Noninvasive real-time temperature imaging during thermal therapies is able to significantly improve clinical outcomes. An optoacoustic (OA) temperature monitoring method is proposed for noninvasive real-time thermometry of vascularized tissue during cryotherapy. The universal temperature-dependent optoacoustic response (ThOR) of red blood cells (RBCs) is employed to convert reconstructed OA images to temperature maps. To obtain the temperature calibration curve for intensity-normalized OA images, we measured ThOR of 10 porcine blood samples in the range of temperatures from 40°C to −16°C and analyzed the data for single measurement variations. The nonlinearity (ΔTmax) and the temperature of zero OA response (T0) of the calibration curve were found equal to 11.4±0.1°C and −13.8±0.1°C, respectively. The morphology of RBCs was examined before and after the data collection confirming cellular integrity and intracellular compartmentalization of hemoglobin. For temperatures below 0°C, which are of particular interest for cryotherapy, the accuracy of a single temperature measurement was ±1°C, which is consistent with the clinical requirements. Validation of the proposed OA temperature imaging technique was performed for slow and fast cooling of blood samples embedded in tissue-mimicking phantoms.
Enhanced delivery of optical clearing agents (OCA) through skin may improve sensitivity of optical and optoacoustic (OA) methods of imaging, sensing, and monitoring. This report describes a two-step method for enhancement of light penetration through skin. Here, we demonstrate that topical application of hyaluronic acid (HA) improves skin penetration of hydrophilic and lipophilic OCA and thus enhances their performance. We examined the OC effect of 100% polyethylene and polypropylene glycols (PPGs) and their mixture after pretreatment by HA, and demonstrated significant increase in efficiency of light penetration through skin. Increased light transmission resulted in a significant increase of OA image contrast in vitro. Topical pretreatment of skin for about 30 min with 0.5% HA in aqueous solution offers effective delivery of low molecular weight OCA such as a mixture of PPG-425 and polyethylene glycol (PEG)-400. The developed approach of pretreatment by HA prior to application of clearing agents (PEG and PPG) resulted in a ∼47-fold increase in transmission of red and near-infrared light and significantly enhanced contrast of OA images.
We developed a new and improved Laser Optoacoustic Imaging System, LOIS-3D for preclinical research applications in small animal models. The advancements include (i) a new stabilized imaging module with a more homogeneous illumination of the mouse yielding a better spatial resolution (<0.2 mm) and (ii) a new low noise amplifier incorporated into the ultrasonic probe and providing the noise equivalent pressure around 2 Pa resulting in increased signal-to-noise ratio and the optical absorption sensitivity of about 0.15 cm-1. We also improved scan time and the image reconstruction times. This prototype has been commercialized for a number of biomedical research applications, such as imaging vascularization and measuring hemoglobin / oxyhemoglobin distribution in the organs as well as imaging exogenous or endogenous optoacoustic contrast agents. As examples, we present in vivo experiments using phantoms and mice with and without tumor injected with contrast agents with indocyanine green (ICG). LOIS-3D was capable of detecting ~1-2 pmole of the ICG, in tissues with relatively low blood content. With its high sensitivity and excellent spatial resolution LOIS-3D is an advanced alternative to fluorescence and bioluminescence based modalities for molecular imaging in live mice.
We found and interpreted the universal temperature-dependent optoacoustic (photoacoustic) response (ThOR) in blood; the normalized ThOR is invariant with respect to hematocrit at the hemoglobin’s isosbestic point. The unique compartmentalization of hemoglobin, the primary optical absorber at 805 nm, inside red blood cells (RBCs) explains the effect. We studied the temperature dependence of Gruneisen parameter in blood and aqueous solutions of hemoglobin and for the first time experimentally observed transition through the zero optoacoustic response at temperature T0, which was proved to be consistent for various blood samples. On the other hand, the hemoglobin solutions demonstrated linear concentration function of the temperature T0. When this function was extrapolated to the average hemoglobin concentration inside erythrocytes, the temperature T0 was found equivalent to that measured in whole and diluted blood. The obtained universal curve of blood ThOR was validated in both transparent and light scattering media. The discovered universal optoacoustic temperature dependent blood response provides foundation for future development of non-invasive in vivo temperature monitoring in vascularized tissues and blood vessels.
