This work was supported in part by a grant from the pilot studies program of the University at Buffalo Clinical and Translational Research Center and the Buffalo Translational Consortium, a grant from Roswell Park Alliance Foundation, startup funds from the University at Buffalo, IT Consilience Creative Program of MKE, and NIPA (C1515-1121-0003) and NRF grant of MEST (2012-0009249).

1. Deep Reflection Mode Photoacoustic Cystography (PAC) System
<ol>
	<li>System configuration17, 18
	<ol>
		<li>A Q-switched Nd:YAG laser (SLII-10; Continuum; 532 nm) pumps a wavelength-tunable laser (Surelite OPO PLUS; Continuum; wavelength tuning range: 680 to 2,500 nm).</li>
		<li>The pulse duration of each laser shot is ~5 nsec, and the laser repetition rate is 10 Hz.</li>
		<li>The wavelength depends on the optical absorption peak of the used contrast agent. If methylene blue serves as the contrast agent, an optical wavelength of 667 nm is used, where the peak absorption is. Plasmonic gold nanostructures can have tunable localized surface plasmon resonance at the NIR spectral region, based on their physical and chemical properties. Further, the broad absorption spectrum of single walled carbon nanotubes can provide a wide option for wavelength selection.</li>
		<li>Light coming out of the tunable laser is delivered to a spherical conical lens through right angle prisms (PS908, Thorlabs).</li>
		<li>A donut-shaped light beam pattern is generated after the light passes through the spherical conical lens. The home-made conical lens is made from a BK7 lens and the cone angle is 152&deg;. The diameter of the lens is 2.5 cm.</li>
		<li>The diverging donut-shaped light beam is redirected via an optical condenser, made of a transparent acrylic sheet. The diameters of the top and bottom surfaces are 6.1 and 4.8 cm, respectively. The thickness of the condenser is 2.5 cm. The redirected ring-shaped light beam forms a donut-shaped with a dark center on the tissue surface.</li>
		<li>Utilize a small water container to boost acoustic coupling. The water container has a bottom opening wrapped with a clear thin polyethylene film which is optically and acoustically transparent. Small animals are positioned under the water container.</li>
		<li>The generated PA waves are detected by a spherically focused ultrasound transducer (V308; Olympus NDT; 5 MHz central frequency), which is mounted in the middle of the optical condenser. The element diameter and focal length of the transducer are 1.9 and 2.5 cm, respectively. Thus, the f-number of the transducer is ~1.3.</li>
		<li>The transverse and axial resolutions are 590 and 144 &mu;m, respectively.</li>
		<li>The detected PA waves are first amplified by a broadband ultrasonic pulser/receiver (5072PR; Olympus NDT; 35 MHz bandwidth and 59 dB rf gain), and then acquired by an oscilloscope (TDS5054; Tektronix).</li>
		<li>Obtain one-dimensional time-resolved images (called as A-line) by measuring the times of arrival of the PA waves. Currently, the speed of PA waves is assumed to be at 1,480 m/sec over the entire image acquisition. Two- (called as B-scans) and three-dimensional PA images can be acquired by mechanically moving the linear raster scanning stage (XY6060; Danaher Dover).</li>
		<li>The imaging time is ~25 min for one volumetric single-wavelength PA image with a field of view (FOV) of 2.5 x 2.4x 1.5 cm3 in the x-y-z planes. We acquired 125 samples along the x direction with a step size of 0.2 mm, 60 samples along the y direction with a step size of 0.4 mm. 500 data points with a 50-MHz sampling rate were obtained along the z direction. The mechanical scanning is controlled by a home-made NI LabVIEW software system.</li>
		<li>The volumetric data is represented by maximum amplitude projection (MAP) using a MathWorks MATLAB software system.</li>
