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Find video protocols related to scientific articles indexed in Pubmed.
Photoacoustic Brain Imaging: from Microscopic to Macroscopic Scales.
Neurophotonics
PUBLISHED: 11-18-2014
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Human brain mapping has become one of the most exciting contemporary research areas, with major breakthroughs expected in the following decades. Modern brain imaging techniques have allowed neuroscientists to gather a wealth of anatomic and functional information about the brain. Among these techniques, by virtue of its rich optical absorption contrast, high spatial and temporal resolutions, and deep penetration, photoacoustic tomography (PAT) has attracted more and more attention, and is playing an increasingly important role in brain studies. In particular, PAT complements other brain imaging modalities by providing high-resolution functional and metabolic imaging. More importantly, PAT's unique scalability enables scrutinizing the brain at both microscopic and macroscopic scales, using the same imaging contrast. In this Review, we present the state-of-the-art PAT techniques for brain imaging, summarize representative neuroscience applications, outline the technical challenges in translating PAT to human brain imaging, and envision potential technological deliverables.
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Breakthrough in Photonics 2013: Photoacoustic Tomography in Biomedicine.
IEEE Photonics J
PUBLISHED: 11-11-2014
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Photoacoustic tomography (PAT) is one of the fastest growing biomedical imaging modalities in the last decade. Building on its high scalability and complementary imaging contrast to other mainstream modalities, PAT has gained substantial momentum in both preclinical and clinical studies. In 2013, PAT has grown markedly in both its technological capabilities and biomedical applications. In particular, breakthroughs have been made in super-resolution imaging, deep blood flow measurement, small animal resting state brain mapping, video rate functional human imaging, and human breast imaging. These breakthroughs have either successfully solved long-standing technical issues in PAT or significantly enhanced its imaging capability. This Review will summarize state-of-the-art developments in PAT and highlight a few representative achievements of the year 2013.
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Amplitude-masked photoacoustic wavefront shaping and application in flowmetry.
Opt Lett
PUBLISHED: 11-01-2014
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Optical-resolution photoacoustic flowmetry (PAF) allows noninvasive single-cell flow measurements. However, its operational depth is limited by optical diffusion, which prevents focusing beyond shallow depths in scattering media, as well as reducing the measurement signal-to-noise ratio (SNR). To overcome this limitation, we used binary-amplitude wavefront shaping to enhance light focusing in the presence of scattering. Here, the transmission modes that contributed constructively to the intensity at the optical focus were identified and selectively illuminated, resulting in a 14-fold intensity increase and a corresponding increase in SNR. This technique can potentially extend the operational depth of optical-resolution PAF beyond 1 mm in tissue.
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Grueneisen relaxation photoacoustic microscopy.
Phys. Rev. Lett.
PUBLISHED: 10-20-2014
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The temperature-dependent property of the Grueneisen parameter has been employed in photoacoustic imaging mainly to measure tissue temperature. Here we explore this property using a different approach and develop Grueneisen relaxation photoacoustic microscopy (GR-PAM), a technique that images nonradiative absorption with confocal optical resolution. GR-PAM sequentially delivers two identical laser pulses with a microsecond-scale time delay. The first laser pulse generates a photoacoustic signal and thermally tags the in-focus absorbers. When the second laser pulse excites the tagged absorbers within the thermal relaxation time, a photoacoustic signal stronger than the first one is produced, owing to the temperature dependence of the Grueneisen parameter. GR-PAM detects the amplitude difference between the two colocated photoacoustic signals, confocally imaging the nonradiative absorption. We greatly improved axial resolution from 45???m to 2.3???m and, at the same time, slightly improved lateral resolution from 0.63???m to 0.41???m. In addition, the optical sectioning capability facilitates the measurement of the absolute absorption coefficient without fluence calibration.
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Near-infrared optical-resolution photoacoustic microscopy.
Opt Lett
PUBLISHED: 08-29-2014
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Compared with visible light (380-700 nm), near-infrared light (700-1400 nm) undergoes weaker optical attenuation in biological tissue; thus, it can penetrate deeper. Herein, we demonstrate near-infrared optical-resolution photoacoustic microscopy (NIR-OR-PAM) with 1046 nm illumination. A penetration depth of 3.2 mm was achieved in chicken breast tissue ex vivo using optical fluence within the American National Standards Institute (ANSI) limit (100??mJ/cm2). Beyond ?0.6??mm deep in chicken breast tissue, NIR-OR-PAM has shown finer resolution than the visible counterpart with 570 nm illumination. The deep imaging capability of NIR-OR-PAM was validated in both a mouse ear and a mouse brain. NIR-OR-PAM of possible lipid contrast was explored as well.
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Handheld photoacoustic microscopy to detect melanoma depth in vivo.
Opt Lett
PUBLISHED: 08-15-2014
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We developed handheld photoacoustic microscopy (PAM) to detect melanoma and determine tumor depth in nude mice in vivo. Compared to our previous PAM system for melanoma imaging, a new light delivery mechanism is introduced to improve light penetration. We show that melanomas with 4.1 and 3.7 mm thicknesses can be successfully detected in phantom and in in vivo experiments, respectively. With its deep melanoma imaging ability and handheld design, this system can be tested for clinical melanoma diagnosis, prognosis, and surgical planning for patients at the bedside.
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Ultralong photonic nanojet formed by a two-layer dielectric microsphere.
Opt Lett
PUBLISHED: 08-15-2014
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A photonic nanojet is a highly focused optical beam with a subwavelength waist on the shadow side of the sphere. Successful far-field applications require long nanojets that extend afar. Using the exact Mie theory, we show that ultralong nanojets can be generated using a simple two-layer microsphere structure, using conventional optical materials that are readily available. In particular, we show that for a glass-based two-layer microsphere, the nanojet has an extension of 22 wavelengths. We also show that long nanojets can be formed using semiconductors at infrared frequencies in free space.
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In vivo optically encoded photoacoustic flowgraphy.
Opt Lett
PUBLISHED: 07-01-2014
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We present an optically encoded photoacoustic (PA) flow imaging method based on optical-resolution PA microscopy. An intensity-modulated continuous-wave laser photothermally encodes the flowing medium, and a pulsed laser generates PA waves to image the encoded heat pattern. Flow speeds can be calculated by cross correlation. The method was validated in phantoms at flow speeds ranging from 0.23 to 11??mm/s. Venous blood flow speed in a mouse ear was also measured.
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Continuous scanning of a time-reversed ultrasonically encoded optical focus by reflection-mode digital phase conjugation.
