Name: hand-held clinical photoacoustic imaging system for real-time non-invasive small animal imaging
Uploaded: 2017-10-16T15:00:00
Description: Watch this Scientific Journal Video about Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging at JoVE.com

The authors would like to acknowledge the financial support from the Tier 1 research grant funded by the Ministry of Education in Singapore (RG48/16: M4011617) and Tier 2 research grant funded by Ministry of Education in Singapore (ARC2/15: M4020238). The authors would like to acknowledge Dr. Rhonnie Austria Dienzo for his help with animal handling.

All animal experiments were performed according to the approved guidelines and regulations by the institutional Animal Care and Use committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263).
1. Handheld Real-time Clinical PA and US Imaging System
<ol>
	<li>The schematic of the handheld clinical PAI system33 is shown in Figure 1a. It consists of a optical parametric oscillator (OPO) laser pumped by...

<ol>
	<li>Yun, S. H., Kwok, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+in+diagnosis,+therapy+and+surgery.">Light in diagnosis, therapy and surgery.</a> Nat. Biomed. Eng. 1, 0008 (2017).</li><li>Tseng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+accuracy+of+preoperative+breast+MRI+for+breast+cancer.">Clinical accuracy of preoperative breast MRI for breast cancer.</a> J. Surg. Oncol. , (2017).</li><li>Baran, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+propagation-based+x-ray+phase-contrast+tomography+for+breast+cancer+imaging.">Optimization of propagation-based x-ray phase-contrast tomography for breast cancer imaging.</a> Phys. Med. Biol. 62 (6), 2315 (2017).</li><li>Huzarski, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+with+magnetic+resonance+imaging,+mammography+and+ultrasound+in+women+at+average+and+intermediate+risk+of+breast+cancer.">Screening with magnetic resonance imaging, mammography and ultrasound in women at average and intermediate risk of breast cancer.</a> Hered. Cancer Clin. Pract. 15 (1), 4 (2017).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+toward+preclinical+and+clinical+translation+of+photoacoustic+tomography:+a+review.">Recent advances toward preclinical and clinical translation of photoacoustic tomography: a review.</a> J. Biomed. Opt. 22 (4), 041006 (2017).</li><li>Wang, L. V., Yao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+photoacoustic+tomography+in+the+life+sciences.">A practical guide to photoacoustic tomography in the life sciences.</a> Nat. Methods. 13 (8), 627-638 (2016).</li><li>Wang, L. V., Gao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+microscopy+and+computed+tomography:+from+bench+to+bedside.">Photoacoustic microscopy and computed tomography: from bench to bedside.</a> Annu Rev Biomed Eng. 16, 155-185 (2014).</li><li>Beard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+photoacoustic+imaging.">Biomedical photoacoustic imaging.</a> Interface Focus. 1 (4), 602-631 (2011).</li><li>Yao, J., Wang, L. 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K., Moothanchery, M., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+laser+diode+photoacoustic+tomography+(PLD-PAT)+system+for+fast+in+vivo+imaging+of+small+animal+brain.">Pulsed laser diode photoacoustic tomography (PLD-PAT) system for fast in vivo imaging of small animal brain.</a> Proc Spie. , (2017).</li><li>Fakhrejahani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+report+on+the+first+prototype+of+a+photoacoustic+tomography+system+with+dual+illumination+for+breast+cancer+imaging.">Clinical report on the first prototype of a photoacoustic tomography system with dual illumination for breast cancer imaging.</a> PLoS One. 10 (10), e0139113 (2015).</li><li>Wang, L. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morbidity+after+sentinel+lymph+node+biopsy+in+primary+breast+cancer:+results+from+a+randomized+controlled+trial.">Morbidity after sentinel lymph node biopsy in primary breast cancer: results from a randomized controlled trial.</a> J. Clin. Oncol. 23 (19), 4312-4321 (2005).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handheld+array-based+photoacoustic+probe+for+guiding+needle+biopsy+of+sentinel+lymph+nodes.">Handheld array-based photoacoustic probe for guiding needle biopsy of sentinel lymph nodes.</a> J. Biomed. Opt. 15 (4), 046010 (2010).</li><li>Garcia-Uribe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-Modality+Photoacoustic+and+Ultrasound+Imaging+System+for+Noninvasive+Sentinel+Lymph+Node+Detection+in+Patients+with+Breast+Cancer.">Dual-Modality Photoacoustic and Ultrasound Imaging System for Noninvasive Sentinel Lymph Node Detection in Patients with Breast Cancer.</a> Sci. Rep. 5, 15748 (2015).</li><li>Kim, C., Song, K. H., Gao, F., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+Lymph+Nodes+and+Lymphatic+Vessels:+Noninvasive+Dual-Modality+in+Vivo+Mapping+by+Using+Indocyanine+Green+in+Rats-Volumetric+Spectroscopic+Photoacoustic+Imaging+and+Planar+Fluorescence+Imaging.">Sentinel Lymph Nodes and Lymphatic Vessels: Noninvasive Dual-Modality in Vivo Mapping by Using Indocyanine Green in Rats-Volumetric Spectroscopic Photoacoustic Imaging and Planar Fluorescence Imaging.</a> Radiology. 255 (2), 442-450 (2010).</li><li>Pan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+photoacoustic+detection+of+sentinel+lymph+nodes+with+gold+nanobeacons.">Near infrared photoacoustic detection of sentinel lymph nodes with gold nanobeacons.</a> Biomaterials. 31 (14), 4088-4093 (2010).</li><li>Song, K. H., Kim, C., Cobley, C. M., Xia, Y., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+gold+nanocages+as+a+new+class+of+tracers+for+photoacoustic+sentinel+lymph+node+mapping+on+a+rat+model.">Near-infrared gold nanocages as a new class of tracers for photoacoustic sentinel lymph node mapping on a rat model.</a> Nano Lett. 9 (1), 183-188 (2009).</li><li>Sivasubramanian, K., Periyasamy, V., Wen, K. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+light+delivery+through+fiber+bundle+in+photoacoustic+imaging+with+clinical+ultrasound+system:+Monte+Carlo+simulation+and+experimental+validation.">Optimizing light delivery through fiber bundle in photoacoustic imaging with clinical ultrasound system: Monte Carlo simulation and experimental validation.</a> J. Biomed. 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Currently the cost of screening, diagnosis, and treatment of cancer is very high. There are different imaging modalities which are being used for cancer screening and diagnosis. However, a lot of these imaging techniques have limitations including bulky machine size, invasive diagnosis, unfriendliness to patients, too expensive, requirement of ionizing radiation, or use of radioactive contrast agents. Therefore, an efficient, cost effective, real-time imaging and guiding system is much needed. Combined US and PA imaging ...

