Name: pulsed laser diode-based desktop photoacoustic tomography for monitoring wash-in and wash-out of dye in rat cortical vasculature
Uploaded: 2019-05-30T15:00:00
Description: Watch this Scientific Journal Video about Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature at JoVE.com

The research is supported by the Singapore Ministry of Health&#8217;s National Medical Research Council (NMRC/OFIRG/0005/2016: M4062012). The authors would like to thank Mr. Chow Wai Hoong Bobby for the machine shop support.

All animal experiments were performed according to the guidelines and regulations approved by the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0331).
1. System description
<ol>
	<li>Mount the PLD laser into the circular scanner and mount the optical diffuser (OD) in front of the PLD exit window to make the output beam homogeneous, as shown in Figure 1A. Connect the PLD to the lase...

<ol>
	<li>Lin, L., 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=Single-breath-hold+photoacoustic+computed+tomography+of+the+breast.">Single-breath-hold photoacoustic computed tomography of the breast.</a> Nature Communications. 9 (1), 2352 (2018).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+photoacoustic+imaging+systems+using+pulsed+laser+diodes:+a+review.">Fast photoacoustic imaging systems using pulsed laser diodes: a review.</a> Biomedical Engineering Letters. 8 (2), 167-181 (2018).</li><li>Yao, J., 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=Recent+progress+in+photoacoustic+molecular+imaging.">Recent progress in photoacoustic molecular imaging.</a> Current Opinion in Chemical Biology. 45, 104-112 (2018).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+toward+preclinical+and+clinical+translation+of+photoacoustic+tomography:+a+review.">Recent advances toward preclinical and clinical translation of photoacoustic tomography: a review.</a> Journal of Biomedical Optics. 22 (4), 041006 (2017).</li><li>Yao, J., 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=Photoacoustic+microscopy.">Photoacoustic microscopy.</a> Laser &amp; Photonics Reviews. 7 (5), 758-778 (2013).</li><li>Awasthi, N., Kalva, S. K., Pramanik, M., Yalavarthy, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Guided+Filtering+for+Improving+Photoacoustic+Tomographic+Image+Reconstruction.">Image Guided Filtering for Improving Photoacoustic Tomographic Image Reconstruction.</a> Journal of Biomedical Optics. 23 (9), 091413 (2018).</li><li>Kalva, S. 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=Experimental+validation+of+tangential+resolution+improvement+in+photoacoustic+tomography+using+a+modified+delay-and-sum+reconstruction+algorithm.">Experimental validation of tangential resolution improvement in photoacoustic tomography using a modified delay-and-sum reconstruction algorithm.</a> Journal of Biomedical Optics. 21 (8), 086011 (2016).</li><li>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=Improving+tangential+resolution+with+a+modified+delay-and-sum+reconstruction+algorithm+in+photoacoustic+and+thermoacoustic+tomography.">Improving tangential resolution with a modified delay-and-sum reconstruction algorithm in photoacoustic and thermoacoustic tomography.</a> The Journal of the Optical Society of America. 31 (3), 621-627 (2014).</li><li>Xu, 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=Universal+back-projection+algorithm+for+photoacoustic+computed+tomography.">Universal back-projection algorithm for photoacoustic computed tomography.</a> Physical Review E. 71 (1), 016706 (2005).</li><li>Wang, L. V., Hu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+Tomography:+In+Vivo+Imaging+from+Organelles+to+Organs.">Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs.</a> Science. 335 (6075), 1458-1462 (2012).</li><li>Sivasubramanian, K., Periyasamy, V., 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=Non-invasive+sentinel+lymph+node+mapping+and+needle+guidance+using+clinical+handheld+photoacoustic+imaging+system+in+small+animal.">Non-invasive sentinel lymph node mapping and needle guidance using clinical handheld photoacoustic imaging system in small animal.</a> Journal of Biophotonics. 11 (1), e201700061 (2018).</li><li>Hu, S., Maslov, K., 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=Second-generation+optical-resolution+photoacoustic+microscopy+with+improved+sensitivity+and+speed.">Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed.</a> Optics Letters. 36 (7), 1134-1136 (2011).</li><li>Stein, E. W., Maslov, K., 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,+in+vivo+imaging+of+blood-oxygenation+dynamics+within+the+mouse+brain+using+photoacoustic+microscopy.">Noninvasive, in vivo imaging of blood-oxygenation dynamics within the mouse brain using photoacoustic microscopy.</a> Journal of Biomedical Optics. 14 (2), 020502 (2009).