Opt Lett
PUBLISHED: 07-01-2014
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Time-reversed ultrasonically encoded (TRUE) optical focusing in turbid media was previously implemented using both analog and digital phase conjugation. The digital approach, in addition to its large energy gain, can improve the focal intensity and resolution by iterative focusing. However, performing iterative focusing at each focal position can be time-consuming. Here, we show that by gradually moving the focal position, the TRUE focal intensity is improved, as in iterative focusing at a fixed position, and can be continuously scanned to image fluorescent targets in a shorter time. In addition, our setup is, to the best of our knowledge, the first demonstration of TRUE focusing using a digital phase conjugate mirror in a reflection mode, which is more suitable for practical applications.
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Microvascular quantification based on contour-scanning photoacoustic microscopy.
J Biomed Opt
PUBLISHED: 06-09-2014
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Accurate quantification of microvasculature remains of interest in fundamental pathophysiological studies and clinical trials. Current photoacoustic microscopy can noninvasively quantify properties of the microvasculature, including vessel density and diameter, with a high spatial resolution. However, the depth range of focus (i.e., focal zone) of optical-resolution photoacoustic microscopy (OR-PAM) is often insufficient to encompass the depth variations of features of interest—such as blood vessels—due to uneven tissue surfaces. Thus, time-consuming image acquisitions at multiple different focal planes are required to maintain the region of interest in the focal zone. We have developed continuous three-dimensional motorized contour-scanning OR-PAM, which enables real-time adjustment of the focal plane to track the vessels’ profile. We have experimentally demonstrated that contour scanning improves the signal-to-noise ratio of conventional OR-PAM by as much as 41% and shortens the image acquisition time by 3.2 times. Moreover, contour-scanning OR-PAM more accurately quantifies vessel density and diameter, and has been applied to studying tumors with uneven surfaces.
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Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast.
J Biomed Opt
PUBLISHED: 06-09-2014
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Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells.
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Microcirculatory changes identified by photoacoustic microscopy in patients with complex regional pain syndrome type I after stellate ganglion blocks.
J Biomed Opt
PUBLISHED: 06-05-2014
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Complex regional pain syndrome (CRPS) is a chronic pain syndrome that causes intractable pain, disability, and poor quality of life for patients. The etiology and pathophysiology of CRPS are still poorly understood. Due to a lack of proper diagnostic tools, the prognosis of CRPS is primarily based on clinical observation. The objective of this work is to evaluate a new imaging modality, photoacoustic microscopy (PAM), for assisting diagnoses and monitoring the progress and treatment outcome of CRPS. Blood vasculature and oxygen saturation (sO?) were imaged by PAM from eight adult patients with CRPS-1. Patients' hands and cuticles were imaged both before and after stellate ganglion block (SGB) for comparison. For all patients, both vascular structure and sO? could be assessed by PAM. In addition, more vessels and stronger signals were observed after SGB. The results show that PAM can help diagnose and monitor CRPS.
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Sensitivity of photoacoustic microscopy.
Photoacoustics
PUBLISHED: 06-01-2014
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Building on its high spatial resolution, deep penetration depth and excellent image contrast, 3D photoacoustic microscopy (PAM) has grown tremendously since its first publication in 2005. Integrating optical excitation and acoustic detection, PAM has broken through both the optical diffusion and optical diffraction limits. PAM has 100% relative sensitivity to optical absorption (i.e., a given percentage change in the optical absorption coefficient yields the same percentage change in the photoacoustic amplitude), and its ultimate detection sensitivity is limited only by thermal noise. Focusing on the engineering aspects of PAM, this Review discusses the detection sensitivity of PAM, compares the detection efficiency of different PAM designs, and summarizes the imaging performance of various endogenous and exogenous contrast agents. It then describes representative PAM applications with high detection sensitivity, and outlines paths to further improvement.
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Photoacoustic microscopy and computed tomography: from bench to bedside.
Annu Rev Biomed Eng
PUBLISHED: 05-28-2014
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Photoacoustic imaging (PAI) of biological tissue has seen immense growth in the past decade, providing unprecedented spatial resolution and functional information at depths in the optical diffusive regime. PAI uniquely combines the advantages of optical excitation and those of acoustic detection. The hybrid imaging modality features high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth. Here we first summarize the fundamental principles underpinning the technology, then highlight its practical implementation, and finally discuss recent advances toward clinical translation.
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Three-dimensional arbitrary trajectory scanning photoacoustic microscopy.
J Biophotonics
PUBLISHED: 05-09-2014
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We have enhanced photoacoustic microscopy with three-dimensional arbitrary trajectory (3-DAT) scanning, which can rapidly image selected vessels over a large field of view (FOV) and maintain a high signal-to-noise ratio (SNR) despite the depth variation of the vessels. We showed that hemoglobin oxygen saturation (sO2 ) and blood flow can be measured simultaneously in a mouse ear in vivo at a frame rate 67 times greater than that of a traditional two-dimensional raster scan. We also observed sO2 dynamics in response to switching from systemic hypoxia to hyperoxia. 3-DAT-scanning photoacoustic microscopy. Schematic diagram of the 3D scanning stage and method.
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Radioactive 198Au-doped nanostructures with different shapes for in vivo analyses of their biodistribution, tumor uptake, and intratumoral distribution.
ACS Nano
PUBLISHED: 04-30-2014
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With Au nanocages as an example, we recently demonstrated that radioactive (198)Au could be incorporated into the crystal lattice of Au nanostructures for simple and reliable quantification of their in vivo biodistribution by measuring the ? radiation from (198)Au decay and for optical imaging by detecting the Cerenkov radiation. Here we extend the capability of this strategy to synthesize radioactive (198)Au nanostructures with a similar size but different shapes and then compare their biodistribution, tumor uptake, and intratumoral distribution using a murine EMT6 breast cancer model. Specifically, we investigated Au nanospheres, nanodisks, nanorods, and cubic nanocages. After PEGylation, an aqueous suspension of the radioactive Au nanostructures was injected into a tumor-bearing mouse intravenously, and their biodistribution was measured from the ? radiation while their tumor uptake was directly imaged using the Cerenkov radiation. Significantly higher tumor uptake was observed for the Au nanospheres and nanodisks relative to the Au nanorods and nanocages at 24 h postinjection. Furthermore, autoradiographic imaging was performed on thin slices of the tumor after excision to resolve the intratumoral distributions of the nanostructures. While both the Au nanospheres and nanodisks were only observed on the surfaces of the tumors, the Au nanorods and nanocages were distributed throughout the tumors.
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Label-free photoacoustic nanoscopy.
J Biomed Opt
PUBLISHED: 04-28-2014
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Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.
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Urogenital photoacoustic endoscope.