	</ol>
	</li>
	<li>System alignment
	<ol>
		<li>After the spherical conical lens, make sure that the beam pattern is a perfect ring shape. If the donut-shaped beam pattern is not properly generated, the PA signals originated from the skin surface are dominant. Thus, it is difficult to achieve deep tissue imaging.</li>
		<li>The line-shaped light focus in water should be coaxially aligned with the ultrasound focal zone. If those are not coaxially aligned, the system suffers from low signal-to-noise ratio.</li>
		<li>The irradiated laser pulse energy on the skin surface is varied from ~1 - 2 mJ/cm2 when the wavelength is tuned from 680 to 1,000 nm, respectively. These laser pulse energies are much lower than the American National Standards Institute safety limit, varying from 20 to 80 mJ/cm2 over the spectral region, respectively.</li>
	</ol>
	</li>
</ol>
2. In vivo and Ex vivo Imaging Procedures
<ol>
	<li>Animal preparation
	<ol>
		<li>Use female Spraque-Dawley rats with a weight of 200 - 250 g in all PA imaging experiments.</li>
		<li>To begin with, anesthetize the rat by intraperitoneal injection of a mixture of ketamine (85 mg/kg of body weight) and xylazine (15 mg/kg).</li>
		<li>Depilate the hairs in the abdominal area.</li>
		<li>Position the rat atop of a custom-made animal holder.</li>
		<li>Coat a 22-gauge catheter with lubricant to improve the catheter insertion.</li>
		<li>Hold the catheter vertically over the urethral opening. Next, insert the distal end of the catheter, horizontally, into the urethra until the hub of the catheter finally reaches the opening.</li>
		<li>Urine in the bladder will void via the catheter.</li>
	</ol>
	</li>
	<li>In vivo PAC imaging
	<ol>
		<li>Position the rat, located on top of the animal holder, below the water container in the PAC system.</li>
		<li>Apply ultrasound gel (Sonotech) between the animal skin surface and plastic membrane to improve acoustic coupling.</li>
		<li>Fully anesthetize the rat using vaporized isoflurane (1 L/min of oxygen and 0.75% isoflurane) during the in vivo PA imaging experiments.</li>
		<li>Obtain a control PA image prior to injection of contrast agents.</li>
		<li>Introduce an aqueous solution of methylene blue (0.8 &mu;l/g body weight with a concentration of 30 mM), gold nanocages (1.2 &mu;l/g body weight with a concentration of 2 nM), or single walled carbon nanotubes (0.8 &mu;l/g body weight with a concentration of 0.3 &mu;M) to the bladder via the catheter. Use a 1-ml-syringe with a 22-gauge catheter.</li>
		<li>Acquire a series of PA images.</li>
	</ol>
	</li>
	<li>Ex vivo PAC imaging
	<ol>
		<li>Sacrifice the rat after in vivo PAC imaging, by injecting an overdose of pentobarbital.</li>
		<li>Remove the two major organs, bladders and kidneys, to investigate biodistribution, and on a glass plate.</li>
		<li>Position the glass plate below the water container in the PAC system.</li>
		<li>Apply ultrasound gel (Sonotech) between the excised organs and plastic membrane to improve acoustic coupling.</li>
		<li>Acquire PA images.</li>
		<li>Photoacoustically image the organs removed from a healthy rat as a control.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Riccabona, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystography+in+infants+and+children:+a+critical+appraisal+of+the+many+forms+with+special+regard+to+voiding+cystourethrography.">Cystography in infants and children: a critical appraisal of the many forms with special regard to voiding cystourethrography.</a> Eur. Radiol. 12 (12), 2910-2918 (2002).</li><li>Khattar, N., Dorairajan, L. N., Kumar, S., Pal, B. C., Elangovan, S., Nayak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+obstructive+megaureter+causing+contralateral+ureteral+obstruction+and+hydronephrosis:+a+first-time+report.">Giant obstructive megaureter causing contralateral ureteral obstruction and hydronephrosis: a first-time report.