Gold-based contrast agents, gold nanorod (GNR), were designed for the enhancement of optoacoustic signal. After synthesis, the GNR-CTAB complexes were modified by pegylation (PEG), or replacement of CTAB (cetyl trimethylammonium bromide) with MTAB (16-mercaptohexadecyl trimethylammonium bromide) for coverage of gold nanorods with heparin (GNR-HP). Modified GNR are purified through centrifugation and filtration. GNRCTAB can be used as a model of positively charged gold surface for quantitative optoacoustic sensing in GNRbacteria interactions, whereas GNR-PEG and GNR-HP can be used as negatively charged gold surface models. We studied controlled agglomeration of contrast agents with the bacteria E.Coli and Vibrio Cholerae. For bacterial sensing, the localized plasmon resonance peak shifts as a function of electrostatic binding, which was detected with two different wavelengths through 3D optoacoustic imaging.
In this report, we demonstrate the feasibility of using optoacoustic tomography for deducing biodistributions of
nanoparticles in animal models. The redistribution of single-walled carbon nanotubes (SWCNTs) was visualized in
living mice. Nanoparticle concentrations in harvested organs were measured spectroscopically using the intrinsic optical
absorption and fluorescence of SWCNTs. Observed increases in optoacoustic signal brightness in tissues were compared
with increases in optical absorptivity coefficients caused by SWCNT accumulation. The methodology presented in this
report paves the way for measuring concentrations of optically absorbing agents in small animals using optoacoustic
tomography.
We used a 3-D optoacoustic (OA) tomography system to create maps of optical absorbance of mice tissues contrasted with gold nanorods (GNRs). Nude mice were scanned before and after injection of GNRs at time periods varying from 1 to 192 h. Synthesized GNRs were purified from hexadecyltrimethylammonium bromide and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications. Intravenous administration of purified GNR-PEG complexes resulted in enhanced OA contrast of internal organs and blood vessels compared to the same mouse before injection of the contrast agent. Maximum enhancement of the OA images was observed 24 to 48 h postinjection, followed by a slow clearance trend for the remaining part of the studied period (eight days). We demonstrate that OA imaging with two laser wavelengths can be used for noninvasive, long-term studies of biological distribution of contrast agents.
Gold nanorods (GNR) with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A
novel protocol has been developed to replace hexadecyltrimethylammonium bromide (CTAB) on the surface of GNR
with 16-mercaptohexadecanoic acid (MHDA) and metoxy-poly(ethylene glycol)-thiol (PEG), and the monoclonal
antibodies: HER2 or CD33. The physical chemistry property of the conjugates was monitored through optical and zetapotential
measurements to confirm surface chemistry. The plasmon resonance is kept in the near infrared area, and
changes from strong positive charge for GNR-CTAB to slightly negative for GNR-PEG-mAb conjugates are observed.
The conjugates were investigated for different cells lines: breast cancer cells and human leukemia lines in vivo
applications. These results demonstrate successful tumor accumulation of our modified PEG-MHDA conjugates of GNR
for HER2/neu in both overexpressed breast tumors in nude mice, and for thermolysis of human leukemia cells in vitro.
The conjugates are non-toxic and can be used in pre-clinical applications, as well as molecular and optoacoustic imaging,
and quantitative sensing of biological substrates.
Using the method of 3D optoacoustic tomography, we studied changes in tissues of the whole body of nude mice as the
changes manifested themselves from live to postmortem. The studies provided the necessary baseline for optoacoustic
imaging of necrotizing tissue, acute and chronic hypoxia, and reperfusion. They also establish a new optoacoustic model
of early postmortem conditions of the whole mouse body. Animals were scanned in a 37°C water bath using a three-dimensional
optoacoustic tomography system previously shown to provide high contrast maps of vasculature and organs
based on changes in the optical absorbance. The scans were performed right before, 5 minutes after, 2 hours and 1 day
after a lethal injection of KCl. The near-infrared laser wavelength of 765 nm was used to evaluate physiological features
of postmortem changes. Our data showed that optoacoustic imaging is well suited for visualization of both live and
postmortem tissues. The images revealed changes of optical properties in mouse organs and tissues. Specifically, we
observed improvements in contrast of the vascular network and organs after the death of the animal. We associated these
with reduced optical scattering, loss of motion artifacts, and blood coagulation.