Opt Lett
PUBLISHED: 04-03-2014
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Photoacoustic (PA) endoscopy for human urogenital imaging has the potential to diagnose many important diseases, such as endometrial and prostate cancers. We have specifically developed a 12.7 mm diameter, rigid, side-scanning PA endoscopic probe for such applications. The key features of this endoscope are the streamlined structure for smooth cavity introduction and the proximal actuation mechanism for fast scanning. Here we describe the probe's composition and scanning mechanism and present in vivo experimental results suggesting its potential for comprehensive clinical applications.
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Fully motorized optical-resolution photoacoustic microscopy.
Opt Lett
PUBLISHED: 04-02-2014
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We have developed fully motorized optical-resolution photoacoustic microscopy (OR-PAM), which integrates five complementary scanning modes and simultaneously provides a high imaging speed and a wide field of view (FOV) with 2.6 ?m lateral resolution. With one-dimensional (1D) motion-mode mechanical scanning, we measured the blood flow through a cross section of a blood vessel in vivo. With two-dimensional (2D) optical scanning at a laser repetition rate of 40 kHz, we achieved a 2 kHz B-scan rate over a range of 50 ?m with 20 A-lines and 50 Hz volumetric-scan rate over a FOV of 50???m × 50???m with 400 A-lines, which enabled real-time tracking of cellular dynamics in vivo. With synchronized 1D optical and 2D mechanical hybrid scanning, we imaged a 10??mm × 8??mm FOV within three minutes, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical contour scanning, we maintained the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is essential for quantitative studies.
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Optical-resolution photoacoustic microscopy for volumetric and spectral analysis of histological and immunochemical samples.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 03-28-2014
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Optical-resolution photoacoustic microscopy (OR-PAM) is an imaging modality with superb penetration depth and excellent absorption contrast. Here we demonstrate, for the first time, that this technique can advance quantitative analysis of conventional chromogenic histochemistry. Because OR-PAM can quantify the absorption contrast at different wavelengths, it is feasible to spectrally resolve the specific biomolecules involved in a staining color. Furthermore, the tomographic capability of OR-PAM allows for noninvasive volumetric imaging of a thick sample without microtoming it. By immunostaining the sample with different chromogenic agents, we further demonstrated the ability of OR-PAM to resolve different types of cells in a coculture sample with imaging depths up to 1?mm. Taken together, the integration of OR-PAM with (immuno)histochemistry offers a simple and versatile technique with broad applications in cell biology, pathology, tissue engineering, and related biomedical studies.
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Fast spatiotemporal image reconstruction based on low-rank matrix estimation for dynamic photoacoustic computed tomography.
J Biomed Opt
PUBLISHED: 03-21-2014
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In order to monitor dynamic physiological events in near-real time, a variety of photoacoustic computed tomography (PACT) systems have been developed that can rapidly acquire data. Previously reported studies of dynamic PACT have employed conventional static methods to reconstruct a temporally ordered sequence of images on a frame-by-frame basis. Frame-by-frame image reconstruction (FBFIR) methods fail to exploit correlations between data frames and are known to be statistically and computationally suboptimal. In this study, a low-rank matrix estimation-based spatiotemporal image reconstruction (LRME-STIR) method is investigated for dynamic PACT applications. The LRME-STIR method is based on the observation that, in many PACT applications, the number of frames is much greater than the rank of the ideal noiseless data matrix. Using both computer-simulated and experimentally measured photoacoustic data, the performance of the LRME-STIR method is compared with that of conventional FBFIR method followed by image-domain filtering. The results demonstrate that the LRME-STIR method is not only computationally more efficient but also produces more accurate dynamic PACT images than a conventional FBFIR method followed by image-domain filtering.
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Calibration-free structured-illumination photoacoustic flowgraphy of transverse flow in scattering media.
J Biomed Opt
PUBLISHED: 03-21-2014
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We propose a calibration-free photoacoustic (PA) method for transverse flow measurements. In this method, a pulsed periodically structured (i.e., grating patterned) optical beam is used to illuminate flowing absorptive particles in an optically scattering medium. The PA signal amplitudes measured over consecutive laser pulses carry an imprint of the illumination structure. The modulation frequency of the imprint is proportional to the component of the flow speed projected onto the normal axis of the striped illumination pattern. This method can tolerate high particle density, and is insensitive to the particle size, thus calibration-free. Bovine blood and microsphere phantoms were used to validate the proposed method. Blood flow in a mouse ear was measured in vivo as well.
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Plasmonics-enhanced and optically modulated delivery of gold nanostars into brain tumor.
Nanoscale
PUBLISHED: 03-11-2014
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Plasmonics-active gold nanostars exhibiting strong imaging contrast and efficient photothermal transduction were synthesized for a novel pulsed laser-modulated plasmonics-enhanced brain tumor microvascular permeabilization. We demonstrate a selective, optically modulated delivery of nanoprobes into the tumor parenchyma with minimal off-target distribution.
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Photoacoustic thermography of tissue.
J Biomed Opt
PUBLISHED: 02-14-2014
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Photoacoustic (PA) techniques can measure temperature in biological tissues because PA signal amplitude is sensitive to tissue temperature. So far, temperature-measuring PA techniques have focused on sensing of temperature changes at a single position. In this work, we photoacoustically measured spatial distribution of temperature in deep tissue. By monitoring the temperature at a single position using a thermocouple, the relationship between the PA signal amplitude and the actual temperature was determined. The relationship was then used to translate a PA image into a temperature map. This study showed that it is possible to calibrate the system for the temperature range of hyperthermia using single-point measurements over a smaller temperature range. Our experimental results showed a precision of -0.8±0.4°C (mean±standard error) in temperature measurement, and a spatial resolution as fine as 1.0 mm. PA techniques can be potentially applied to monitor temperature distribution deep in tissue during hyperthermia treatment of cancer.
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Handheld photoacoustic tomography probe built using optical-fiber parallel acoustic delay lines.
J Biomed Opt
PUBLISHED: 02-13-2014
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The development of the first miniaturized parallel acoustic delay line (PADL) probe for handheld photoacoustic tomography (PAT) is reported. Using fused-silica optical fibers with low acoustic attenuation, we constructed two arrays of eight PADLs. Precision laser micromachining was conducted to produce robust and accurate mechanical support and alignment structures for the PADLs, with minimal acoustic distortion and interchannel coupling. The 16 optical-fiber PADLs, each with a different time delay, were arranged to form one input port and two output ports. A handheld PADL probe was constructed using two single-element transducers and two data acquisition channels (equal to a channel reduction ratio of 8?1). Photoacoustic (PA) images of a black-ink target embedded in an optically scattering phantom were successfully acquired. After traveling through the PADLs, the eight channels of differently time-delayed PA signals reached each single-element ultrasonic transducer in a designated nonoverlapping time series, allowing clear signal separation for PA image reconstruction. Our results show that the PADL technique and the handheld probe can potentially enable real-time PAT, while significantly reducing the complexity and cost of the ultrasound receiver system.