</a> Urology. 74 (6), 1306-1308 (2009).</li><li>Lim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicoureteral+reflux+and+urinary+tract+infection:+evolving+practices+and+current+controversies+in+pediatric+imaging.">Vesicoureteral reflux and urinary tract infection: evolving practices and current controversies in pediatric imaging.</a> AJR Am. J. Roentgenol. 192 (5), 1197-1208 (2009).</li><li>Scardapane, A., Pagliarulo, V., Ianora, A. A., Pagliarulo, A., Angelelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast-enhanced+multislice+pneumo-CT-cystography+in+the+evaluation+of+urinary+bladder+neoplasms.">Contrast-enhanced multislice pneumo-CT-cystography in the evaluation of urinary bladder neoplasms.</a> Eur. J. Radiol. 66 (2), 246-252 (2008).</li><li>Verpoorten, C., Buyse, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurogenic+bladder:+medical+treatment.">The neurogenic bladder: medical treatment.</a> Pediatr. Nephrol. 23 (5), 717-725 (2008).</li><li>Ron, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let's+not+relive+the+past:+a+review+of+cancer+risk+after+diagnostic+or+therapeutic+irradiation.">Let's not relive the past: a review of cancer risk after diagnostic or therapeutic irradiation.</a> Pediatr. Radiol. 32 (10), 739-744 (2002).</li><li>Berrington De Gonzalez, A., Darby, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+cancer+from+diagnostic+X-rays:+estimates+for+the+UK+and+14+other+countries.">Risk of cancer from diagnostic X-rays: estimates for the UK and 14 other countries.</a> Lancet. 363 (9406), 345-351 (2004).</li><li>Kim, C., Favazza, C., 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=In+vivo+photoacoustic+tomography+of+chemicals:+high-resolution+functional+and+molecular+optical+imaging+at+new+depths.">In vivo photoacoustic tomography of chemicals: high-resolution functional and molecular optical imaging at new depths.</a> Chem. Rev. 110 (5), 2756-2782 (2010).</li><li>Ke, H., Erpelding, T. N., Jankovic, L., Liu, C., 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=Performance+characterization+of+an+integrated+ultrasound,+photoacoustic,+and+thermoacoustic+imaging+system.">Performance characterization of an integrated ultrasound, photoacoustic, and thermoacoustic imaging system.</a> J. Biomed. Opt. 17 (5), 056010 (2012).</li><li>Akers, W. J., Kim, C., Berezin, , 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=Noninvasive+Photoacoustic+and+Fluorescence+Sentinel+Lymph+Node+Identification+using+Dye-Loaded+Perfluorocarbon+Nanoparticles.">Noninvasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-Loaded Perfluorocarbon Nanoparticles.</a> Acs Nano. 5 (1), 173-182 (2011).</li><li>Jiao, S. L., Jiang, M. S., Hu, 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=Photoacoustic+ophthalmoscopy+for+in+vivo+retinal+imaging.">Photoacoustic ophthalmoscopy for in vivo retinal imaging.</a> Opt. Express. 18 (4), 3967-3972 (2010).</li><li>Kim, C., Cho, E. C., Chen, 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=In+vivo+molecular+photoacoustic+tomography+of+melanomas+targeted+by+bioconjugated+gold+nanocages.">In vivo molecular photoacoustic tomography of melanomas targeted by bioconjugated gold nanocages.</a> Acs Nano. 4 (8), 4559-4564 (2010).</li><li>Kim, C., Song, H. M., Cai, X., Yao, J., Wei, A., 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=In+vivo+photoacoustic+mapping+of+lymphatic+systems+with+plasmon-resonant+nanostars.">In vivo photoacoustic mapping of lymphatic systems with plasmon-resonant nanostars.</a> J. Mater. Chem. 21 (9), 2841-2844 (2011).