KEYWORDS: Optical coherence tomography, Transducers, Ultrasonography, 3D image processing, In vitro testing, Cavitation, Image segmentation, In vivo imaging, Tissues, Blood
Several in vitro and in vivo studies have established accelerated thrombolysis using ultrasound (US) induced microbubble (MB) cavitation. However, the mechanisms underlying MB mediated sonothrombolysis are still not completely elucidated. We performed three-dimensional (3-D) volumetric optical coherence tomography (OCT) imaging before and after the application of contrast US to thrombus. The most dramatic reduction in clot volume was observed with US + MB + recombinant tissue plasminogen activator (rt-PA). Thrombus surface erosion in this group on the side of the thrombus exposed to MB and ultrasound was evident on the OCT images. This technique may assist in clarifying the mechanisms underlying sonothrombolysis, especially regarding the importance of US transducer orientation on lytic efficacy and the effects of MB cavitation on thrombus structure.
KEYWORDS: Tissues, Monte Carlo methods, Photoacoustic spectroscopy, Diffusion, Optical simulations, Acoustics, Signal generators, Interfaces, Signal detection, Chemical elements
A finite element (FE)-based simulation model for photoacoustic (PA) has been developed incorporating light
propagation, PA signal generation, and sound wave propagation in soft tissues using a commercial FE simulation
package, COMSOL Multiphysics. The developed simulation model is evaluated by comparing with other known
simulation models such as Monte Carlo method and heat-pressure model. In this in silico simulation, FE model is
composed of three parts of 1) homogeneous background soft tissues submerged in water, 2) target tissue inclusion (or
PA contrast agents), and 3) short pulsed laser source (pulse length of 5-10 ns). The laser point source is placed right
above the tissues submerged in water. This laser source light propagation through the multi-layer tissues using the
diffusion equation is compared with Monte Carlo solution. Photoacoustic signal generation by the target tissue inclusion
is simulated using bioheat equation for temperature change, and resultant stress and strain. With stress-strain model, the
process of the PA signal generation can be simulated further in details step by step to understand and analyze the photothermal
properties of the target tissues or PA contrast agents. The created wide-band acoustic pressure (band width > 150
MHz) propagates through the background tissues to the ultrasound detector located at the tissue surface, governed by
sound wave equation. Acoustic scattering and absorption in soft tissues also have been considered. Accuracy and
computational time of the developed FE-based simulation model of photoacoustics have been quantitatively analyzed.
A new design of light illumination scheme for deep tissue photoacoustic (PA) imaging, a light catcher, is proposed and
evaluated by in silico simulation. Finite element (FE)-based numerical simulation model was developed for
photoacoustic (PA) imaging in soft tissues. In this in silico simulation using a commercially available FE simulation
package (COMSOL MultiphysicsTM, COMSOL Inc., USA), a short-pulsed laser point source (pulse length of 5 ns) was
placed in water on the tissue surface. Overall, four sets of simulation models were integrated together to describe the
physical principles of PA imaging. Light energy transmission through background tissues from the laser source to the
target tissue or contrast agent was described by diffusion equation. The absorption of light energy and its conversion to
heat by target tissue or contrast agent was modeled using bio-heat equation. The heat then causes the stress and strain
change, and the resulting displacement of the target surface produces acoustic pressure. The created wide-band acoustic
pressure will propagate through background tissues to the ultrasound detector, which is governed by acoustic wave
equation. Both optical and acoustical parameters in soft tissues such as scattering, absorption, and attenuation are
incorporated in tissue models. PA imaging performance with different design parameters of the laser source and energy
delivery scheme was investigated. The laser light illumination into the deep tissues can be significantly improved by up
to 134.8% increase of fluence rate by introducing a designed compact light catcher with highly reflecting inner surface
surrounding the light source. The optimized parameters through this simulation will guide the design of PA system for
deep tissue imaging, and help to form the base protocols of experimental evaluations in vitro and in vivo.
