Spectroscopic Photoacoustic Imaging Research Articles

In-vivo ultrasound and photoacoustic image- guided photothermal cancer therapy using silica-coated gold nanorods

In nanoparticle-augmented photothermal therapy, evaluating the delivery and spatial distribution of nanoparticles, followed by remote temperature mapping and monitoring, is essential to ensure the optimal therapeutic outcome. The utility of ultrasound and photoacoustic imaging to assist photothermal therapy has been previously demonstrated. Here, using a mouse xenograft tumor model, it is demonstrated in vivo that ultrasound-guided photoacoustic imaging can be used to plan the treatment and to guide the therapy. To evaluate nanoparticle delivery and spatial distribution, three-dimensional ultrasound and spectroscopic photoacoustic imaging of a mouse with a tumor was performed before and after intravenous injection of silica-coated gold nanorods. After injection and sufficient circulation of nanoparticles, photothermal therapy was performed for 5 min using an 808-nm continuouswave laser. During the photothermal therapy, photoacoustic images were acquired continuously and used to measure the temperature changes within tissue. A heterogeneous distribution of temperature, which was spatially correlated with the measured distribution of nanoparticles, indicated that peak temperatures of 53°C were achieved in the tumor. An Arrhenius thermal damage model determined that this thermal deposition would result in significant cell death. The results of this study suggest that ultrasound and photoacoustic imaging can effectively guide photothermal therapy to achieve the desired thermal treatment.

Optimization of in vivo spectroscopic photoacoustic imaging by smart optical wavelength selection

Spectroscopic photoacoustic (sPA) imaging is an emerging biomedical imaging modality which can be used to simultaneously identify multiple optical absorbers in tissue. With current technology, the image acquisition time is limited primarily by the laser repetition rate, and sPA image acquisition can take seconds to minutes, depending on the system and imaging volume. We have developed an algorithm that can be used to eliminate extraneous wavelengths and decrease image acquisition time while maintaining image quality. Here, we show the effect the wavelength selection has on in vivo sPA imaging. As an example, dye draining to the lymph node of a mouse was imaged. We demonstrate that sPA image quality is more accurately preserved when wavelengths are chosen based on the spectral features of the absorbers compared to often-used selection of evenly spaced wavelengths. In fact, only three wavelengths are needed to accurately spectrally unmix the dye from oxygenated and deoxygenated hemoglobin.

In vitro photoacoustic visualization of myocardial ablation lesions

Radiofrequency (RF) ablation to treat atrial arrhythmia is limited by the inability to reliably assess lesion durability and transmurality. The purpose of this study was to determine the feasibility of photoacoustic characterization of myocardial ablation lesions in vitro. In this study, we investigated the feasibility of combined ultrasound (US) and spectroscopic photoacoustic imaging to visualize RF ablation lesions in three dimensions (3D) based on unique differences in the optical absorption spectra between normal and ablated myocardial tissue. Tissue samples were excised from the ventricles of fresh porcine hearts. Lesions were generated using an RF catheter ablation system using 20 to 30 W of power applied for 40 to 60 seconds. Ablated samples were imaged in the near-infrared regime (740-780 nm) using a combined PA/US imaging system. Measured PA spectra were correlated to the absorption spectra of deoxyhemoglobin and ablated tissue to produce a tissue characterization map (TCM) identifying 3D lesion location and extent. Tissue samples were stained and photographed for gross pathology. TCM and gross pathology images were coregistered to assess TCM accuracy. TCM reliably characterized ablated and non-ablated tissue up to depths of 3 mm. TCM also assessed lesion position and extent with submillimeter accuracy in multiple dimensions. Segmented TCMs achieved >69% agreement with gross pathology. The study results suggest that spectroscopic photoacoustic imaging has the potential to accurately assess RF ablation lesion size and position with submillimeter precision and may be well suited to guide transcatheter RF atrial ablation in clinical practice.

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/cm(2) (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

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.