We report on the optoacoustic (OA) imaging of the whole mouse body using a biocompatible contrast agent - highly
purified, pegylated gold nanorods (GNR) - which has strong optical absorption in the near-infrared region and low level
of toxicity. In vitro toxicity studies showed no significant change in survival rates of the cultured normal epithelium
IEC-6 cells when incubated for 24 hours with up to 1 nM of GNR. In vivo toxicity studies in nude mice showed no
pathological changes in liver 1 month after the IV injection of GNR with intra-body concentration around 0.25-0.50 nM.
In order to study the enhancement of the OA contrast and accumulation of GNR in different tissues, we performed 3D
OA imaging of live nude mice with IV-injected GNR. The enhancement of the OA contrast in comparison with the
images of the untreated mice was visible starting 1 hour after the GNR injection. The OA contrast of kidneys, liver, and
spleen peaked at about 2-3 days after the administration of the GNR, and then was gradually reducing.
We introduce a novel experimental design for non-invasive scanning optoacoustic microscopy that utilizes a parabolic
surface for ultrasound focusing. We demonstrate that off-axis parabolic mirrors made of sufficiently high acoustic
impedance materials work as ideal reflectors in a wide range of apertures and provide lossless conversion of a spherical
acoustic wavefront into a plane wave. We further test the performance of a custom optoacoustic imaging setup which
was developed and built based on these principles. The achieved resolution limit of 0.3 mm, with NA of 0.5 and the
transducer bandwidth of 5 MHz, matches the resolution limit defined by diffraction. Although further improvements of
current experimental setup are required to achieve resolution similar to leading microscopy systems, this proof-of-concept
work demonstrates the viability of the proposed design for optoacoustic microscopy applications.
We used a three-dimensional optical tomogaphy system that was previously developed to create high contrast maps of
optical absorbance of mice tissues. In this study, animals were scanned before and after injection of gold nanorods
(GNRs) at different time periods. As-synthesized GNRs were purified from hexadecyltrimethylammonium bromide
(CTAB) and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications.
Intravenous administration of the purified GNR-PEG complexes to mice resulted in an enhanced contrast of normal
tissues and blood vessels as compared to ordinary nude mice. In parallel with optoacoustic imaging we investigated the
accumulation of GNRs in liver using invasive analytical techniques. Maximum levels of GNRs in liver macrophages
were observed after 48-72 hours post-injection, followed by slow clearance trend after 8 days. Optoacoustic imaging
revealed redistribution of GNR in mouse organ and tissues: in the initial hours, accumulation of GNRs is seen
predominantly in the periphery of the mouse, while a gradual increase of GNR levels in liver, spleen and kidneys is seen
in 1 and 24 hours.
We developed an optoacoustic biosensor intended for the detection of bloodborne microorganisms using
immunoaffinity reactions of antibody-coupled gold nanorods as contrast agents specifically targeted to the antigen
of interest. Optoacoustic responses generated by the samples are detected using a wide band ultrasonic transducer.
The sensitivity of the technique has been assessed by determining minimally detectable optical density which
corresponds to the minimum detectable concentration of the target viral surface antigens. Both ionic solutions and
gold nanorods served as the contrast agent generating the optoacoustic response. The sensitivity of Nano-LISA is at
least OD=10-6 which allows reliable detection of 1 pg/ml (depending on the commercial antibodies that are used).
Adequate detection sensitivity, as well as lack of non-specific cross-reaction between antigens favors NanoLISA as
a viable technology for biosensor development.
In the present work, we demonstrate a potential use of gold nanorods as a contrast agent for selective photothermal
therapy of human acute leukemia cells (HL-60) using a near-infrared laser. Gold Nanorods (GNR) are synthesized
and conjugated to CD33, a 67 kDa glycoprotein found on the surface of myeloid cells that belongs to the
sialoadhesin family of proteins. After pegylation, or conjugation with CD33 antibody, GNR were non-toxic for
acute and chronic leukemia cells. We used a Quanta System q-switched titanium sapphire laser emitting at a center
wavelength of 755 nm. Each sample was illuminated with 1 laser shot at either high or low fluence. Both laser
modes were used in 3 independent cell probes. HL-60 cells were treated for 45 min with GNR conjugated with mAb
CD33, or with GNR-Pegylated particles. After laser application, the cells were resuspended and analyzed to cell
viability with Trypan blue exclusion assay. GNR-CD33 conjugates significantly increase the percentage of cell
death as compared with a control group after laser illumination: a 3 fold increase is observed.
