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 a frequency doubled nanosecond pulsed Nd:YAG pump laser, a bifurcated optical fiber bundle (Figure 1b), a custom-made 3D printed handheld probe holder (Figure 1c)33, clinical dual modal US and PA system, and a clinically compatible linear array UST (see Table of Materials).</li>
	<li>Run the software provided by the manufacturer in the clinical US system by clicking on the icon for the software from the desktop.</li>
	<li>From the touch screen, select the &#39;research&#39; button to operate the US system in the research mode. Click on the combined US and PA imaging script from the list of scripts and click the run button for imaging in the combined mode.</li>
	<li>Synchronize the clinical US system with the laser using the laser trigger out or using a photodiode. 
	NOTE: Connect the fixed sync out from the laser to the US system sync in. Make sure to provide positive Transistor-transistor logic (TTL) signal as the sync signal. A photodiode signal can also be used for sync purpose. The US system sync in is connected to the photodiode detector using a photodiode bias module. Whenever the laser is ON, the photodiode gives a signal to trigger and sync both the laser and the US system. Perform this step every time.</li>
	<li>To turn on the laser, switch on AC power, and turn the key to the left on the laser controller. Start the laser after ensuring the repetition rate is 10 Hz (F10 will be displayed on the display) and the Q-switch delay is as low as 170 &#181;s to ensure low laser energy. To set the delay, press the select key till you see the delay value and increase it to 170. 
	NOTE: The laser takes approximately 20 min to warm up.</li>
	<li>Open the software interface on a computer and in the goto menu enter the wavelength as 675 nm and press the &#39;start&#39; button to set the wavelength at 675 nm. 
	NOTE: The laser can be tuned from 670 to 2,500 nm, however, it is unstable at 670 nm.</li>
	<li>Press the shutter button and turn the laser on using the switch to align the laser beam to the fiber input.</li>
	<li>Using a 1 inch (2.5 cm) diameter plano-convex lens of focal length 15 mm, focus the laser beam to the fiber bundle such that all the light falls on the fiber input end. 
	NOTE: The optical fiber has 1,600 small fibers bundled together. It is bifurcated in the middle with two rectangular output ends which have 800 optical fibers each. The 800 fibers are packed into an area of 0.1 x 4 cm, to match with the dimensions of the UST. The core diameter of each optical fiber is 185 &#181;m, with a numerical aperture of 0.22.</li>
	<li>Switch OFF the laser after alignment.</li>
	<li>From the 4 probe holders with different angles of illumination (0&#176;, 5&#176;, 10&#176;, and 15&#176;) choose the appropriate probe holder based on the application (depth of imaging, size of the object, shape of the object, and location of the object). 
	NOTE: The probe holders were designed and 3D printed in the lab. It has three slots, two for the bifurcated optical fibers and the central one for the UST. The dimension of the probe holder was based on the dimensions of the optical fiber and the UST. Monte Carlo simulations were done to study the light illumination required for imaging SLNs at higher depth. SNR was higher at lower depths for an illumination of 15&#176;33.</li>
	<li>Fit the bifurcated optical fiber into the 3D printed probe holder at a light illumination angle of 15&#730;.</li>
	<li>Insert the UST into the center slot of the holder. 
	NOTE: Figure 1d shows the photograph of the probe holder with the optical fiber and UST. The linear array UST has 128 array elements. The center frequency of the UST is 8.5 MHz and the fractional bandwidth is 95%. The length of the UST is 3.85 cm. However, the system has only 64 parallel data-acquisition hardware and requires two laser pulses to collect data from all the 128 elements. Therefore, the effective frame rate of the system is half the pulse repetition rate of the laser, which is 5 frames per second34.</li>
	<li>Adjust the distance between the UST and the fiber end to 1 cm by loosening the screws on the side and tighten it after adjusting the exact distance. 
	NOTE: The parameters are optimized for SLN imaging with simulation and phantom experiments33. The UST can be secured with the two screws on the probe holder. This gives the flexibility to vary the distance between the optical fiber and the UST.</li>
	<li>Switch ON the laser and make sure to obtain a rectangular laser beam spot in front of the UST.</li>
	<li>Switch OFF the laser. Increase the laser energy (by increasing the delay) to the desired value for the imaging purpose. 
	NOTE: Refer to the manual for the maximum delay that can be set for the Nd:YAG laser. The desired delay value for this system for SLN imaging was set to 210.</li>
</ol>
2. Resolution Characterization
<ol>
	<li>Take the commercially available chicken tissue slab and cut it into a 6 x 6 cm2 slab. Using a knife cut it into 0.5 cm thick slices.</li>
	<li>Place a point object, like a 23 G needle of diameter 0.6 mm, on top of chicken breast tissue.</li>
	<li>Switch ON the laser. 
	Caution: Safety goggles should be worn when working with the laser for the remainder of the protocol. An exception was made during the alignment process, since the laser energy was weak.</li>
	<li>Take PA images of the needle at different depths by stacking multiple chicken breast tissue slices of thickness 0.5 cm one-by-one up to 3 cm. Apply US gel between chicken breast tissue layers to improve US coupling.</li>
	<li>Save and store the beam-formed images as .mat file.</li>
	<li>Switch OFF the laser.</li>
	<li>Process the data with the in-house code using image processing software17. 
	NOTE: To determine the axial and lateral resolution, find the point-spread function from the normalized PA signals along the respective directions and fit them to a Gaussian distribution function17. Obtain the full width at half maximum.To obtain the point spread function, it is required to image a point. However, there is another way of obtaining a point spread function when imaging a point target is difficult (as in our case, for a very tiny point target the signal is quite small and therefore we use a slightly larger target). If the target is large, instead of directly getting the point spread function, one can obtain an edge spread function. Then by taking the first derivative of the edge spread function, one can get the point spread function. Therefore, it is not absolutely necessary to use a point target to calculate the resolution22.</li>