Optical wavelength selection for improved spectroscopic photoacoustic imaging

Spectroscopic photoacoustic imaging has the potential to become a powerful tool that can estimate distributions of optically absorbing chromophores in the body. We have developed an algorithm to select imaging wavelengths for spectroscopic photoacoustics given the spectra of expected chromophores. The algorithm uses the smallest singular value of a matrix constructed from the absorption spectra as a criterion to remove extraneous wavelengths. The method performed significantly better than an approach where evenly spaced wavelengths were used in the presence of noise and wavelength-dependent attenuation of light in tissue. Finally, the algorithm was applied to photoacoustic imaging of a phantom containing indocyanine green dye and silica-coated gold nanorods, demonstrating significant improvements in the ability to estimate relative contrast agent concentrations compared to the case where evenly spaced wavelengths were chosen. In summary, our work provides a versatile framework to select optical wavelengths and evaluate sets of absorbers for spectroscopic photoacoustic imaging.

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 photography, confocal scanning laser ophthalmoscopy (cSLO), and optical coherence tomography (OCT), 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 sensitivity. 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. More importantly, based on the well-developed spectroscopic photoacoustic imaging, 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 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 contrasts. 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.

Spectroscopic photoacoustic imaging of lipid-rich plaques in the human aorta in the 740 to 1400 nm wavelength range

Spectroscopic photoacoustic imaging has the potential to discriminate between normal and lipid-rich atheromatous areas of arterial tissue by exploiting the differences in the absorption spectra of lipids and normal arterial tissue in the 740 to 1400 nm wavelength range. Identification of regions of high lipid concentration would be useful to identify plaques that are likely to rupture (vulnerable plaques). To demonstrate the feasibility of visualizing lipid-rich plaques, samples of human aortas were imaged in forward mode, at wavelengths of 970 and 1210 nm. It was shown that the structure of the arterial wall and the boundaries of lipid-rich plaques obtained from the photoacoustic images were in good agreement with histology. The presence of lipids was also confirmed by comparing the photoacoustic spectra (740 to 1400 nm) obtained in a region within the plaque to the spectral signature of lipids. Furthermore, a lipid-rich plaque was successfully imaged while illuminating the sample through 2.8 mm of blood demonstrating the possibility of implementing the photoacoustic technique in vivo.

Noninvasive detection of intimal xanthoma using combined ultrasound, strain rate and photoacoustic imaging

Background and motivation The structure, composition and mechanics of carotid artery are good indicators of early progressive atherosclerotic lesions. The combination of three imaging modalities (ultrasound, strain rate and photoacoustic imaging) which could provide corroborative information about the named arterial properties could enhance the characterization of intimal xanthoma. Methods The experiments were performed using a New Zealand white rabbit model of atherosclerosis. The aorta excised from an atherosclerotic rabbit was scanned ex vivo using the three imaging techniques: (1) ultrasound imaging of the longitudinal section: standard ultrasound B-mode (74 Hz frame rate); (2) strain rate imaging: the artery was flushed with blood and a 1.5 Hz physiologic pulsation was induced, while the ultrasound data were recorded at higher frame rate (296 Hz); (3) photoacoustic imaging: the artery was irradiated with nanosecond pulsed laser light of low fluence in the 1210–1230 nm wavelength range and the photoacoustic data was recorded at 10 Hz frame rate. Post processing algorithms based on cross-correlation and optical absorption variation were implemented to derive strain rate and spectroscopic photoacoustic images, respectively. Results Based on the spatio-temporal variation in displacement of different regions within the arterial wall, strain rate imaging reveals differences in tissue mechanical properties. Additionally, spectroscopic photoacoustic imaging can spatially resolve the optical absorption properties of arterial tissue and identify the location of lipid pools. Conclusions The study demonstrates that ultrasound, strain rate and photoacoustic imaging can be used to simultaneously evaluate the structure, the mechanics and the composition of atherosclerotic lesions to improve the assessment of plaque vulnerability.

In vivo three-dimensional spectroscopic photoacoustic imaging for monitoring nanoparticle delivery.

In vivo monitoring of nanoparticle delivery is essential to better understand cellular and molecular interactions of nanoparticles with tissue and to better plan nanoparticle-mediated therapies. We developed a three-dimensional ultrasound and photoacoustic (PA) imaging system and a spectroscopic PA imaging algorithm to identify and quantify the presence of nanoparticles and other tissue constituents. Using the developed system and approach, three-dimensional in vivo imaging of a mouse with tumor was performed before and after intravenous injection of gold nanorods. The developed spectroscopic PA imaging algorithm estimated distribution of nanoparticle as well as oxygen saturation of blood. Moreover, silver staining of excised tumor tissue confirmed nanoparticle deposition, and showed good correlation with spectroscopic PA images. The results of our study suggest that three-dimensional ultrasound-guided spectroscopic PA imaging can monitor nanoparticle delivery in vivo.