We developed a 3D whole-body optoacoustic tomography system for applications in preclinical research on mice. The
system is capable of generating images with resolution better than 0.6 mm. Two pulsed lasers, an Alexandrite laser
operating at 755 nm and a Nd:YAG laser operating at 532 nm and 1064nm were used for light delivery. The
tomographic images were obtained while the objects of study (phantoms or mice) were rotated within a sphere outlined
by a concave arc-shaped array of 64 piezo-composite transducers. During the scan, the mouse was illuminated
orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. Illumination at 532 nm showed
superficial vasculature, but limited penetration depth at this wavelength prevented the detection of deeper structures.
Illumination at 755 and 1064 nm showed organs and blood vessels, respectively. Filtering of the optoacoustic signals
using high frequency enhancing wavelets further emphasized the smaller blood vessels.
A three-dimensional laser optoacoustic imaging system was developed, which combines the advantages of optical
spectroscopy and high resolution ultrasonic detection, to produce high contrast maps of optical absorbance in tissues.
This system was tested in a nude mouse model of breast cancer and produced tissue images of tumors and vasculature.
The imaging can utilize either optical properties of hemoglobin and oxyhemoglobin, which are the main endogenous
tissue chromophores in the red and near-infrared spectral ranges, or exogenous contrast agent based on gold nanorods.
Visualization of tissue molecules targeted by the contrast agent provides molecular information. Visulization of blood at
multiple colors of light provides functional information on blood concentration and oxygen saturation. Optoacoustic
imaging, using two or more laser illumination wavelengths, permits an assessment of the angiogenesis-related
microvasculature, and thereby, an evaluation of the tumor stage and its metastatic potential.
The optoacoustic imaging system was also used to generate molecular images of the malignancy-related receptors
induced by the xenografts of BT474 mammary adenocarcinoma cells in nude mice. The development of the latter images
was facilitated by the use of an optoacoustic contrast agent that utilizes gold nanorods conjugated to monoclonal
antibody raised against HER2/neu antigens. These nanorods possess a very strong optical absorption peak that can be
tuned in the near-infrared by changing their aspect ratio. The effective conversion of the optical energy into heat by the
gold nanorods, followed by the thermal expansion of the surrounding water, makes these nanoparticles an effective
optoacoustic contrast agent. Optical scattering methods and x-ray tomography may also benefit from the application of
this contrast agent. Administration of the gold nanorod bioconjugates to mice resulted in an enhanced contrast of breast
tumors relative the background of normal tissues in the nude mouse model. The combination of this novel contrast agent
and optoacoustic imaging has the potential to become a useful imaging modality, for preclinical research in murine
models of cancer and other human diseases.
In this work, we demonstrate the use of Two-Photon Fluorescence Lifetime Imaging Microscopy (TP-FLIM) for
intracellular calcium ([Ca2+]i) measurement with a calcium sensitive fluorescent dye in a neuronal cell model and brain
tissue. Calcium perturbations were induced via chemical stimulation and our results signify the potential of TP-FLIM
for quantitative ([Ca2+]i) measurement within the physiological range of intracellular [Ca2+].
We proposed and have been developing real-time, noninvasive monitoring of blood oxygenation, total hemoglobin concentration, and thermotherapy including hyperthermia, coagulation, and cryotherapy. In this paper we propose to use the optoacoustic technique for monitoring of nanoparticle-mediated photothermal therapy (NPT) of tumors. NPT is based on heating exogenous strongly-absorbing nanoparticles selectively delivered in tumors. Real-time monitoring of NPT is necessary for precise tumor therapy with minimal damage to normal tissues. In this study we injected PEGylated and non-PEGylated carbon nanoparticles in nude mice bearing human tumors (5-15 mm) and irradiated the tumors for 10 minutes with nanosecond Nd:YAG laser pulses which produced both thermal damage to the tumors and optoacoustic signals for monitoring NPT in real time. Irradiation of tumors was performed during or after (3 or 24 hours) nanoparticle injection. Amplitude and temporal parameters of optoacoustic signals (measured with a custom-made wide-band optoacoustic probe) correlated well with nanoparticle injection, temperature rise in tumors, and tumor coagulation. Substantial thermal damage in large areas of the tumors was produced when optimal irradiation parameters were used. Monte Carlo modeling of light distribution in tumors and optoacoustic theory were applied to study kinetics of nanoparticle concentration in the tumors. Our results demonstrated that the optoacoustic technique can be used for real-time monitoring of NTP and provide precise tumor therapy with minimal damage to normal tissues.
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