</li><li>Ku, G., Wang, X. D., Xie, X. Y., 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=Imaging+of+tumor+angiogenesis+in+rat+brains+in+vivo+by+photoacoustic+tomography.">Imaging of tumor angiogenesis in rat brains in vivo by photoacoustic tomography.</a> Applied Optics. 44 (5), 770-775 (2005).</li><li>Ma, R., Taruttis, A., Ntziachristos, V., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+optoacoustic+tomography+(MSOT)+scanner+for+whole-body+small+animal+imaging.">Multispectral optoacoustic tomography (MSOT) scanner for whole-body small animal imaging.</a> Optics Express. 17 (24), 21414-21426 (2009).</li><li>Zhou, Y., 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=A+Phosphorus+Phthalocyanine+Formulation+with+Intense+Absorbance+at+1000+nm+for+Deep+Optical+Imaging.">A Phosphorus Phthalocyanine Formulation with Intense Absorbance at 1000 nm for Deep Optical Imaging.</a> Theranostics. 6 (5), 688-697 (2016).</li><li>Li, L., 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=Single-impulse+panoramic+photoacoustic+computed+tomography+of+small-animal+whole-body+dynamics+at+high+spatiotemporal+resolution.">Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution.</a> Nature Biomedical Engineering. 1 (1), 0071 (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> Biomedical Optics Express. 7 (2), 312-323 (2016).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+in+vivo+imaging+of+small+animal+brain+using+pulsed+laser+diode-based+photoacoustic+tomography+system.">Dynamic in vivo imaging of small animal brain using pulsed laser diode-based photoacoustic tomography system.</a> Journal of Biomedical Optics. 22 (9), 090501 (2017).</li><li>Upputuri, P. K., Periyasamy, V., Kalva, S. 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=A+High-performance+compact+photoacoustic+tomography+system+for+in+vivo+small-animal+brain+imaging.">A High-performance compact photoacoustic tomography system for in vivo small-animal brain imaging.</a> Journal of Visualized Experiments. (124), e55811 (2017).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+laser+diode+based+optoacoustic+imaging+of+biological+tissues.">Pulsed laser diode based optoacoustic imaging of biological tissues.</a> Biomedical Physics &amp; Engineering Express. 1 (4), 045010-045017 (2015).</li><li>Arabul, M. U., 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=Toward+the+detection+of+intraplaque+hemorrhage+in+carotid+artery+lesions+using+photoacoustic+imaging.">Toward the detection of intraplaque hemorrhage in carotid artery lesions using photoacoustic imaging.</a> Journal of Biomedical Optics. 22 (4), (2016).</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> Optics Express. 22 (21), 26365-26374 (2014).</li><li>Allen, J. S., Beard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+near-infrared+laser+diode+excitation+system+for+biomedical+photoacoustic+imaging.">Pulsed near-infrared laser diode excitation system for biomedical photoacoustic imaging.</a> Optics Letters. 31 (23), 3462-3464 (2006).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+characterization+of+low-cost,+high-speed,+portable+pulsed+laser+diode+photoacoustic+tomography+(PLD-PAT)+system.">Performance characterization of low-cost, high-speed, portable pulsed laser diode photoacoustic tomography (PLD-PAT) system.</a> Biomed Opt Express. 6 (10), 4118-4129 (2015).</li><li>Kalva, S. K., Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed,+low-cost,+pulsed-laser-diode-based+second-generation+desktop+photoacoustic+tomography+system.">High-speed, low-cost, pulsed-laser-diode-based second-generation desktop photoacoustic tomography system.</a> Optics Lett. 44 (1), 81-84 (2019).</li><li>Kalva, S. K., Hui, Z. Z., 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=Calibrating+reconstruction+radius+in+a+multi+single-element+ultrasound-transducer-based+photoacoustic+computed+tomography+system.">Calibrating reconstruction radius in a multi single-element ultrasound-transducer-based photoacoustic computed tomography system.</a> J Opt Soc Am A. 35 (5), 764-771 (2018).</li><li>Kalva, S. 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=Use+of+acoustic+reflector+to+make+compact+photoacoustic+tomography+system.">Use of acoustic reflector to make compact photoacoustic tomography system.</a> Journal of Biomedical Optics. 22 (2), 026009 (2017).</li><li>. <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></ol>

This work presents a protocol to use a desktop PLD-PAT system for conducting experiments on small animals like rats for in vivo brain imaging and dynamic fast-uptake and clearance process of contrast agents like ICG. Bulky, expensive OPO-PAT systems take several minutes (2-5 min) to acquire a single cross-sectional in vivo image. A compact, low-cost, first generation portable PLD-PAT system provides single cross-sectional in vivo images in 5 s. In contrast, a high-speed, compact, low-cost desktop PLD-PAT system renders a...