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Dependence of optical scattering from Intralipid in gelatin-gel based tissue-mimicking phantoms on mixing temperature and time.
J Biomed Opt
PUBLISHED: 02-12-2014
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Intralipid is widely used as an optical scattering agent in tissue-mimicking phantoms. Accurate control when using Intralipid is critical to match the optical diffusivity of phantoms to the prescribed value. Currently, most protocols of Intralipid-based hydrogel phantom fabrication focus on factors such as Intralipid brand and concentration. In this note, for the first time to our knowledge, we explore the dependence of the optical reduced scattering coefficient (at 532 nm optical wavelength) on the temperature and the time of mixing Intralipid with gelatin-water solution. The studied samples contained 1% Intralipid and were measured with oblique-incidence reflectometry. It was found that the reduced scattering coefficient increased when the Intralipid-gelatin-water mixture began to solidify at room temperature. For phantoms that had already solidified completely, the diffusivity was shown to be significantly influenced by the temperature and the duration of the mixing course. The dependence of the measured diffusivity on the mixing conditions was confirmed by experimental observations. Moreover, the mechanism behind the dependence behavior is discussed.
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Calibration-free absolute quantification of particle concentration by statistical analyses of photoacoustic signals in vivo.
J Biomed Opt
PUBLISHED: 01-28-2014
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Currently, laser fluence calibration is typically required for quantitative measurement of particle concentration in photoacoustic imaging. Here, we present a calibration-free method to quantify the absolute particle concentration by statistically analyzing photoacoustic signals. The proposed method is based on the fact that Brownian motion induces particle count fluctuation in the detection volume. If the count of particles in the detection volume is assumed to follow the Poisson distribution, its expected value can be calculated by the photoacoustic signal mean and variance. We first derived a theoretical model for photoacoustic signals. Then, we applied our method to quantitative measurement of different concentrations of various particles, including red blood cells. Finally, we performed in vivo experiments to demonstrate the potential of our method in biological applications. The experimental results agreed well with the predictions from the theoretical model suggesting that our method can be used for noninvasive measurement of absolute particle concentrations in deep tissue without fluence calibration.
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Integrated photoacoustic, confocal, and two-photon microscope.
J Biomed Opt
PUBLISHED: 01-28-2014
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The invention of green fluorescent protein and other molecular fluorescent probes has promoted applications of confocal and two-photon fluorescence microscopy in biology and medicine. However, exogenous fluorescence contrast agents may affect cellular structure and function, and fluorescence microscopy cannot image nonfluorescent chromophores. We overcome this limitation by integrating optical-resolution photoacoustic microscopy into a modern Olympus IX81 confocal, two-photon, fluorescence microscope setup to provide complementary, label-free, optical absorption contrast. Automatically coregistered images can be generated from the same sample. Imaging applications in ophthalmology, developmental biology, and plant science are demonstrated. For the first time, in a familiar microscopic fluorescence imaging setting, this trimodality microscope provides a platform for future biological and medical discoveries.
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Catheter-based photoacoustic endoscope.
J Biomed Opt
PUBLISHED: 01-20-2014
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We report a flexible shaft-based mechanical scanning photoacoustic endoscopy (PAE) system that can be potentially used for imaging the human gastrointestinal tract via the instrument channel of a clinical video endoscope. The development of such a catheter endoscope has been an important challenge to realize the technique's benefits in clinical settings. We successfully implemented a prototype PAE system that has a 3.2-mm diameter and 2.5-m long catheter section. As the instrument's flexible shaft and scanning tip are fully encapsulated in a plastic catheter, it easily fits within the 3.7-mm diameter instrument channel of a clinical video endoscope. Here, we demonstrate the intra-instrument channel workability and in vivo animal imaging capability of the PAE system.
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High-resolution photoacoustic tomography of resting-state functional connectivity in the mouse brain.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-23-2013
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The increasing use of mouse models for human brain disease studies presents an emerging need for a new functional imaging modality. Using optical excitation and acoustic detection, we developed a functional connectivity photoacoustic tomography system, which allows noninvasive imaging of resting-state functional connectivity in the mouse brain, with a large field of view and a high spatial resolution. Bilateral correlations were observed in eight functional regions, including the olfactory bulb, limbic, parietal, somatosensory, retrosplenial, visual, motor, and temporal regions, as well as in several subregions. The borders and locations of these regions agreed well with the Paxinos mouse brain atlas. By subjecting the mouse to alternating hyperoxic and hypoxic conditions, strong and weak functional connectivities were observed, respectively. In addition to connectivity images, vascular images were simultaneously acquired. These studies show that functional connectivity photoacoustic tomography is a promising, noninvasive technique for functional imaging of the mouse brain.
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Wide-field two-dimensional multifocal optical-resolution photoacoustic-computed microscopy.
Opt Lett
PUBLISHED: 12-11-2013
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Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technique that directly images optical absorption in tissue at high spatial resolution. To date, the majority of OR-PAM systems are based on single-focused optical excitation and ultrasonic detection, limiting the wide-field imaging speed. While 1D multifocal OR-PAM (1D-MFOR-PAM) has been developed, the potential of microlens and transducer arrays has not been fully realized. Here we present the development of 2D multifocal optical-resolution photoacoustic-computed microscopy (2D-MFOR-PACM), using a 2D microlens array and a full-ring ultrasonic transducer array. The 10??mm×10??mm microlens array generates 1800 optical foci within the focal plane of the 512-element transducer array, and raster scanning the microlens array yields optical-resolution photoacoustic images. The system has improved the in-plane resolution of a full-ring transducer array from ?100 to 29 ?m and achieved an imaging time of 36 s over a 10??mm×10??mm field of view. In comparison, the 1D-MFOR-PAM would take more than 4 min to image over the same field of view. The imaging capability of the system was demonstrated on phantoms and animals both ex vivo and in vivo.
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Absolute photoacoustic thermometry in deep tissue.