Spectral analysis of photoacoustic (PA) molecular imaging (PMI) of ferritin expressed in human melanoma cells
(SK-24) was performed in vitro. Ferritin is a ubiquitously expressed protein which stores iron that can be detected by PA
imaging, allowing ferritin to act as a reporter gene. To
over-express ferritin, SK-24 cells were co-transfected with plasmid
expressing Heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using
LipofectamineTM 2000. Non-transfected SK-24 cells served as a negative control. Fluorescent imaging of EGFP
confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation in SK-24 cells, a
focused high frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (<20mJ/cm2), was used to
scan the PA signal from 680 nm to 950 nm (in 10 nm increments) from the surface of the 6-well culturing plate. PA
signal intensity from H-FT transfected SK-24 cells was not different from that of non-transfected SK-24 cells at
wavelengths less than 770 nm, but was over 4 dB higher than
non-transfected SK-24 cells at 850 ~ 950 nm. Fluorescent
microscopy indicates significant accumulation of ferritin in H-FT transfected SK-24 cells, with little ferritin expression
in non-transfected SK-24 cells. The PA spectral analysis clearly differentiates transfected SK-24 cells from nontransfected
SK-24 cells with significantly increased iron signal at 850 ~ 950 nm, and these increased signals were
associated with transfection of H-FT plasmid. As such, the feasibility of ferritin as a reporter gene for PMI has been
demonstrated in vitro. The use of ferritin as a reporter gene represents a new concept for PA imaging, and may provide
various opportunities for molecular imaging and basic science research.
Multiple cardiovascular inflammatory biomarkers were simultaneously imaged in vivo using antibody conjugated
gold nanorods (GNRs) injected into a mouse model of vascular injury stimulated by a photochemical reaction of Rose
Bengal dye to green light. Mixed solutions of ICAM-1 antibody conjugated GNRs (715 nm) and E-selectin antibody
conjugated GNRs (800 nm) were injected to bind to their respective inflammatory markers on the luminal surface of the
inferior vena cava of a mouse. Photoacoustic intensity was measured by a commercial ultrasound probe synchronized to
a pulsed laser (10-18 mJ/cm2) at 715 nm or 800 nm clearly identified the upregulation of targeted biomarkers.
Histopathology on the harvested tissues confirmed inflammation. The feasibility of simultaneous photoacoustic
molecular imaging of inflammation responses in cardiovascular system using a commercial ultrasound system has been
demonstrated in vivo.
Cardiovascular inflammatory activity was imaged in vivo. Inflammation is known to be a major cause of
cardiovascular disease. Photoacoustic (PA) imaging was employed using bio-conjugated gold nanorods (GNR) as a
contrast agent. A mouse model based on vascular endothelium injury by a photochemical reaction of Rose Bengal (RB)
dye to green light laser was used. Following a mid-line laparotomy under an approved animal protocol, anti-ICAM-1
conjugated GNR was injected through the dorsal penile vein followed by RB injection through the same vein. The
inferior vena cava immediately distal to the renal veins of a C57BL/6 mouse was exposed to the green light laser for 10
minutes. The peak absorption of GNR was tuned to be 700 nm to minimize possible background absorption by blood
and RB. The stability of GNR in the blood plasma was tested in vitro. Photoacoustic images were obtained through an
ultrasound gel pouch in the mouse abdomen using a commercial ultrasound probe to evaluate inflammatory changes to
the vascular endothelium, confirmed by histology. Preliminary results demonstrate the feasibility of in vivo
photoacoustic imaging by a commercial ultrasound scanner of inflammation using GNR as a contrast agent.