For the detection and staging of cancer, different imaging techniques are available. Some of the widely used imaging modalities are magnetic resonance imaging (MRI), X-Ray computed tomography (CT), X-Ray, ultrasound (US), positron emission tomography (PET), fluorescence imaging, etc.1,2,3,4. But, some of the existing imaging techniques are either invasive, have harmful radiation, or are slow, expensive, bulky, or unfriendly to patients. Thus, there is a constant need to develop new, fast, and cost-effective imaging techniques for diagnostics and therapy5.
Photoacoustic imaging (PAI) is an emerging imaging technique, which combines rich optical contrast with high ultrasonic resolution at a deeper imaging depth5,6,7,8,9. In PAI, a short laser pulse is used for tissue irradiation. The light gets absorbed by the tissue which leads to a small temperature rise. Due to thermoelastic expansion, pressure waves (in the form of acoustic waves) are generated within the tissue. The generated acoustic waves (also known as photoacoustic (PA) waves) are acquired with a wideband ultrasound transducer (UST) outside the tissue boundary. These acquired PA signals can be used to reconstruct PA images, revealing the structural and functional information inside the tissue. PAI has a wide array of applications, including: blood vessel imaging, sentinel lymph node imaging, brain vasculature imaging, tumor imaging, molecular imaging, etc.10,11,12,13,14,15 PAI has numerous applications because of its advantages, namely: deeper penetration depth, good spatial resolution, and high soft tissue contrast. The contrast in PAI can be endogenous from blood, melanin, etc. When the endogenous contrast is not strong enough, exogenous contrast agents like organic dyes, nanoparticles, quantum dots, etc.16,17,18,19,20,21 can be used for improving the contrast.
Although PAI has numerous benefits relative to other imaging techniques, clinical translation is still a very big challenge. The main limitations are the bulky nature of the lasers being used, most of the USTs used for data acquisition are not compatible with clinical US systems, and the non-availability of commercially available clinical US imaging systems which give access to raw channel data. Only recently, commercial clinical US machines with access to raw data have become available22. In this work, we aim to demonstrate the feasibility of PAI with a handheld set-up using a clinical US platform. We aim to demonstrate this by showing non-invasive imaging of sentinel lymph nodes (SLNs) in a small animal model.
Invasive breast tumors are one of the leading causes of cancer death among women. Diagnosing and staging breast cancer early is vital for deciding treatment strategies, which play an important role in the prognosis of the patient. For breast cancer staging sentinel lymph node biopsies (SLNB) are usually used23,24. SLN is the primary lymph node where the possibility of finding cancer cells is the highest due to metastasis. SLNBs involve injecting a dye or a radioactive tracer, followed by cutting open the area with a small incision, and then locating the SLN visually in case of dyes or with the help of a Geiger counter, in case of a radioactive tracer. After identification, a few SLN are removed for histopathological studies24,25. Positive SLNB indicates that the tumor has metastasized to nearby lymph nodes and maybe to other organs. Negative SLNB indicates that the probability of metastasis is negligible26. SLNB has numerous complications associated with it like arm numbness, lymphedema, etc.27 To eliminate the SLNB associated complications, a non-invasive imaging technique is needed.
For SLN mapping in small animals and humans, PA imaging has been explored extensively with the help of different contrast agents15,28,29,30,31,32. However, the systems used currently cannot be used in a clinical scenario as pointed out earlier. Another concern to be addressed is the surgical procedure involved in SLNB28. Adapting minimally invasive procedures for fine needle aspiration biopsy (FNAB) was needed to reduce the recovery time and the side effects of the patients. In this work, a clinical US system was used for combined US and PA imaging was used. For ease of use in clinical setup, a custom made handheld holder for housing optical fiber and UST was designed. Methylene blue (MB) was used for identifying and mapping SLNs. Additionally, to eliminate the complications associated with the SLNB surgery, non-invasive real-time needle tracking is also demonstrated.

