We present a study of the functional photoacoustic imaging of tumor hypoxia in mice in vivo. Based on spectroscopic photoacoustic tomography that detects the optical absorption of oxy- and deoxy-hemoglobins, the blood oxygen saturation and the vascularization of brain tumors were visualized. U87 glioblastoma tumor cells were inoculated intracranially at a 3-mm depth from the surface of the nude mouse head seven days before the experiment. Increased blood content and hypoxia inside the tumor vasculature were detected through the intact skin and skull. This technique will be useful for future studies on tumor metabolic activities in the brain and hypoxia-related therapeutic resistance.
KEYWORDS: Tumors, Molecular imaging, Spectroscopy, Brain, Imaging spectroscopy, Acquisition tracking and pointing, In vivo imaging, Photoacoustic spectroscopy, Neuroimaging, Near infrared
Molecular imaging is a newly emerging field in which the modern tools of molecular and cell biology have been
married to state-of-the-art technologies for noninvasive imaging. The study of molecular imaging will lead to better
methods for understanding biological processes as well as diagnosing and managing disease. Here we present
noninvasive in vivo spectroscopic photoacoustic tomography (PAT)-based molecular imaging of αvβ3 integrin in a
nude mouse U87 brain tumor. PAT combines high optical absorption contrast and high ultrasonic resolution by
employing short laser pulses to generate acoustic waves in biological tissues through thermoelastic expansion.
Spectroscopic PAT-based molecular imaging offers the separation of the contributions from different absorbers based
on the differences in optical absorption spectra among those absorbers. In our case, in the near infrared (NIR) range,
oxy-heamoglobin (O2Hb), deoxy-heamoglobin (HHb) and the injected αvβ3-targeted peptide-ICG conjugated NIR
fluorescent contrast agent are the three main absorbers. Therefore, with the excitation by multiple wavelength laser
pulses, spectroscopic PAT-based molecular imaging not only provides the level of the contrast agent accumulation in
the U87 glioblastoma tumor, which is related to the metabolism and angiogenesis of the tumor, but also offers the
information on tumor angiogenesis and tumor hypoxia.
Simultaneous transcranial imaging of two functional parameters, the total concentration of hemoglobin and the hemoglobin oxygen saturation, in the rat brain in vivo is realized noninvasively using laser-based photoacoustic tomography (PAT). As in optical diffusion spectroscopy, PAT can assess the optical absorption of endogenous chromophores, e.g., oxygenated and deoxygenated hemoglobins, at multiple optical wavelengths. However, PAT can provide high spatial resolution because its resolution is diffraction-limited by photoacoustic signals rather than by optical diffusion. Laser pulses at two wavelengths are used sequentially to acquire photoacoustic images of the vasculature in the cerebral cortex of a rat brain through the intact skin and skull. The distributions of blood volume and blood oxygenation in the cerebral cortical venous vessels, altered by systemic physiological modulations including hyperoxia, normoxia, and hypoxia, are visualized successfully with satisfactory spatial resolution. This technique, with its prominent sensitivity to endogenous contrast, can potentially contribute to the understanding of the interrelationship between neural, hemodynamic, and metabolic activities in the brain.
Based on the multi-wavelength laser-based photoacoustic tomography, non-invasive in vivo imaging of functional parameters, including the hemoglobin oxygen saturation and the total concentration of hemoglobin, in small-animal brains was realized. The high sensitivity of this technique is based on the spectroscopic differences between oxy- and deoxy-hemoglobin while its spatial resolution is bandwidth-limited by the photoacoustic signals rather than by the optical diffusion as in optical imaging. The point-by-point distributions of blood oxygenation and blood volume in the cerebral cortical venous vessels, altered by systemic physiological modulations including hyperoxia, normoxia and hypoxia, were visualized successfully through the intact skin and skull. This technique, with its prominent intrinsic advantages, can potentially accelerate the progress in neuroscience and provide important new insights into cerebrovascular physiology and brain function that are of great significance to the neuroscience community.
KEYWORDS: Tumors, Luminescence, Acquisition tracking and pointing, In vivo imaging, Brain, Photoacoustic tomography, Neuroimaging, Near infrared, Head, Signal detection
We present a dual modality imaging technique by combining photoacoustic tomography (PAT) and near-infrared (NIR) fluorescence imaging for the study of animal model tumors. PAT provides high-resolution structural images of tumor angiogenesis, and fluorescence imaging offers high sensitivity to molecular probes for tumor detection. Coregistration of the PAT and fluorescence images was performed on nude mice with M21 human melanoma cell lines with αvβ3 integrin expression. An integrin αvβ3-targeted peptide-ICG conjugated NIR fluorescent contrast agent was used as the molecular probe for tumor detection. PAT was employed to noninvasively image the brain structures and the angiogenesis associated with tumors in nude mice. Coregistration of the PAT and fluorescence images was used in this study to visualize tumor location, angiogenesis, and brain structure simultaneously.