Photoacoustic computed tomography (PACT/PAT) is a promising non-invasive biomedical imaging modality combining rich&#160;optical contrast with high&#160;ultrasond&#160;resolution1,2,3,4,5. When a nanosecond pulsed laser deposits energy onto light absorbing chromophores&#160;present inside any biological tissue, local temperature increases leading to thermoelastic expansion and contraction of the tissue, resulting in generation of pressure waves. These pressure waves are known as ultrasound waves or photoacoustic (PA) waves, which can be detected by ultrasound transducers around the sample. The detected PA signals are reconstructed using various reconstruction algorithms6,7,8,9 to generate cross-sectional PA images. PA imaging provides structural and functional information from macroscopic organs to microscopic organelles due to the wavelength dependence of endogenous chromophores present inside the body10. PAT imaging has been successfully used for breast cancer detection1, sentinel lymph node imaging11, mapping of oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), total hemoglobin concentration (HbT), oxygen saturation (SO2)12,13, tumor angiogenesis14, small animal whole body imaging15, and other applications.
Nd:YAG/OPO lasers are conventional excitation sources for first generation PAT systems that are widely used in photoacoustic community for small animal imaging and deep tissue imaging16. These lasers provide ~100 mJ energy pulses at low repetition rates of ~10-100 Hz. The PAT imaging systems using these costly and bulky lasers are not suitable for high-speed imaging with single-element ultrasound transducers (SUTs), due to the limited pulse repetition rate. This inhibits real-time monitoring of physiological changes occurring at high speeds inside the animal. Using array-based transducers like linear, semi-circular, circular, and volumetric arrays with Nd:YAG laser excitation, high-speed imaging is possible. However, these array transducers are expensive and provide lower sensitivities compared to SUTs; yet, the imaging speed is limited by the low repetition rate of the laser. State-of-the-art single-impulse PACT systems with customized full-ring array transducer obtain the PA data at 50 Hz frame rates17. These array transducers need complex back-end receiving electronics and signal amplifiers, making the overall system more expensive and difficult for clinical use.
Their compact size, lower cost requirements, and higher pulse repetition rate (order of KHz) make pulsed laser diodes (PLDs) more promising for real-time imaging. Due to these advantages, PLDs are actively used as an alternate excitation source in second generation PAT systems. PLD-based PAT systems have been demonstrated successfully for high-frame rate imaging using array transducers18, deep-tissue and brain imaging19,20,21, cardiovascular disease diagnosis22, and rheumatology diagnosis23. As SUTs are highly sensitive and less expensive compared to array transducers, they are still extensively used for PAT imaging. Fiber-based PLD system have been demonstrated for phantom imaging24. A portable PLD-PAT system has been demonstrated previously by mounting the PLD inside the PAT scanner25. With one SUT circular scanner, phantom imaging was performed during 3 s of scan time, and in vivo rat brain imaging was performed during a 5 s period using this PLD-PAT system19.
Furthermore, improvements have been made to this PLD-PAT system to make it more compact and create a desktop model using eight acoustic reflector-based single-element ultrasound transducers (SUTRs)26,27. Here, SUTs were placed in a vertical instead of horizontal direction with the aid of a 90&#176; acoustic reflector28. This system can be employed for scan times of up to 0.5 s and ~3 cm deep in tissue imaging and in vivo small animal brain imaging. In this work, this desktop PLD-PAT system is used to provide the visual demonstration of experiments for in vivo brain imaging in small animals and for dynamic visualization of uptake and clearance process of Food and Drug Administration (FDA)-approved indocyanine green (ICG) dye in rat brains.