<table>
	<tbody>
		<tr>
			<td>Q-switched Nd:YAG laser</td>
			<td>Continuum</td>
			<td>Surelite</td>
			<td>Pump laser</td>
		</tr>
		<tr>
			<td>Optical parametric oscillator</td>
			<td>Continuum</td>
			<td></td>
			<td>OPO laser</td>
		</tr>
		<tr>
			<td>Clinical ultrasound imaging system</td>
			<td>Alpinion</td>
			<td>E-CUBE 12R</td>
			<td>Dual modal ultrasound and photoacoustic imaging system</td>
		</tr>
		<tr>
			<td>Linear array ultrasound transducer</td>
			<td>Alpinion</td>
			<td>L3-12</td>
			<td>128 element linear array transducer with centre frequency of 8.5 MHz, fractional bandwidth of 95%,</td>
		</tr>
		<tr>
			<td>Bifurcated optical fiber</td>
			<td>CeramOptec</td>
			<td>Custom made</td>
			<td>To couple the light from the laser to the handheld fiber holder</td>
		</tr>
		<tr>
			<td>Lens</td>
			<td>Thorlabs</td>
			<td>LB1869</td>
			<td>Focus light from the laser to the optical fiber</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Acoustic coupling</td>
		</tr>
		<tr>
			<td>Image Processing software</td>
			<td>Mathworks</td>
			<td>Matlab</td>
			<td>Home made program using Matlab</td>
		</tr>
		<tr>
			<td>Anesthetic Machine</td>
			<td>medical plus pte ltd</td>
			<td>Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td>
			<td>Supplies oxygen and isoflurane to animal</td>
		</tr>
		<tr>
			<td>Pulse Oxymeter portable</td>
			<td>Medtronic</td>
			<td>PM10N with veterinary sensor</td>
			<td>Monitors the pulse oxymetry of the animal</td>
		</tr>
		<tr>
			<td>Animal distributor</td>
			<td>In Vivos Pte Ltd, Singapore</td>
			<td></td>
			<td>Animal distributor that supplies small animals for research purpose.</td>
		</tr>
		<tr>
			<td>Breathing mask</td>
			<td>Custom made</td>
			<td></td>
			<td>Used along with animal holder to supply anesthesia mixture to the animal</td>
		</tr>
		<tr>
			<td>chicken breast tissue</td>
			<td>Pasar</td>
			<td></td>
			<td>Used to add depth to mimic human imaging scenario</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td>BD Precisionglide</td>
			<td>23G,1 and half inch</td>
			<td>Used for realtime needle guidance</td>
		</tr>
		<tr>
			<td>Holder for the fiber optic cable</td>
			<td>Custom made</td>
			<td></td>
			<td>To hold the input end of the bifurcated cable</td>
		</tr>
		<tr>
			<td>Handheld probe</td>
			<td>Custom made 3D printed</td>
			<td></td>
			<td>With two slots for the two output ends of the optical fiber and one slot for the ultrasound transducer</td>
		</tr>
		<tr>
			<td>Methylene blue (10 mg/mL)</td>
			<td>Sterop</td>
			<td></td>
			<td>Contrast agent for PA imaging</td>
		</tr>
		<tr>
			<td>Laser tuning software</td>
			<td>Surelite OPO PLUS</td>
			<td>SLOPO</td>
			<td>Software to tune the wavelength of OPO laser</td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>SP05/M</td>
			<td>To detect the laser pulse to trigger the ultrasound system</td>
		</tr>
		<tr>
			<td>Photodiode bias module</td>
			<td>Thorlabs</td>
			<td>PBM42</td>
			<td>To amplify the photodiode signal to tigger ultrasound signal</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Reckitt Benckiser</td>
			<td>Veet</td>
			<td>Used to remove hair from the imaging area</td>
		</tr>
		<tr>
			<td>Laser power meter</td>
			<td>Ophir</td>
			<td>Starlite, p/n: 7Z01565</td>
			<td>Used to measure the laser power</td>
		</tr>
	</tbody>
</table>

hand-held clinical photoacoustic imaging system for real-time non-invasive small animal imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56649/56649fig1.jpg" /> 
Figure 1: System description. (a) Schematic representation of the PAI system with dual modal clinical US system. OPO - optical parametric oscillator, OF - optical fiber bundle, FH - fiber holder, USM- clinical US machine. The fiber holder integrates the UST and two output optical fiber bundle. The anesthesia machine supplying isoflurane and oxygen is used to keep the ...

Watch this Scientific Journal Video about Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging at JoVE.com

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

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.

Research

JoVE Journal

Bioengineering

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