</ol>
3. Animal Preparation for SLN Imaging
NOTE: The handheld clinical imaging system described above was demonstrated for imaging small animal SLN. For experiments, 6- to 8-week-old healthy, female rats (NTac:Sprague Dawley, 220 &#177; 30 g) were procured. Female rats are used because the occurrence of breast cancer in male rats is less frequent. However, male rats can also be used for the studies. Additionally, in the literature, female rats are used more widely for the SLN imaging.
<ol>
	<li>Rat anesthetization
	<ol>
		<li>Before imaging, anesthetize the rat with the anesthesia solution, which contains a cocktail of ketamine (100 mg/mL), xylazine (20 mg/mL), and injectable saline at a proportion of 2:1:1. Add 0.2 mL of the cocktail per 100 g of animal weight to a sterile surgical 1 mL syringe with needle (27G, &#189; inch).</li>
		<li>Scruff the neck of the rat by hand and disinfect the right-lower quadrant of the abdomen with an alcohol swab. Inject anesthesia solution into the animal body.</li>
		<li>Ensure that the animal is anaesthetized by checking for reflex on toe pinch.</li>
	</ol>
	</li>
	<li>For SLN imaging, remove hair from the region of interest gently with commercially available hair removal cream. Use as much as required to cover the area completely. Remove the cream with a wet cotton swab after 3 - 5 min of application. To prevent eyes from dryness and accidental laser damage, apply artificial tear ointment.</li>
	<li>Place blue underpad on the table and position the animal sideways on it. Administer inhalation anesthesia through a nose cone (0.75% of isoflurane along with oxygen (1.2 L/min)) to maintain the animal under anesthesia during experiments. Clip the pulse oximeter to the hind paw of the rat to monitor the heart rate and peripheral oxygen saturation throughout experiments. 
	&#160;NOTE: Make sure the animal is warm using a heating pad approved for animal use.</li>
</ol>
4. In Vivo SLN Imaging of Rats
<ol>
	<li>Before imaging, apply 0.5 to 1 mL of US gel on the skin using a syringe and spread it well using an applicator. Place a 0.5 cm thick chicken breast tissue slice of size 6 cm x 6 cm on the imaging area and apply more US gel on the chicken tissue to improve coupling. 
	NOTE:&#160;LNs are located under the skin (within 2-3 mm) in rats. In humans, LNs are at a depth of 1 cm. Hence, chicken tissue is placed in the imaging area of rat to mimic the human imaging scenario. Alternatively, tissue mimicking phantoms can be used in place of chicken tissue.</li>
	<li>Switch on the laser. Place the handheld probe holder on top of the chicken tissue and scan it (move the holder right to left) in the combined US and PA mode. 
	NOTE: The area of the laser beam is approximately 3 cm2 and fluence is calculated to be approximately 8 mJ/cm2, which is less than the American National Standards Institute (ANSI) safety limit (20 mJ/cm2)35. The imaging depth is set to 2 cm in the clinical US system. Safety goggles should be worn at all times when the laser is switched on.</li>
	<li>Set the imaging depth to 2 cm for PA imaging.</li>
	<li>Obtain a control image of the region of interest, above the front leg in the thoracic area, by moving the handheld probe from side to side before injection of the contrast agent. 
	NOTE: All the data is saved in the beam-formed data type for further processing.</li>
	<li>Inject 0.2 mL of the contrast agent, (i.e., MB (10 mg/mL)) in the forepaw of the animal and massage it well for 2 min to facilitate contrast agent movement to the lymph nodes through the lymph vessels.</li>
	<li>Scan after 5 min with the handheld probe along the chicken tissue to locate the SLN with the help of the PA signal. 
	NOTE: All the frames are saved in the beam-formed data type.</li>
	<li>Click on the &#39;freeze&#39; button from the controls on the US system and click the &#39;export selected frames&#39; button from the touch screen monitor to export the recorded data. 
	NOTE: Data can be stored in different formats namely, beam formed, scan converted, channel, and I.Q.</li>
	<li>Add 2 more layers of 0.5 cm thick chicken tissue slices on top of the animal one by one and locate the SLN to demonstrate the feasibility of depth imaging up to 1.5 cm.</li>
	<li>After imaging, remove all the chicken tissue slices</li>
	<li>Switch off the laser.</li>
</ol>
5. PA Spectroscopy of SLN
<ol>
	<li>Place a 0.5 cm thick chicken breast tissue slice on the rat.</li>
	<li>Set the wavelength of the laser as 670 nm using the software.</li>
	<li>Switch on the laser. Scan with the probe along the chicken breast tissue on the area to locate the SLN with the help of the PA signal.</li>
	<li>Hold the probe stable after identifying the SLN.</li>
	<li>On the laser tuning software provided, enter the wavelength as 800 nm in the laser software, set the speed as 10 nm/s, and click the &#39;start&#39; button. 
	NOTE: This will vary the wavelength from 670 nm to 800 nm at a speed of 10 nm/s. The wavelength range to be varied depends on the absorption spectrum of the contrast agent used. MB has a sharp peak around 670 nm.</li>
	<li>Observe the change in PA signal with wavelength change.</li>
	<li>Switch off the laser.</li>
	<li>Remove the chicken tissue slice.</li>
</ol>
6. Real-time Needle Tracking Using PAI
<ol>
	<li>Place a 0.5 cm thick chicken breast tissue slice on the animal. Set the wavelength to 675 nm.</li>
	<li>Switch on the laser. Move the probe to locate and identify SLN on screen with the help of the PA signal.</li>
	<li>Real-time needle tracking
	<ol>
		<li>Inject a 23 G needle, of dimensions 0.6 x 32 mm2, parallel to the UST through the chicken tissue into the animal to reach the SLN, while guiding it in real-time on the clinical US system monitor.</li>
	</ol>
	</li>
	<li>After experiments, turn the laser off. Remove the chicken tissue and pulse oximeter from the animal and move the animal to the work bench. Clean the ultrasound gel on the rat with cotton wipes.</li>
	<li>Place the animal on its bedding and monitor it until it regains consciousness.</li>
	<li>Return the animal to its cage after its behavior is normal.</li>
</ol>

