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>
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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.

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

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

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

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...