<table>
	<tbody>
		<tr>
			<td>12 V power supply</td>
			<td>Voltcraft</td>
			<td>PPS-11810</td>
			<td>To supply operating voltage for PLD</td>
		</tr>
		<tr>
			<td>Acoustic reflector</td>
			<td>Olympus</td>
			<td>F102</td>
			<td>45 degree reflector augmented to the ultrasound transducer</td>
		</tr>
		<tr>
			<td>Acrylic water tank</td>
			<td>NTU workshop</td>
			<td>Custom-made</td>
			<td>It is used to hold water that acts as an acoustic coupling medium between animal brain and detector</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>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>Animal holder</td>
			<td>NTU workshop</td>
			<td>Custom-made</td>
			<td>Used for holding animal on its abdomen</td>
		</tr>
		<tr>
			<td>Breathing mask</td>
			<td>NTU workshop</td>
			<td>Custom-made</td>
			<td>Used along with animal holder to supply anesthesia mixture to the animal</td>
		</tr>
		<tr>
			<td>Circular Scanner</td>
			<td>NTU workshop</td>
			<td>Custom-made</td>
			<td>Scanner is made out of aluminum</td>
		</tr>
		<tr>
			<td>DAQ (Data acquisition) Card</td>
			<td>Spectrum</td>
			<td>M2i.4932-exp</td>
			<td>16 bit, 30 Ms/s, 8 channels, 1 Gs, PCIe</td>
		</tr>
		<tr>
			<td>Data acqusition software</td>
			<td>National Instruments Corporation,Austin,TX,USA)</td>
			<td>NI LabVIEW 2015 SP1 (32 bit)</td>
			<td>LabVIEW based program developed in our laboratory for controlling the stepper motor and acquring the PA singnals from the detector</td>
		</tr>
		<tr>
			<td>Data processing software</td>
			<td>Matlab (Mathworks, Natick, MA, USA)</td>
			<td>Matlab R2015b</td>
			<td>Matlab code developed in our laboratory for reconstructing cross-sectional PA images</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>RIGOL</td>
			<td>DG1022</td>
			<td>To change the repetition rate of the PLD. It will provide TTL signal to synchronize the DAQ with the laser excitation.</td>
		</tr>
		<tr>
			<td>Low noise signal amplifier</td>
			<td>Genetron</td>
			<td>Custom-made using Mini-circuits, ZFL-500LN-BNC</td>
			<td>To receive, and amplify the PA signal from SUTR. Its gain is 24 dB.</td>
		</tr>
		<tr>
			<td>Optical diffuser</td>
			<td>Thorlabs</td>
			<td>DG-120</td>
			<td>Used to to make the laser beam homogeneous</td>
		</tr>
		<tr>
			<td>Pulsed laser diode</td>
			<td>Quantel, France</td>
			<td>QD-Q1924-ILO-WATER</td>
			<td>It is the excitation laser source with specifications of 816 nm wavelength, 3.4 mJ per pulse energy, 107 ns pulse width, 2 KHz maximum pulse repitition rate, dimensions : 13.0 x 7.6 x 5.0 cm</td>
		</tr>
		<tr>
			<td>Rats</td>
			<td>In Vivos Pte Ltd, Singapore</td>
			<td>NTac:SD, Sprague Dawley / SD</td>
			<td>Female, weight 100&#177;10g, strain of rats: Sprague Dawley, age: 4-5 weeks</td>
		</tr>
		<tr>
			<td>Stepper motor with gearbox</td>
			<td>LIN Engineering (Servo Dynamics)</td>
			<td>Motor: CO-5718L-01P-RO, Gearbox: DPL64/1; Power supply PW-100-24</td>
			<td>To move the detector holder in a circular geometry. Torque: 2.08 N-m, Rotor inertia: 2.6 kg-cm2</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Clear ultrasound gel</td>
		</tr>
		<tr>
			<td>Ultrasound Transducer</td>
			<td>Olympus</td>
			<td>V309-SU/ U8423013</td>
			<td>Ultrasonic sensors used for photoacoustic detection. Central freqency 5 MHz, 0.5 in</td>
		</tr>
		<tr>
			<td>Variable high voltage power supply</td>
			<td>Elektro-Automatik</td>
			<td>EA-PS 8160-04 T</td>
			<td>To change the laser output power</td>
		</tr>
	</tbody>
</table>

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;

The potentiality of the described desktop PLD-PAT system for dynamic in vivo brain imaging has been showcased in this protocol with corresponding results. High-speed imaging capability of the desktop PLD-PAT system was demonstrated by performing in vivo brain imaging of healthy female rats. PA signals were collected using eight SUTRs rotating in 360&#176; and 45&#176; around the rat brain at scan speeds of 4 s and 0.5 s, respectively. Figure 2A,B show brain images of a fe...