</li><li>Wang, X., Pang, Y., Ku, G., Xie, X., Stoica, G., 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=Noninvasive+laser-induced+photoacoustic+tomography+for+structural+and+functional+in+vivo+imaging+of+the+brain.">Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain.</a> Nat. Biotechnol. 21 (7), 803-806 (2003).</li><li>Xie, Z., Roberts, W., Carson, P., Liu, X., Tao, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+bladder+microvasculature+with+high-resolution+photoacoustic+imaging.">Evaluation of bladder microvasculature with high-resolution photoacoustic imaging.</a> Opt. Lett. 36 (24), 4815-4817 (2011).</li><li>Zhang, H. F., Maslov, K., Stoica, G., 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=Functional+photoacoustic+microscopy+for+high-resolution+and+noninvasive+in+vivo+imaging.">Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging.</a> Nat. Biotechnol. 24 (7), 848-851 (2006).</li><li>Kim, C., Jeon, M., 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=Nonionizing+photoacoustic+cystography+in+vivo.">Nonionizing photoacoustic cystography in vivo.</a> Opt. Lett. 36 (18), 3599-3601 (2011).</li><li>Homan, K., Kim, S., Chen, Y. S., Wang, B., Mallidi, S., Emelianov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+of+molecular+photoacoustic+imaging+at+1064+nm+wavelength.">Prospects of molecular photoacoustic imaging at 1064 nm wavelength.</a> Opt. Lett. 35 (15), 2663-2665 (2010).</li><li>Chang, S. L., Shortliffe, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+urinary+tract+infections.">Pediatric urinary tract infections.</a> Pediatr. Clin. N. Am. 53 (3), 379 (2006).</li><li>Stratton, K. L., Pope, J. C., Adams, M. C., Brock, J. W., Thomas, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Implications of Ionizing Radiation in the Pediatric Urology. 183 (6), 2137-2142 (2010).</li><li>Ermilov, S. A., Khamapirad, T., Conjusteau, 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=Laser+optoacoustic+imaging+system+for+detection+of+breast+cancer.">Laser optoacoustic imaging system for detection of breast cancer.</a> J. Biomed. Opt. 14 (2), 024007 (2009).</li><li>Erpelding, T. N., Kim, C., Pramanik, M., 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=Sentinel+lymph+nodes+in+the+rat:+noninvasive+photoacoustic+and+US+imaging+with+a+clinical+US+system.">Sentinel lymph nodes in the rat: noninvasive photoacoustic and US imaging with a clinical US system.</a> Radiology. 256 (1), 102-110 (2010).</li><li>Kim, C., Erpelding, T. N., Jankovic, L., 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=Performance+benchmarks+of+an+array-based+hand-held+photoacoustic+probe+adapted+from+a+clinical+ultrasound+system+for+non-invasive+sentinel+lymph+node+imaging.">Performance benchmarks of an array-based hand-held photoacoustic probe adapted from a clinical ultrasound system for non-invasive sentinel lymph node imaging.</a> Philos. Transact. A. Math Phys. Eng. Sci. 369 (1955), 4644-4650 (1955).</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>Kruger, R. A., Kiser, W. L., Reinecke, D. R., Kruger, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoacoustic+computed+tomography+using+a+conventional+linear+transducer+array.">Thermoacoustic computed tomography using a conventional linear transducer array.</a> Medical Physics. 30 (5), 856-860 (2003).</li><li>Kruger, R. A., Lam, R. B., Reinecke, D. R., Del Rio, S. P., Doyle, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+angiography+of+the+breast.">Photoacoustic angiography of the breast.</a> Med. Phys. 37 (11), 6096-6100 (2010).</li><li>Manohar, S., Kharine, A., Van Hespen, J. C., Steenbergen, W., Van Leeuwen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Twente+Photoacoustic+Mammoscope:+system+overview+and+performance.">The Twente Photoacoustic Mammoscope: system overview and performance.</a> Phys. Med. Biol. 50 (11), 2543-2557 (2005).</li></ol>