Opt Lett
PUBLISHED: 12-11-2013
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Photoacoustic thermography is a promising tool for temperature measurement in deep tissue. Here we propose an absolute temperature measurement method based on the dual temperature dependences of the Grüneisen parameter and the speed of sound in tissue. By taking ratiometric measurements at two adjacent temperatures, we can eliminate the factors that are temperature irrelevant but difficult to correct for in deep tissue. To validate our method, absolute temperatures of blood-filled tubes embedded ?9??mm deep in chicken tissue were measured in a biologically relevant range from 28°C to 46°C. The temperature measurement accuracy was ?0.6°C. The results suggest that our method can be potentially used for absolute temperature monitoring in deep tissue during thermotherapy.
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Photobleaching imprinting microscopy: seeing clearer and deeper.
J. Cell. Sci.
PUBLISHED: 12-06-2013
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We present a generic sub-diffraction-limited imaging method - photobleaching imprinting microscopy (PIM) - for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a two-fold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community. Moreover, we show that PIM can increase the image contrast of biological tissue, effectively extending the fundamental depth limit of multi-photon fluorescence microscopy.
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Enhancement of photoacoustic tomography by ultrasonic computed tomography based on optical excitation of elements of a full-ring transducer array.
Opt Lett
PUBLISHED: 10-10-2013
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Photoacoustic computed tomography (PACT) is a hybrid technique that combines optical excitation and ultrasonic detection to provide high-resolution images in deep tissues. In the image reconstruction, a constant speed of sound (SOS) is normally assumed. This assumption, however, is often not strictly satisfied in deep tissue imaging, due to acoustic heterogeneities within the object and between the object and the coupling medium. If these heterogeneities are not accounted for, they will cause distortions and artifacts in the reconstructed images. In this Letter, we incorporated ultrasonic computed tomography (USCT), which measures the SOS distribution within the object, into our full-ring array PACT system. Without the need for ultrasonic transmitting electronics, USCT was performed using the same laser beam as for PACT measurement. By scanning the laser beam on the array surface, we can sequentially fire different elements. As a first demonstration of the system, we studied the effect of acoustic heterogeneities on photoacoustic vascular imaging. We verified that constant SOS is a reasonable approximation when the SOS variation is small. When the variation is large, distortion will be observed in the periphery of the object, especially in the tangential direction.
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Calibration-free in vivo transverse blood flowmetry based on cross correlation of slow time profiles from photoacoustic microscopy.
Opt Lett
PUBLISHED: 10-02-2013
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We propose a cross-correlation-based method to measure blood-flow velocity by using photoacoustic microscopy. Unlike in previous autocorrelation-based methods, the measured flow velocity here is independent of particle size. Thus an absolute flow velocity can be obtained without calibration. We first measured the flow velocity ex vivo, using defibrinated bovine blood. Then flow velocities in vessels with different structures in a mouse ear were quantified in vivo. We further measured the flow variation in the same vessel and at a vessel bifurcation. All the experimental results indicate that our method can be used to accurately quantify blood velocity in vivo.
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Small-animal whole-body photoacoustic tomography: a review.
IEEE Trans Biomed Eng
PUBLISHED: 09-25-2013
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With the wide use of small animals for biomedical studies, in vivo small-animal whole-body imaging plays an increasingly important role. Photoacoustic tomography (PAT) is an emerging whole-body imaging modality that shows great potential for preclinical research. As a hybrid technique, PAT is based on the acoustic detection of optical absorption from either endogenous tissue chromophores, such as oxy-hemoglobin and deoxy-hemoglobin, or exogenous contrast agents. Because ultrasound scatters much less than light in tissue, PAT generates high-resolution images in both the optical ballistic and diffusive regimes. Using near-infrared light, which has relatively low blood absorption, PAT can image through the whole body of small animals with acoustically defined spatial resolution. Anatomical and vascular structures are imaged with endogenous hemoglobin contrast, while functional and molecular images are enabled by the wide choice of exogenous optical contrasts. This paper reviews the rapidly growing field of small-animal whole-body PAT and highlights studies done in the past decade.
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Time-reversed ultrasonically encoded optical focusing using two ultrasonic transducers for improved ultrasonic axial resolution.
J Biomed Opt
PUBLISHED: 09-18-2013
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Focusing light inside highly scattering media is a challenging task in biomedical optical imaging, manipulation, and therapy. A recent invention has overcome this challenge by time reversing ultrasonically encoded diffuse light to an ultrasound-modulated volume inside a turbid medium. In this technique, a photorefractive (PR) crystal or polymer can be used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the PR response time of the PR material, is usually used to encode the diffuse light. This long burst results in poor focusing resolution along the acoustic axis. In this work, we propose to use two intersecting ultrasound beams, emitted from two ultrasonic transducers at different frequencies, to modulate the diffuse light at the beat frequency within the intersection volume. We show that the time reversal of the light encoded at the beat frequency can converge back to the intersection volume. Experimentally, an acoustic axial resolution of ~1.1 mm was demonstrated inside turbid media, agreeing with theoretical estimation.
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Optical sectioning by wide-field photobleaching imprinting microscopy.
Appl Phys Lett
PUBLISHED: 09-10-2013
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We present a generic wide-field optical sectioning scheme, photobleaching imprinting microscopy (PIM), for depth-resolved cross-sectional fluorescence imaging. Wide-field PIM works by extracting a nonlinear component that depends on the excitation fluence as a result of photobleaching-induced fluorescence decay. Since no specific fluorescent dyes or illumination modules are required, wide-field PIM is easy to implement on a standard microscope. Moreover, wide-field PIM is superior to deconvolution microscopy in removing background fluorescence, yielding a six-fold improvement in image contrast.
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Improving limited-view photoacoustic tomography with an acoustic reflector.
J Biomed Opt
PUBLISHED: 09-04-2013
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The versatility and real-time imaging capability of commercial linear array transducers make them widely used in clinical ultrasound and photoacoustic imaging. However, they often suffer from limited detection view. For instance, acoustic waves traveling at a grazing angle to the transducer surface are difficult to detect. In this letter, we propose a simple and easy approach to ameliorate this problem by using a 45-deg acoustic reflector. The reflector forms a virtual array that is perpendicular to the physical array, thereby doubling the detection coverage. The improvement in image quality in photoacoustic tomography was demonstrated through a hair phantom, a leaf skeleton phantom, and an ex vivo mouse ear experiment.
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A brief account of nanoparticle contrast agents for photoacoustic imaging.