Watch this Scientific Journal Video about Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature at JoVE.com

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

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. ...

Photoacoustic tomography, or PAT, is an imaging hybrid biomedical imaging modality, combining both optical and ultrasound imaging for monitoring dynamic changes in small animal brains. This is a cost-effective, portable high speed imaging system that uses imaging speed as short as half a second. Demonstrating the procedure with Sandeep will be Praveen, a PhD student, Dr.Paul, a research fellow, and Rhonnie, a research assistant.Begin by mounting all single element ultrasound transducers onto each transducer holder one at a time, such that the surface of each acoustic reflector faces toward the center of the scanning area. Use connecting cables to connect each transducer cable to the low noise signal amplifier. Switch on the power supply of the chiller and turn on the chiller switch to set the temperature between 20 to 25 degrees Celsius.Switch on the low voltage power supply and slowly turn the current control to 0.3 amps. Set the voltage to 12 volts and verify that the current does not exceed 0.1 amp. Switch on the high voltage power supply and press the preset button to set the current to one amp and the voltage to zero volts.Enable the output button and switch on the power supply of the function generator. Press the recall button and select a two kilohertz configuration to generate the laser pulses at this repetition rate. Place the acrylic tank inside the scanner and fill the tank with water until the detecting surfaces of the transducers are completely immersed.Then switch on the power supply of the low noise signal amplifier. Place the anesthetized rat in the prone position on the workbench. After confirming a lack of response to toe pinch, use a hair trimmer to remove the fur on the scalp.Gently apply depilatory cream to remove the fur completely from the exposed skin using a cotton swab to remove the cream after four to five minutes. Apply ointment to animal's eyes and mount a custom made animal holder equipped with a breathing mask on a lab jack. Place the rat in the prone position on the holder with the head resting on the horizontal platform of the holder.Use surgical tape to secure the animal to the holder. Clamp the pulse oximeter to one hind limb to monitor its physiological condition. Use a cotton tipped applicator to apply a layer of colorless ultrasound gel to the scalp.Then adjust the lab jack position to the center of the scanner. Adjust the height of the jack manually so that the imaging plane is at the center of the acoustic reflector. After setting the parameters in the data acquisition software for a 360 degree acquisition scan, enable the output of the function generator to turn on the pulsed laser diode laser emission.Slowly increase the voltage of the variable high voltage power supply to 120 volts for maximum per pulse energy. Run the data acquisition software program to rotate all eight of the transducers 360 degrees over a four second scan time. Disable the output of the function generator to turn off the laser emission.Using the reconstruction algorithm in the data processing software, determine the scanning radius of all eight of the transducers by trial and error, using the back-projection algorithm. Set the parameters in the data acquisition software for a 45 degree acquisition over a 0.5 second scan time. Enable the output of the function generator to turn on the laser emission.Run the data acquisition software program to rotate all eight of the transducers 45 degrees to obtain the initial control data. Disable the output of the function generator to turn off the laser emission. Next, inject 0.3 milliliters of indocyanine green into the tail vein of the rat.Enable the output of the function generator to turn on the laser emission. Then run the data acquisition software to acquire A-lines over a 0.5 second scan time in a 45 degree rotation. At the end of the acquisition, use the back-projection algorithm to reconstruct the cross sectional brain image from the saved A-lines.Here, brain images from a 98 gram female rat, collected at four and 0.5 second scan speeds are shown. The sagittal sinus and the transfer sinus are clearly visible in both images. The photographs show the same region of the rat brain before and after removing the scalp.Photoacoustic tomography imaging can achieve the same visualization noninvasively with the skin and skull intact. In this plot, increases in the average photoacoustic signal in the sagittal sinus due to increases and subsequently decreases in the optical absorption by indocyanine green at 860 nanometer wavelengths over time can be observed. Always take extra care when injecting the ICG dye into the tail vein of the animal.This low cost desktop photoacoustic tomography system can be used for the dynamic imaging of small animal brain vasculatures with high special resolution at 0.5 seconds scan speed. Other applications of this imaging system include brain tumor imaging, the imaging of different organs in small animals and other therapeutic applications.

pulsed-laser-diode-based-desktop-photoacoustic-tomography-for

Research

JoVE Journal

Bioengineering

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 ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

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 ...

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 ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

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 ...