Photoacoustic (PA) imaging provides excellent optical contrast with decent penetration and high spatial resolution, making it attractive for a variety of neural applications. We evaluated optical contrast agents with high absorption in the near infrared (NIR) as potential enhancers for PA neuroimaging: optical dyes, gold nanorods (GNRs) and PEBBLEs loaded with indocyanine green. Two PA systems were developed to test these agents in excised neural tissue and in vivo mouse brain. Lobster nerves were stained with the agents for 30 minutes and placed in a hybrid nerve chamber capable of electrical stimulation and recording, optical spectroscopy and PA imaging. Contrast agents boosted the PA signal by at least 30 dB using NIR
illumination from a tunable pulsed laser. Photobleaching may be a limiting factor for optical dyes-the PA signal decreased steadily with laser illumination. The second setup enabled in vivo transcranial imaging of the mouse brain. A custom clinical ultrasound scanner and a 10-MHz linear array provided near real-time
images during and after an injection of 2 nM gold nanorods into the tail vein. The peak PA signal from the brain vasculature was enhanced by up to 2 dB at 710 nm. Temporal dynamics of the PA signal were also consistent with mixing of the GNRs in the blood. These studies provide a baseline for enhanced PA imaging in neural tissue. The smart contrast agents employed in this study can be further engineered for molecular targeting and controlled drug delivery with potential treatment for a myriad of neural disorders.
Photoacoustic imaging provides optical contrast with good penetration and high spatial resolution,
making it an attractive tool for noninvasive neural applications. We chose a commercial dye (NK2761)
commonly used for optical imaging of membrane potential to enhance photoacoustic images of the live
lobster nerve cord. The abdominal segment of the nerve cord was excised, stained and positioned in a
custom neural recording system, enabling electrical stimulation and recording of compound action
potentials. Photoacoustic and pulse echo images were also collected using a commercial ultrasound scanner
and a 10-MHz linear probe. A wavelength-tunable pulsed laser source (SureliteTM, 5 ns, ~15 mJ, 30
mJ/cm2) operating at 20 Hz produced photoacoustic waves. Longitudinal photoacoustic scans of a 25-mm
segment of the excised nerve cord, including ganglionic and axonal processes, were collected and displayed
every 7 seconds. Without the contrast agent, an average of 10 scans produced a peak photoacoustic signal 6
dB over background noise. An additional 29 dB was obtained after the nerve was submerged in the dye for
20 minutes. The gain decreased to 23 dB and 14 dB at 810 nm and 910 nm, respectively - consistent with
the dye's optical absorbance measured using a portable spectrometer. The contrast-enhanced photoacoustic
signal had a broad spectrum peaking at 4 MHz, and, after high pass filtering, images approached 200-&mgr;m
spatial resolution. The hybrid imaging system, which provided several hours of electrical stimulation and
recording, represents a robust testbed to develop novel photoacoustic contrast for neural applications.
Conventional methods for mapping cardiac current fields lack either spatial resolution (e.g. ECG) or are time consuming (e.g., intra-cardiac catheter electrode mapping). We present a method based on the acousto-electric effect (AEE) with potential for rapid mapping of current fields in the heart with high spatial resolution. The AEE is a pressure-induced conductivity modulation, in which focused ultrasound can be used as a spatially localized pressure source. When an ultrasound beam is focused between a pair of recording electrodes in a homogeneous conductive medium, an induced voltage will be produced due to the pressure-modulated conductivity and local current density. The amplitude of the voltage change should be proportional to fluctuations in current density, such as those generated during the cardiac cycle, in the region of focused ultrasound. Preliminary experiments demonstrate the feasibility of this method. A 540 kHz ultrasound transducer is focused between two tin electrodes lying parallel to the beam axis. These electrodes inject current into a 0.9% saline solution. A pair of insulated stainless steel electrodes exposed at the tip is used to record voltage. To simulate a cardiac current, a low frequency current waveform is injected into the sample such that the peak current density (8 mA/cm2) approximates cardiac currents. The transducer is pulsed at different delays after waveform initiation. Delays are chosen such that the low frequency waveform is adequately sampled. Using this approach an emulated ECG waveform has been successfully reconstructed from the ultrasound modulated voltage traces.