TU‐C‐220‐08: Assessing Intra‐Tumor Hemodynamics and Oxygen Concentration Using Photoacoustic Computed Tomography

Purpose: The goal is to develop a method to non‐invasively assess intra‐tumor pO2 using photoacoustic imaging. It is well established that the oxygen status of a tumor plays a critical role in the therapeutic response and resistance, resulting in treatment failure and poor disease‐free and overall survival. Clinical techniques, such as polarographic electrode, photoluminescence‐quenching, or pimonidazole, are invasive and lack the patho‐physiological etiology leading to hypoxia. A biophysical model is proposed to determine local pO2 based on fusing hemodynamic measurements acquired from dynamic contrast‐enhanced and spectroscopic photoacoustic imaging (DCE‐PCT and PCT‐S). Methods: MCF7 and MCF7/VEGF xenograft breast tumors were first scanned within a prototype PCT scanner to obtain parametric maps of oxygen saturation and hemoglobin concentration and within a clinical CT scanner to obtain dynamic contrast‐enhanced data (DCE‐CT) to obtain parametric maps of perfusion, fractional‐plasma‐volume, and fractional‐interstitial‐volume. These maps were fused based on a mathematical model of tissue oxygen delivery, and compared to measurements taken with an OxyLite probe. To replace DCE‐CT, a new generation of PCT scanner was developed to acquire DCE‐PCT data and realize physiological maps. Mice with MDA‐ MB‐231 xenograft breast tumors were i.v. injected with ICG, and scanned prior to and every 12‐seconds for up to 4‐minutes and at 15‐minutes post‐injection. These contrast‐enhance curves were fit compartmental models to obtain maps of tumor perfusion and fractional plasma volumes. Results: Preliminary results comparing pO2 measurements implementing biophysical models and PCT‐S and DCE‐CT and OxyLite probe measured a linear correlation of R2=0.82, and a slope of 0.975(±0.10) and an intercept of −0.093(±2.2)mmHg. The first parametric maps of tumor perfusion and fractional plasma volume using DCE‐PCT will be presented and compared to DCE‐CT, and ICG kinematics identifying regions of high extravasation. Conclusions: A biophysical model of pO2 based on hemodynamic data and physiological maps in breast tumors obtained from photoacoustic imaging has been demonstrated.

MO-D-220-02: Assessing Intra-Tumor Hemodynamics and Oxygen Concentration Using Photoacoustic Computed Tomography

Purpose:The goal is to develop a method to non-invasively assess intra- tumor pO2 using photoacoustic imaging. It is well established that the oxygen status of a tumor plays a critical role in the therapeutic response and resistance, resulting in treatment failure and poor disease-free and overall survival. Clinical techniques, such as polarographic electrode, photoluminescence-quenching, or pimonidazole, are invasive and lack the patho-physiological etiology leading to hypoxia. A biophysical model is proposed to determine local pO2 based on fusing hemodynamic measurements acquired from dynamic contrast-enhanced and spectroscopic photoacoustic imaging (DCE-PCT and PCT-S). Methods: MCF7 and MCF7/VEGF xenograft breast tumors were first scanned within a prototype PCT scanner to obtain parametric maps of oxygen saturation and hemoglobin concentration and within a clinical CT scanner to obtain dynamic contrast-enhanced data (DCE-CT) to obtain parametric maps of perfusion, fractional-plasma-volume, and fractional-interstitial-volume. These maps were fused based on a mathematical model of tissue oxygen delivery, and compared to measurements taken with an OxyLite probe. To replace DCE-CT, a new generation of PCT scanner was developed to acquire DCE-PCT data and realize physiological maps. Mice with MDA- MB-231 xenograft breast tumors were i.v. injected with ICG, and scanned prior to and every 12-seconds for up to 4-minutes and at 15-minutes post- injection. These contrast-enhance curves were fit compartmental models to obtain maps of tumor perfusion and fractional plasma volumes. Results: Preliminary results comparing pO2 measurements implementing biophysical models and PCT-S and DCE-CT and OxyLite probe measured a linear correlation of R2=0.82, and a slope of 0.975(±0.10) and an intercept of − 0.093(±2.2)mmHg. The first parametric maps of tumor perfusion and fractional plasma volume using DCE-PCT will be presented and compared to DCE-CT, and ICG kinematics identifying regions of high extravasation. Conclusions: A biophysical model of pO2 based on hemodynamic data and physiological maps in breast tumors obtained from photoacoustic imaging has been demonstrated. NIH/SBIR Funding from OptoSonics, Inc, OptoSonics, Inc., 108 Straight Road, Oriental, NC 28571; Consulting for Endra, Inc, 35 Research Drive, Suite 100, Ann Arbor, MI 48103.

