrometers. Before adding the contrast agent, methylene blue, the sentinel lymph node blends in with the other tissue. Following injection, the sentinel lymph node can be very easily identified due to the strong photoacoustic signal from the accumulated methylene blue.

Photoacoustic imaging is also useful for needle guidance as ultrasound alone provides poor contrast. With photoacoustic imaging, the contrast obtained from the needle is very high and can be easily monitored and tracked in vivo. Once the needle reaches the sentinel lymph node, a small portion of the lymph tissue can be taken out for further histological examination.

The visibility of the photoacoustic needle guidance is demonstrated with the clinical ultrasound system. Once mastered, the technique of photoacoustic sentinel lymph node and needle imaging can be done within an hour if it performed properly. Laser light delivery integrated with clinical ultrasound system enables hand held photoacoustic imaging.

This will help in the translation of photoacoustic imaging into the clinic. Potential applications of handheld photoacoustic imaging system incudes imaging of blood vessels, tumor, sentinel lymph node, urinary bladder, and circulating tumor cells.