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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. Opt. 22 (4), 041008 (2017).</li><li>Sivasubramanian, 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=High+frame+rate+photoacoustic+imaging+at+7000+frames+per+second+using+clinical+ultrasound+system.">High frame rate photoacoustic imaging at 7000 frames per second using clinical ultrasound system.</a> Biomed. Opt. Express. 7 (2), 312-323 (2016).</li><li>Laser Institute of America. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+National+Standard+for+Safe+Use+of+Lasers.">American National Standard for Safe Use of Lasers.</a> ANSI Standard Z136.1-2007. , (2007).</li><li>Chapman, G. A., Johnson, D., Bodenham, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+needle+position+using+ultrasonography.">Visualisation of needle position using ultrasonography.</a> Anaesthesia. 61 (2), 148-158 (2006).</li><li>Daoudi, K., 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+probe+integrating+laser+diode+and+ultrasound+transducer+array+for+ultrasound/photoacoustic+dual+modality+imaging.">Handheld probe integrating laser diode and ultrasound transducer array for ultrasound/photoacoustic dual modality imaging.</a> Opt. Express. 22 (21), 26365-26374 (2014).</li></ol>

The authors have no relevant financial interests in the manuscript and no other potential conflicts of interest to disclose.

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 ...

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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, ...

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Bioengineering

11.4K Views.  Nanyang Technological University. Een klinische handheld photoacoustic imaging systeem zullen worden gedemonstreerd voor real-time niet-invasieve kleine dierlijke imaging.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neuroscience

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

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

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