Detection of macrophages and lipid using ultrasound guided spectroscopic intravascular photoacoustic imaging.

Macrophages and lipid are two key components in the development and characterization of atherosclerotic plaques. Imaging the activity and distribution of macrophages and lipid in the vessel wall would help identify the vulnerable plaques. In this study, spectroscopic intravascular photoacoustic (sIVPA) imaging was used to detect phagocytically active macrophages and deposits of lipid simultaneously. Polyethylene glycol coated spherical gold nanoparticles (Au NPs) were intravenously injected in vivo into an atherosclerotic rabbit allowing phagocytically active macrophages within the plaques to be labeled with Au NPs. Atherosclerotic aorta was harvested and scanned with a bench-top sIVPA imaging system. Au NP-labeled macrophages were detected in the 710–770 nm wavelength range because of the plasmon resonance coupling effect. Lipid was imaged in 1210–1230 nm wavelength range based on the characteristic optical absorption spectrum of fatty acids. An image processing method was developed to identify Au-NP-labeled macrophages and lipid deposits from the multiwavelength IVPA data. Coregistered sIVPA images were combined with corresponding IVUS images to demonstrate the location of Au NP-labeled macrophages and lipid within the vessel wall. Finally, histochemistry stains confirmed that combined sIVPA and IVUS imaging can successfully detect the distribution of phagocytically active macrophages and lipid deposits.

Photoacoustic tomography of foreign bodies in soft biological tissue

In detecting small foreign bodies in soft biological tissue, ultrasound imaging suffers from poor sensitivity (52.6%) and specificity (47.2%). Hence, alternative imaging methods are needed. Photoacoustic (PA) imaging takes advantage of strong optical absorption contrast and high ultrasonic resolution. A PA imaging system is employed to detect foreign bodies in biological tissues. To achieve deep penetration, we use near-infrared light ranging from 750 to 800 nm and a 5-MHz spherically focused ultrasonic transducer. PA images were obtained from various targets including glass, wood, cloth, plastic, and metal embedded more than 1 cm deep in chicken tissue. The locations and sizes of the targets from the PA images agreed well with those of the actual samples. Spectroscopic PA imaging was also performed on the objects. These results suggest that PA imaging can potentially be a useful intraoperative imaging tool to identify foreign bodies.

TU-E-201C-09: Photoacoustic Tomography for Imaging in the Mammographic Geometry

Purpose: To add spectroscopicphotoacoustictomography (S‐PAT) to an ultrasound imagingsystem in the mammographic geometry to test whether this minimally invasive and ultimately inexpensive modality can detect and interpret small vascular anomalies, providing information supplemental to that of x‐ray and ultrasound much as is done with MRI. Blood oxygenation and blood volume information from S‐PAT might replace or exceed information provided by MRI resolution, possibly without contrast agents. Method and Materials: The specimen surface is flooded with a <25 ns pulse of light from a tunable, 720–900 nm plus 1064 nm, laser. An 8 cm diameter, 570 element PVDF array in a square grid of 3.2 mm spacing serves as the ultrasound receiver of the thermoacoustic signals produced by optical absorption. 20 channels were multiplexed to acquire the 570 signals. Delay‐and‐sum beamforming produces an image of the thermoacoustic sources. The receiver bandwidth, here 0.4 to ∼3 MHz at −12 dB is critical, as the center frequency from a spherical target is inversely proportional to the object diameter. A 3 mm ID tube of blood was imbedded in porcine tissues, 24mm of fat and 28mm of loin to the 725 nm illuminated surface and 45mm of loin to the receiver. 256 pulses of 70 mJ from a preliminary laser were averaged. Results: The blood tube could be easily discerned at the resolution anticipated for the acoustic frequency from the blood tube and the available receiver aperture. Conclusion: This described example is through optical and acoustical path lengths greater than that required for imaging from each side of most breasts at modest compression. This and similar results in vitro give hope of adequate sensitivity to blood collections in the breast of appropriate spatial frequencies. S‐PAT should be compatible for integration in a combined tomosynthesis/ultrasound/optical breast imagingsystem. Supported by R01_CA91713,