Wiley Interdiscip Rev Nanomed Nanobiotechnol
PUBLISHED: 08-23-2013
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Photoacoustic imaging (PAI) is a hybrid, nonionizing modality offering excellent spatial resolution, deep penetration, and high soft tissue contrast. In PAI, signal is generated based on the absorption of laser-generated optical energy by endogenous tissues or exogenous contrast agents leading to acoustic emissions detected by an ultrasound transducer. Research in this area over the years has shown that PAI has the ability to provide both physiological and molecular imaging, which can be viewed alone or used in a hybrid modality fashion to extend the anatomic and hemodynamic sensitivities of clinical ultrasound. PAI may be performed using inherent contrast afforded by light absorbing molecules such as hemoglobin, myoglobin, and melanin or exogenous small molecule contrast agent such as near infrared dyes and porphyrins. However, this review summarizes the potential of exogenous nanoparticle-based agents for PAI applications including contrast based on gold particles, carbon nanotubes, and encapsulated copper compounds.
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Optical clearing-aided photoacoustic microscopy with enhanced resolution and imaging depth.
Opt Lett
PUBLISHED: 08-14-2013
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Due to strong light scattering in tissue, both the spatial resolution and maximum penetration depth of optical-resolution photoacoustic microscopy (OR-PAM) deteriorate sharply with depth. To reduce tissue scattering, we propose to use glycerol as an optical clearing agent in OR-PAM. Our results show that the imaging performance of OR-PAM can be greatly enhanced by optical clearing both in vitro and in vivo.
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Calibration-free quantification of absolute oxygen saturation based on the dynamics of photoacoustic signals.
Opt Lett
PUBLISHED: 08-02-2013
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Photoacoustic tomography (PAT) is a hybrid imaging technique that has broad preclinical and clinical applications. Based on the photoacoustic effect, PAT directly measures specific optical absorption, which is the product of the tissue-intrinsic optical absorption coefficient and the local optical fluence. Therefore, quantitative PAT, such as absolute oxygen saturation (sO?) quantification, requires knowledge of the local optical fluence, which can only be estimated through invasive measurements or sophisticated modeling of light transportation. In this Letter, we circumvent this requirement by taking advantage of the dynamics in sO?. The new method works when the sO? transition can be simultaneously monitored with multiple wavelengths. For each wavelength, the ratio of photoacoustic amplitudes measured at different sO? states is utilized. Using the ratio cancels the contribution from optical fluence and allows calibration-free quantification of absolute sO?. The new method was validated through both phantom and in vivo experiments.
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Random-access optical-resolution photoacoustic microscopy using a digital micromirror device.
Opt Lett
PUBLISHED: 08-02-2013
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We developed random-access optical-resolution photoacoustic microscopy using a digital micromirror device. This system can rapidly scan arbitrarily shaped regions of interest within a 40 ?m×40 ?m imaging area with a lateral resolution of 3.6 ?m. To identify a region of interest, a global structural image is first acquired, then the selected region is scanned. The random-access ability was demonstrated by imaging two static samples, a carbon fiber cross and a monolayer of red blood cells, with an acquisition rate up to 4 kHz. The system was then used to monitor blood flow in vivo in real time within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 9.2 times.
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Deep-tissue photoacoustic tomography of Förster resonance energy transfer.
J Biomed Opt
PUBLISHED: 07-26-2013
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Förster resonance energy transfer (FRET) is a distance-dependent process that transfers excited state energy from a donor molecule to an acceptor molecule without the emission of a photon. The FRET rate is determined by the proximity between the donor and the acceptor molecules; it becomes significant only when the proximity is within several nanometers. Therefore, FRET has been applied to visualize interactions and conformational changes of biomolecules, such as proteins, lipids, and nucleic acids that cannot be resolved by optical microscopy. Here, we report photoacoustic tomography of FRET efficiency at a 1-cm depth in chicken breast tissue, whereas conventional high-resolution fluorescence imaging is limited to <0.1??cm. Photoacoustic tomography is expected to facilitate the examination of FRET phenomena in living organisms.
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Non-invasive and in?situ characterization of the degradation of biomaterial scaffolds by volumetric photoacoustic microscopy.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 07-19-2013
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Degradation is among the most important properties of biomaterial scaffolds, which are indispensable for regenerative medicine. The currently used method relies on the measurement of mass loss across different samples and cannot track the degradation of an individual scaffold in?situ. Here we report, for the first time, the use of multiscale photoacoustic microscopy to non-invasively monitor the degradation of an individual scaffold. We could observe alterations to the morphology and structure of a scaffold at high spatial resolution and deep penetration, and more significantly, quantify the degradation of an individual scaffold as a function of time, both in?vitro and in?vivo. In addition, the remodeling of vasculature inside a scaffold can be visualized simultaneously using a dual-wavelength scanning mode in a label-free manner. This optoacoustic method can be used to monitor the degradation of individual scaffolds, offering a new approach to non-invasively analyze and quantify biomaterial-tissue interactions in conjunction with the assessment of in?vivo vascular parameters.
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Ultrasonically encoded photoacoustic flowgraphy in biological tissue.
Phys. Rev. Lett.
PUBLISHED: 07-08-2013
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Blood flow speed is an important functional parameter. Doppler ultrasound flowmetry lacks sufficient sensitivity to slow blood flow (several to tens of millimeters per second) in deep tissue. To address this challenge, we developed ultrasonically encoded photoacoustic flowgraphy combining ultrasonic thermal tagging with photoacoustic imaging. Focused ultrasound generates a confined heat source in acoustically absorptive fluid. Thermal waves propagate with the flow and are directly visualized in pseudo color using photoacoustic computed tomography. The Doppler shift is employed to calculate the flow speed. This method requires only acoustic and optical absorption, and thus is applicable to continuous fluid. A blood flow speed as low as 0.24??mm·s^{-1} was successfully measured. Deep blood flow imaging was experimentally demonstrated under 5-mm-thick chicken breast tissue.
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A Green Synthesis of Carbon Nanoparticle from Honey for Real-Time Photoacoustic Imaging.
Nano Res
PUBLISHED: 07-05-2013
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Imaging sentinel lymph nodes (SLN) could provide us with critical information about the progression of a cancerous disease. Real-time high-resolution intraoperative photoacoustic imaging (PAI) in conjunction with a near infrared (NIR) probe may offer the opportunities for the immediate imaging for direct identification and resection of SLN or collecting tissue samples. In this work a commercially amenable synthetic methodology is revealed for developing luminescent carbon nanoparticles with rapid clearance properties. A one-pot "green" technique is pursued, which involved rapid surface passivation of carbon nanoparticles with organic macromolecules (e.g. polysorbate, polyethyleneglycol) in a solvent free condition. Interestingly, the naked carbon nanoparticles are derived for the first time, from commercial food grade honey. Surface coated particles are markedly smaller (~7 nm) than the previously explored particles (gold, SWNT, copper) for SLN imaging. Results indicate an exceptionally rapid signal enhancement (~2 min) of the SLN. Owing to their strong optical absorption in the near infrared region, tiny size and rapid lymphatic transport, this platform offers great potential for faster resection of SLN and may lower complications caused by axillary investigation for mismarking with dyes or low-resolution imaging techniques.