For decades, visual, tactile and radiographic examinations have been the standard for diagnosing caries. Nonetheless, the extent of variation in the diagnosis of dental caries is substantial among dental practitioners using these traditional techniques. Therefore, a more reliable standard for detecting incipient caries would be desirable. Using photoacoustics, near-infrared (NIR) optical contrast between sound and carious dental tissues can be relatively easily and accurately detected at ultrasound resolution. In this paper, a pulsed laser (Nd:YAG, Quanta-Ray) was used to probe extracted human molars at different disease stages determined from periapical radiographs. Both fundamental (1064nm) and first harmonic (532nm) pulses (15ns pulse length, 100mJ at fundamental and 9mJ at first harmonic , 10Hz pulse repetition rate) were used to illuminate the occlusal surface of tooth samples placed in a water tank. The photoacoustic signal was recorded with an unfocused wideband single-element piezoelectric transducer (centered at 12 MHz, bandwidth 15 MHz) positioned at small angle (less than 30 degrees) to the laser beam close to the occlusal surface. At the fundamental wavelength, total photoacoustic energy increases from normal to incipient stage disease by as much as a factor of 10. Differences between photoacoustic energy at the fundamental and first harmonic wavelength further indicate spectral absorption changes of the underlying structure with disease progression. Using a focused laser beam, an extracted molar with suspected incipient caries was scanned along the occulusal surface to help localize the caries inside enamel and dentin. The significantly increasing photoacoustic signal at a specific scan line both at fundamental and first harmonic indicates the local development of the incipient caries. The photoacoustic results compare well with visual inspection after layer by layer dissection. Preliminary results demonstrate the feasibility of detecting incipient occlusal and proximal caries. This technique may ultimately allow for continuous monitoring of caries before and during treatment.
An optoacoustic detector denotes the detection of acoustic signals by optical devices. Recent advances in fabrication techniques and the availability of high power tunable laser sources have greatly accelerated the development of efficient optoacoustic detectors. The unique advantages of optoacoustic technology are of special interest in applications that require high resolution imaging. For these applications optoacoustic technology enables high frequency transducer arrays with element size on the order of 10 μm. Laser generated ultrasound (photoacoustic effect) has been studied since the early observations of A.G. Bell (1880) of audible sound generated by light absorption . Modern studies have demonstrated the use of the photoacoustic effect to form a versatile imaging modality for medical and biological applications. A short laser pulse illuminates a tissue creating rapid thermal expansion and acoustic emission. Detection of the resulting acoustic field by an array enables the imaging of the tissue optical absorption using ultrasonic imaging methods. We present an integrated imaging system that employs photoacoustic sound generation and 2D optoacoustic reception. The optoacoustic receiver consists of a thin polymer Fabry-Perot etalon. The etalon is an optical resonator of a high quality factor (Q = 750). The relatively low elasticity modulus of the polymer and the high Q-factor of the resonator combine to yield high ultrasound sensitivity. The etalon thickness (10 μm) was optimized for wide bandwidth (typically above 50 MHz). An optical scanning and focusing system is used to create a large aperture and high density 2D ultrasonic receiver array. High resolution 3D images of phantom targets and biological tissue samples were obtained.
Recently, it was suggested that ultrasound elasticity imaging can be used to age deep vein thrombosis (DVT) since blood clot hardness changes with fibrin content. The main components of ultrasound elasticity imaging are deformation of the object, speckle or internal boundary tracking and evaluation of tissue motion, measurement of strain tensor components, and reconstruction of the spatial distribution of elastic modulus using strain images. In this paper, we investigate a technique for Young's modulus reconstruction to quantify ultrasound elasticity imaging of DVT. In-vivo strain imaging experiments were performed using Sprague-Dawley rats with surgically induced clots in the inferior vena cavas (IVC). In this model, the clot matures from acute to chronic in less than 10 days. Therefore, nearly every 24 hours the strain imaging experiments were performed to reveal temporal transformation of the clot. The measured displacement and strain images were then converted into maps of elasticity using model-based elasticity reconstruction where the blood clot within an occluded vein was approximated as a layered elastic cylinder surrounded by incompressible tissue. Results of this study demonstrate that Young's modulus gradually increases with clot maturity and can be used to differentiate clots providing a desperately needed clinical tool of DVT staging.
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