TU‐A‐201C‐02: Multi‐modality Breast Imaging Systems: Tomo/Ultrasound/Optics, Ultrasound

Automated 3D ultrasound is being developed extensively to make screening for breast cancer more practical within the US and other medical communities. Ultrasound is sensitive to certain cancers that are not well detected by mammography, particularly in dense breasts, and characterizes cysts that require no further diagnosis. But US screening sees many apparent abnormalities, increasing callbacks. US misses many cancers detected by their microcalcifications. Colocated ultrasound and tomosynthesis essentially eliminates these barriers by allowing rapid clear identification of structures of interest in corresponding modalities, if sufficient US image quality and coverage can be achieved in the mammographic geometry.FDA approval of breast tomosynthesis (BT) has been delayed because of dose considerations and poor resolution for calcifications. With equipment now in prototypes — much better x‐ray detectors at lower doses, stable tube positions and larger viewing angles, the improved diagnosis should outweigh dose considerations in most or all usual breast types. BT is clearly a leading platform for multimodality 3D breast imaging.MRI is very sensitive to abnormalities of vascular volume, permeability, filling and drainage. Time consuming, expensive, with contrast injections, it finds a high percentage of cancers earlier than do other modalities and is recommended for screening of high risk cases. Ultrasonic CT performed carefully in the same geometry, if not the same system, should provide the same roll for MRI detected lesions that US does combined in a system with BT.Doppler ultrasound, without contrast agents, is not sensitive to the slow moving blood in most veins and smaller vessels. Diffuse optical imaging and photoacoustic tomography (PAT) are sensitive to vascular anomalies, with higher resolution, but optical penetration limitations for PAT. The penetration may be adequate with PAT imaging from both sides of the compressed breast. The ability of such optical absorption imaging at different wavelengths (spectroscopic PAT, S‐PAT) to distinguish oxy‐ and deoxyhemoglobin appears to add to discrimination of benign from malignant masses and might find use in selecting and tracking therapies. Coregistered BT, US and optical imaging might well provide similar screening and diagnostic effectiveness in a single, 15 minute examination, as the combination of current mammography, ultrasound and contrast MRI examinations, a combination that can hardly be described as screening.In addition to the considerably orthogonal information provided by BT, conventional US and S‐PAT, other US imaging information such as scatterer size and density and elasticity is available. The mammographic geometry and acoustic array for S‐PAT are compatible with transmission ultrasound imaging, which opens the possibility of attenuation, speed of sound and density imaging as well as reflection mode.Learning Objectives:1. Learn the different information available from modalities with screening potential.2. Understand near term possibilities for combined systems in prone and mammographic positions.3. Understand which modalities offer unique possibilities for long term noninvasive and contrast imaging, as well as therapies, and are therefore worth investments in early, simple equipment and education.Research collaboration: General Electric Company, Sonetics Corp., Zonari Medical Systems, Light Age, Inc. Supported by NIH Grants R01CA91713 and R01CA115267.

Sentinel Lymph Nodes and Lymphatic Vessels: Noninvasive Dual-Modality in Vivo Mapping by Using Indocyanine Green in Rats—Volumetric Spectroscopic Photoacoustic Imaging and Planar Fluorescence Imaging