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Structured-illumination photoacoustic Doppler flowmetry of axial flow in homogeneous scattering media.
Appl Phys Lett
PUBLISHED: 07-03-2013
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We propose a method for photoacoustic flow measurement based on the Doppler effect from a flowing homogeneous medium. Excited by spatially modulated laser pulses, the flowing medium induces a Doppler frequency shift in the received photoacoustic signals. The frequency shift is proportional to the component of the flow speed projected onto the acoustic beam axis, and the sign of the shift reflects the flow direction. Unlike conventional flowmetry, this method does not rely on particle heterogeneity in the medium; thus, it can tolerate extremely high particle density. A red-ink phantom flowing in a tube immersed in water was used to validate the method in both the frequency and time domains. The phantom flow immersed in an intralipid solution was also measured.
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Optical-resolution photoacoustic microscopy: auscultation of biological systems at the cellular level.
Biophys. J.
PUBLISHED: 06-16-2013
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Photoacoustic microscopy (PAM) offers unprecedented sensitivity to optical absorption and opens a new window to study biological systems at multiple length- and timescales. In particular, optical-resolution PAM (OR-PAM) has pushed the technical envelope to submicron length scales and millisecond timescales. Here, we review the state of the art of OR-PAM in biophysical research. With properly chosen optical wavelengths, OR-PAM can spectrally differentiate a variety of endogenous and exogenous chromophores, unveiling the anatomical, functional, metabolic, and molecular information of biological systems. Newly uncovered contrast mechanisms of linear dichroism and Förster resonance energy transfer further distinguish OR-PAM. Integrating multiple contrasts and advanced scanning mechanisms has capacitated OR-PAM to comprehensively interrogate biological systems at the cellular level in real time. Two future directions are discussed, where OR-PAM holds the potential to translate basic biophysical research into clinical healthcare.
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Photoacoustic Microscopy in Tissue Engineering.
Mater Today (Kidlington)
PUBLISHED: 06-15-2013
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Photoacoustic tomography (PAT) is an attractive modality for noninvasive, volumetric imaging of scattering media such as biological tissues. By choosing the ultrasonic detection frequency, PAT enables scalable spatial resolution with desired imaging depth up to ~7 cm while maintaining a high depth-to-resolution ratio of ~200 and consistent optical absorption contrasts. Photoacoustic microscopy (PAM), the microscopic embodiment of PAT, aims to image at millimeter depth and micrometer-scale resolution. PAM is well-suited for characterizing three-dimensional scaffold-based samples, including scaffolds themselves, cells, and blood vessels, both qualitatively and quantitatively. Here we review our previous work on applications of PAM in tissue engineering and then discuss its future developments.
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Cross-correlation-based transverse flow measurements using optical resolution photoacoustic microscopy with a digital micromirror device.
J Biomed Opt
PUBLISHED: 05-29-2013
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A cross-correlation-based method is proposed to quantitatively measure transverse flow velocity using optical resolution photoacoustic (PA) microscopy enhanced with a digital micromirror device (DMD). The DMD is used to alternately deliver two spatially separated laser beams to the target. Through cross-correlation between the slow-time PA profiles measured from the two beams, the speed and direction of transverse flow are simultaneously derived from the magnitude and sign of the time shift, respectively. Transverse flows in the range of 0.50 to 6.84??mm/s are accurately measured using an aqueous suspension of 10-?m-diameter microspheres, and the root-mean-squared measurement accuracy is quantified to be 0.22??mm/s. The flow measurements are independent of the particle size for flows in the velocity range of 0.55 to 6.49??mm/s, which was demonstrated experimentally using three different sizes of microspheres (diameters: 3, 6, and 10 ?m). The measured flow velocity follows an expected parabolic distribution along the depth direction perpendicular to the flow. Both maximum and minimum measurable velocities are investigated for varied distances between the two beams and varied total time for one measurement. This technique shows an accuracy of 0.35??mm/s at 0.3-mm depth in scattering chicken breast, making it promising for measuring flow in biological tissue.
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Noise-equivalent sensitivity of photoacoustics.
J Biomed Opt
PUBLISHED: 05-13-2013
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The fundamental limitations of photoacoustic microscopy for detecting optically absorbing molecules are investigated both theoretically and experimentally. We experimentally demonstrate noise-equivalent detection sensitivities of 160,000 methylene blue molecules (270 zeptomol or 2.7×10-19??mol) and 86,000 oxygenated hemoglobin molecules (140 zeptomol) using narrowband continuous-wave photoacoustics. The ultimate sensitivity of photoacoustics is fundamentally limited by thermal noise, which can present in the acoustic detection system as well as in the medium itself. Under the optimized conditions described herein and using commercially available detectors, photoacoustic microscopy can detect as few as 100s of oxygenated hemoglobin molecules. Realizable improvements to the detector may enable single molecule detection of select molecules.
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Photoacoustic recovery after photothermal bleaching in living cells.
J Biomed Opt
PUBLISHED: 04-22-2013
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We present an innovative method, photoacoustic recovery after photothermal bleaching (PRAP), for studying particle dynamics at micron scale via photoacoustic imaging. As an intuitive way to visualize and quantify dynamic processes, PRAP is demonstrated first in a simple phantom study and then in a more complex measurement involving live cells. Compared with the conventional fluorescence-based approach, PRAP provides high signal-to-noise ratio (SNR) imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic processes inside a cell.
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Ultrasound-heated photoacoustic flowmetry.
J Biomed Opt
PUBLISHED: 03-08-2013
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We report the development of photoacoustic flowmetry assisted by high-intensity focused ultrasound (HIFU). This novel method employs HIFU to generate a heating impulse in the flow medium, followed by photoacoustic monitoring of the thermal decay process. Photoacoustic flowmetry in a continuous medium remains a challenge in the optical diffusive regime. Here, both the HIFU heating and photoacoustic detection can focus at depths beyond the optical diffusion limit (~1 mm in soft tissue). This method can be applied to a continuous medium, i.e., a medium without discrete scatterers or absorbers resolvable by photoacoustic imaging. Flow speeds up to 41 mm·s-1 have been experimentally measured in a blood phantom covered by 1.5-mm-thick tissue.
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Intracellular temperature mapping with fluorescence-assisted photoacoustic-thermometry.