Photoacoustic tomography (PAT) in a circular scanning configuration was developed to image the deeply embedded optical heterogeneity in biological tissues. Based on the intrinsic contrast between blood and chicken breast muscle, an embedded blood object that was 5 cm deep in the tissue was detected using pulsed laser light at a wavelength of 1064 nm. Compared with detectors for flat active surfaces, cylindrically focused ultrasonic transducers can reduce the interference generated from the off-plane photoacoustic sources and make the image in the scanning plane clearer. While the optical penetration was optimized with near-infrared laser pulses of 800 nm in wavelength, the optical contrast was enhanced by indocyanine green (ICG) whose absorption peak matched the laser wavelength. This optimized PAT was able to image fine objects embedded at a depth of up to 5.2-cm, which is 6.2 times the 1/e optical penetration depth, in chicken breast muscle, at a resolution of < ~750 microns with a sensitivity of <7 pmol of ICG in blood. The resolution was found to deteriorate slowly with increasing imaging depth.
Since optical contrast is sensitive to functional parameters, including the hemoglobin oxygen saturation and the total concentration of hemoglobin, imaging based on optical contrast has been widely employed for the real-time monitoring of tissue oxygen consumption and hemodynamics in biological tissues. However, due to the overwhelming scattering of light in tissues, traditional optical imaging modalities cannot provide satisfactory spatial resolution. Functional photoacoustic tomography is a novel technique that combines high optical contrast and high ultrasonic resolution. Here, we present our study of a laser-based photoacoustic technique that, for the first time to our knowledge, monitors blood oxygenation in the rat brain in vivo. The cerebral blood oxygenation in the rat brain was imaged by photoacoustic detection at two wavelengths. The change in the hemoglobin oxygen saturation in the brain vessels as a result of the alternation from hyperoxia status to hypoxia status was visualized successfully with satisfactory spatial resolution. This work demonstrates that photoacoustic technique, based on the spectroscopic absorption of oxy- and deoxy-hemoglobin, can provide accurate functional imaging of cerebral blood oxygenation in the small-animal brain non-invasively with the skin and skull intact.
Photoacoustic tomography employs short laser pulses to generate acoustic waves. The photoacoustic image of a test sample can be reconstructed using the detected acoustic signals. The reconstructed image is characterized by the convolution of the sample structure in optical absorption, the laser pulse, and the impulse response of the ultrasonic transducer used for detection. Although laser-induced ultrasonic waves cover a wide spectral range, a single transducer can receive only part of the spectrum because of its limited bandwidth. To systematically analyze this problem, we constructed a photoacoustic tomographic system that uses multiple ultrasonic transducers, each at a different central frequency, to simultaneously receive the induced acoustic waves. The photoacoustic images associated with the different transducers were compared and analyzed. The system was used to detect the vascular system of the rat brain. The vascular vessels in the brain cortex were revealed by all of the transducers, but the image resolutions differed. The higher frequency detectors with wider bandwidths provided better image resolution.
Optical contrast agents, such as indocyanine dyes, nano-particles and their functional derivatives, have been widely applied to enhance the sensitivity and specificity of optical imaging. However, due to the overwhelming scattering of light in biological tissues, the spatial resolution of traditional optical imaging degrades drastically as the imaging depth increases. For the first time to our knowledge, non-invasive in vivo photoacoustic imaging of an optical contrast agent, distributed in the rat brain, was implemented with near-infrared light. Injection of indocyanine green polyethylene glycol, a contrast agent with a high absorption at the 805-nm wavelength, into the circulatory system of a rat enhanced the absorption contrast between the blood vessels and the background brain tissues. Because near-infrared light can penetrate deep into the brain tissues through the skin and skull, we were able to successfully reconstruct the vascular distribution in the rat brain from the detected photoacoustic signals. The dynamic concentration of this contrast agent in the brain blood after the intravenous injection was also studied. This work proved that the distribution of an exogenous contrast agent in biological tissues can be imaged clearly and accurately by photoacoustic tomography. This new technology has high potential for application in dynamic and molecular medical imaging.
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