To noninvasively map sentinel lymph nodes (SLNs) and lymphatic vessels in rats in vivo by using dual-modality nonionizing imaging-volumetric spectroscopic photoacoustic imaging, which measures optical absorption, and planar fluorescence imaging, which measures fluorescent emission-of indocyanine green (ICG). Institutional animal care and use committee approval was obtained. Healthy Sprague-Dawley rats weighing 250-420 g (age range, 60-120 days) were imaged by using volumetric photoacoustic imaging (n = 5) and planar fluorescence imaging (n = 3) before and after injection of 1 mmol/L ICG. Student paired t tests based on a logarithmic scale were performed to evaluate the change in photoacoustic signal enhancement of SLNs and lymphatic vessels before and after ICG injection. The spatial resolutions of both imaging systems were compared at various imaging depths (2-8 mm) by layering additional biologic tissues on top of the rats in vivo. Spectroscopic photoacoustic imaging was applied to identify ICG-dyed SLNs. In all five rats examined with photoacoustic imaging, SLNs were clearly visible, with a mean signal enhancement of 5.9 arbitrary units (AU) + or - 1.8 (standard error of the mean) (P < .002) at 0.2 hour after injection, while lymphatic vessels were seen in four of the five rats, with a signal enhancement of 4.3 AU + or - 0.6 (P = .001). In all three rats examined with fluorescence imaging, SLNs and lymphatic vessels were seen. The average full width at half maximum (FWHM) of the SLNs in the photoacoustic images at three imaging depths (2, 6, and 8 mm) was 2.0 mm + or - 0.2 (standard deviation), comparable to the size of a dissected lymph node as measured with a caliper. However, the FWHM of the SLNs in fluorescence images widened from 8 to 22 mm as the imaging depth increased, owing to strong light scattering. SLNs were identified spectroscopically in photoacoustic images. These two modalities, when used together with ICG, have the potential to help map SLNs in axillary staging and to help evaluate tumor metastasis in patients with breast cancer.

Simultaneous Molecular and Hypoxia Imaging of Brain Tumors In Vivo Using Spectroscopic Photoacoustic Tomography

Noninvasive molecular and functional imaging in vivo is promising for detecting and monitoring various physiological conditions in animals and ultimately humans. To this end, we present a novel noninvasive technology, spectroscopic photoacoustic tomography (SPAT), which offers both strong optical absorption contrast and high ultrasonic spatial resolution. Optical contrast allows spectroscopic separation of signal contributions from multiple optical absorbers (e.g., oxyhemoglobin, deoxyhemoglobin, and a molecular contrast agent), thus enabling simultaneous molecular and functional imaging. SPAT successfully imaged with high resolution the distribution of a molecular contrast agent targeting integrin overexpressed in human U87 glioblastomas in nude mouse brains. Simultaneously, SPAT also imaged the hemoglobin oxygen saturation and the total hemoglobin concentration of the vasculature, which revealed hypoxia in tumor neovasculature. Therefore, SPAT can potentially lead to better understanding of the interrelationships between hemodynamics and specific biomarkers associated with tumor progression.

In vivo imaging and characterization of hypoxia-induced neovascularization and tumor invasion

Hypoxia is a critical event in tumor progression and angiogenesis. Hypoxia can be detected noninvasively by a novel spectroscopic photoacoustic tomography technology (SPAT) and this finding is supported by our molecular biology investigation aimed to elucidate the etiopathogenesis of SPAT detected hypoxia and angiogenesis. The present study provides an integrated approach to define oxygen status (hypoxia) of intracranial tumor xenografts using spectroscopic photoacoustic tomography. Brain tumors can be identified based on their distorted vascular architecture and oxygen saturation (SO2) images. Noninvasive in vivo tumor oxygenation imaging using SPAT is based on the spectroscopic absorption differences between oxyhemoglobin (O2Hb) and deoxyhemoblobin (HHb). Sprague-Dawley rats inoculated intracranially with ENU1564, a carcinogen-induced rat mammary adenocarcinoma cell line, were imaged with SPAT three weeks post inoculation. Proteins important for tumor angiogenesis and invasion were detected in hypoxic brain foci identified by SPAT and were elevated compared with control brain. Immunohistochemistry, Western blotting, and semi-quantitative RT-PCR showed that HIF-1 alpha, VEGF-A, and VEGFR2 (Flk-1) protein and mRNA expression levels were significantly higher (P < 0.05) in brain tumor tissues compared to normal brain. Gelatin zymography and RT-PCR demonstrated the upregulation of MMP-9 in tumor foci compared with brain control. Together these results suggest the critical role of hypoxia in driving tumor angiogenesis and invasion through upregulation of target genes important for these functions. Moreover this report validates our hypothesis that a novel noninvasive technology (SPAT) developed in our laboratory is suitable for detection of tumors, hypoxia, and angiogenesis.