All animal experiments were in compliance with the State University of New York at Buffalo Institutional Animal Care and Use Committee. The authors have nothing to disclose.

In conclusion, we have shown the possibility of nonionizing PAC using nontoxic optical absorbers in a rat model in vivo. We have successfully imaged a rat bladder filled with optical absorbents using our nonionizing and noninvasive PAC system. Two critical safety issues have been resolved in our approach: (1) the use of nonionizing radiation for cystographic applications and (2) no accumulation of contrast agents in the body.
Our clinical interest includes monitoring vesicoureteral re...

X-ray cystography1 is an imaging process to identify bladder-related diseases such as bladder cancer, vesicoureteral reflux, blockage of ureters, neurogenic bladder, etc.2-5 Typically, urines are voided and a radio-opaque agent is injected through a catheter. Then, fluoroscopic X-ray images are acquired to delineate urinary bladders. However, the key safety issue is that harmful ionizing radiation is used in this procedure. The percentage of cumulative cancer risk to age 75 years owing to diagnostic X-rays ranges from 0.6 to 1.8%.6 In addition, the carcinogenic threat is significant in pediatric patients. A UK study showed that among 9 major internal organs, the average annual radiation dose from diagnostic X-rays was highest in the bladders in female children less than 4 and second highest in male children less than 4.7 This indicates that the bladder cancer risk is most significant in pediatric patients. Although pediatric radiologists endeavor to reduce the radiation exposure rate as low as reasonably achievable, ionizing radiation cannot be completely excluded. Therefore, the limitation creates a need for a completely radiation-free, sensitive, cost-effective, and high-resolution imaging modality with nonradioactive contrast agents in cystography.
Recently, photoacoustic tomography (PAT) has become a premier biomedical imaging modality because PAT can provide strong optical absorption contrasts and a high ultrasonic spatial resolution in biological tissues.8 The principle of PAT is that ultrasonic waves are induced due to thermoelastic expansion of a target followed by light absorption. By detecting time-resolved acoustic waves travelling via a medium, two- or three-dimensional photoacoustic (PA) images are formed. Because ultrasound (US) is much less scattered in tissues compared to light (typically two or three orders of magnitude), the imaging depth of PAT can reach up to ~8 cm in tissues, while the spatial resolution is maintained to 1/200 of the imaging depth.9 The key advantages of PAT for the cystographic application include: (1) PAT is completely free from ionizing radiation. (2) ClinicalUSimaging systems can be easily adapted to supply dual-modal PA and US imaging capabilities. Thus, the dual-modal PA/US imaging system can be relatively portable, cost-effective, and fast, which are key criteria for fast clinical translation. Using both endogenous and exogenous contrasts, PAT has provided high-resolution morphological, functional, and molecular imaging of tissues to study tumor physiopathology, brain hemodynamics, internal organs, ophthalmology, angiography, and etc. 10-16
In this article, we demonstrate the experimental protocols of nonionizing photoacoustic cystography (PAC) using near-infrared (NIR) optical absorbents (i.e. methylene blue, gold nanocages, or single walled carbon nanotubes) as nontoxic optical-turbid tracers. Rat bladders filled with the contrast agents were photoacoustically and spectroscopically delineated in vivo. No agents persistently accumulated in the bladders and kidneys of the rats. Thus, long-term toxicity which may be caused by agent accumulation can be excluded. This result implies that PAC with combination of the optical absorbers can potentially be a truly harmless cystographic modality for pediatric patients. The system configuration, system alignment, and in vivo / ex vivo imaging procedures are discussed in this article.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Q-switched Nd:YAG laser</td>
			<td>Continuum</td>
			<td>SLII-10</td>
			<td>pump laser</td>
		</tr>
		<tr>
			<td>OPO laser</td>
			<td>Continuum</td>
			<td>Surelite OPO PLUS</td>
			<td>tunable laser</td>
		</tr>
		<tr>
			<td>Prisms</td>
			<td>Thorlabs</td>
			<td>PS908</td>
			<td>light deliver</td>
		</tr>
		<tr>
			<td>Ultrasound transducer</td>
			<td>Olympus NDT</td>
			<td>V308</td>
			<td>5 MHz</td>
		</tr>
		<tr>
			<td>Ultraoundpulser/receiver</td>
			<td>Olympus NDT</td>
			<td>5072PR</td>
			<td>amplifier</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS5054</td>
			<td>data acquisition</td>
		</tr>
		<tr>
			<td>Scanning stage</td>
			<td>Danaher Dover</td>
			<td>XY6060</td>
			<td>raster scanning</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140-25G</td>
			<td>contrast agent</td>
		</tr>
		<tr>
			<td>Rats</td>
			<td>Harlan</td>
			<td>Spague-Dawley</td>
			<td>animal subject</td>
		</tr>
		<tr>
			<td>Isoflourane vaporizer</td>
			<td>Euthanex</td>
			<td>EZ-155</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Sonotech</td>
			<td>Clear Image singles</td>
			<td>acoustic coupling</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows the In vivo nonionizing and noninvasive PAC using optically turbid methylene blue (MB). The control PA image was obtained at 667 nm, at the peak optical absorption for MB (Figure 1A). Although the blood vessels in the FOV are clearly visualized, the bladder is invisible because it is optically transparent at this wavelength. As shown in Figure 1B, the bladder is clearly revealed in the PA image acquired at 0.2 hr post-injection of MB. To confirm t...

Watch this Scientific Journal Video about Photoacoustic Cystography at JoVE.com

Photoacoustic Cystography

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 ...

Research

JoVE Journal

Bioengineering

13.2K 조회수.  University at Buffalo, The State University of New York. 광 음향 cystography (PAC)는 어떤 전리 방사선이나 독성 조영제를 사용하지 않고 요도 방광, 소아 환자에서 방사선에 민감한 내부 장기를 매핑 할 큰 잠재력을 가지고 있습니다. 여기에서 우리는 쥐 광학 불투명 추적자의 주입 요도 방광을 매핑 PAC의 사용을 보여줍니다 생체 내.

비디오: Photoacoustic Cystography

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 ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

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 ...

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.

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, ...

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. ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

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 ...