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

Summary

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.

Abstract

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. 

Introduction

Photoacoustic computed tomography (PACT/PAT) is a promising non-invasive biomedical imaging modality combining rich optical contrast with high ultrasond resolution^1,2,3,4,5. When a nanosecond pulsed laser deposits energy onto light absorbing chromophores 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 photoaco....

Protocol

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

1. 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 laser driver unit (LDU).
   NOTE: The PLD generates ~816 nm wavelength pulses, pulses of ~107 ns in duration, and up to a 2 KHz repetition rate with a m....

Results

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° and 45° around the rat brain at scan speeds of 4 s and 0.5 s, respectively. Figure 2A,B show brain images of a female rat (.......

Discussion

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.......

Materials

List of materials used in this article

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