Appl Phys Lett
PUBLISHED: 03-08-2013
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Measuring intracellular temperature is critical to understanding many cellular functions but still remains challenging. Here, we present a technique-fluorescence-assisted photoacoustic thermometry (FAPT)-for intracellular temperature mapping applications. To demonstrate FAPT, we monitored the intracellular temperature distribution of HeLa cells with sub-degree (0.7?°C) temperature resolution and sub-micron (0.23??m) spatial resolution at a sampling rate of 1?kHz. Compared to traditional fluorescence-based methods, FAPT features the unique capability of transforming a regular fluorescence probe into a concentration- and excitation-independent temperature sensor, bringing a large collection of commercially available generic fluorescent probes into the realm of intracellular temperature sensing.
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Slow-sound photoacoustic microscopy.
Appl Phys Lett
PUBLISHED: 02-25-2013
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We propose to enhance the axial resolution of photoacoustic microscopy (PAM) by reducing the speed of sound within the imaging region of interest. With silicone oil immersion, we have achieved a finest axial resolution of 5.8??m for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. When tissue-compatible low-speed liquid becomes available, this approach may find broad applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging.
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Up-regulation of hypoxia-inducible factor 1 alpha and hemodynamic responses following massive small bowel resection.
J. Pediatr. Surg.
PUBLISHED: 02-19-2013
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Massive small bowel resection (SBR) results in an adaptive response within the remnant bowel. We have previously shown an immediate reduction in intestinal blood flow and oxygen saturation (sO2) after SBR. We therefore sought to determine the duration of resection-induced intestinal hypoxia and expression of hypoxia-inducible factors (HIFs) following SBR.
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Labeling human mesenchymal stem cells with gold nanocages for in vitro and in vivo tracking by two-photon microscopy and photoacoustic microscopy.
Theranostics
PUBLISHED: 01-01-2013
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Stem cell tracking is a highly important subject. Current techniques based on nanoparticle-labeling, such as magnetic resonance imaging, fluorescence microscopy, and micro-computed tomography, are plagued by limitations including relatively low sensitivity or penetration depth, involvement of ionizing irradiation, and potential cytotoxicity of the nanoparticles. Here we introduce a new class of contrast agents based on gold nanocages (AuNCs) with hollow interiors and porous walls to label human mesenchymal stem cells (hMSCs) for both in vitro and in vivo tracking using two-photon microscopy and photoacoustic microscopy. As demonstrated by the viability assay, the AuNCs showed negligible cytotoxicity under a reasonable dose, and did not alter the differentiation potential of the hMSCs into desired lineages. We were able to image the cells labeled with AuNCs in vitro for at least 28 days in culture, as well as to track the cells that homed to the tumor region in nude mice in vivo.
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Multiscale photoacoustic microscopy of single-walled carbon nanotube-incorporated tissue engineering scaffolds.
Tissue Eng Part C Methods
PUBLISHED: 12-22-2011
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Three-dimensional polymeric scaffolds provide structural support and function as substrates for cells and bioactive molecules necessary for tissue regeneration. Noninvasive real-time imaging of scaffolds and/or the process of tissue formation within the scaffold remains a challenge. Microcomputed tomography, the widely used technique to characterize polymeric scaffolds, shows poor contrast for scaffolds immersed in biological fluids, thereby limiting its utilities under physiological conditions. In this article, multiscale photoacoustic microscopy (PAM), consisting of both acoustic-resolution PAM (AR-PAM) and optical-resolution PAM (OR-PAM), was employed to image and characterize single-walled carbon-nanotube (SWNT)-incorporated poly(lactic-co-glycolic acid) polymer scaffolds immersed in biological buffer. SWNTs were incorporated to reinforce the mechanical properties of the scaffolds, and to enhance the photoacoustic signal from the scaffolds. By choosing excitation wavelengths of 570 and 638 nm, multiscale PAM could spectroscopically differentiate the photoacoustic signals generated from blood and from carbon-nanotube-incorporated scaffolds. OR-PAM, providing a fine lateral resolution of 2.6 ?m with an adequate tissue penetration of 660 ?m, successfully quantified the average porosity and pore size of the scaffolds to be 86.5%±1.2% and 153±15 ?m in diameter, respectively. AR-PAM further extended the tissue penetration to 2 mm at the expense of lateral resolution (45 ?m). Our results suggest that PAM is a promising tool for noninvasive real-time imaging and monitoring of tissue engineering scaffolds in vitro, and in vivo under physiological conditions.
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In vivo quantitative evaluation of the transport kinetics of gold nanocages in a lymphatic system by noninvasive photoacoustic tomography.
ACS Nano
PUBLISHED: 11-15-2011
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Sentinel lymph node (SLN) biopsy has emerged as a preferred method for axillary lymph node staging of breast cancer, and imaging the SLN in three-dimensional space is a prerequisite for the biopsy. Conventional SLN mapping techniques based on the injection of an organic dye or a suspension of radioactive colloids suffer from invasive surgical operation for visual detection of the dye or hazardous radioactive components and low spatial resolution of Geiger counters in detecting the radioactive colloids. This work systematically investigates the use of gold nanocages (AuNCs) as a novel class of optical tracers for noninvasive SLN imaging by photoacoustic (PA) tomography in a rat model. The transport of AuNCs in a lymphatic system and uptake by the SLN were evaluated by PA tomography on the axillary region of a rat. Quantification of AuNCs accumulated in the lymph node was achieved by correlating the data from PA imaging with the results from inductively coupled plasma mass spectrometry. Several parameters were systematically evaluated and optimized, including the concentration, size, and surface charge of the AuNCs. These results are critical to the further development of this AuNC-based PA tomography system for noninvasive SLN imaging, providing valuable information for metastatic cancer staging.
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Functional photoacoustic microscopy of pH.
J Biomed Opt
PUBLISHED: 10-28-2011
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pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy.
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Recent advances in colloidal gold nanobeacons for molecular photoacoustic imaging.
Contrast Media Mol Imaging
PUBLISHED: 10-26-2011
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Photoacoustic imaging (PAI) represents a hybrid, nonionizing modality, which has been of particular interest because of its satisfactory spatial resolution and high soft tissue contrast. PAI has the potential to provide both functional and molecular imaging in vivo since optical absorption is sensitive to physiological parameters. In this review we summarize our effort to advance molecular PAI with colloidal gold nanobeacons (GNB). GNB represents a robust nanoparticle platform that entraps multiple copies of tiny gold nanoparticles (2-4?nm) within a larger colloidal particle encapsulated by biocompatible synthetic or natural amphilines. The utilization of numerous small gold particles greatly amplifies the signal without exceeding the renal elimination threshold size. With fibrin-targeted GNB, the robust detection of microthrombus formed over a ruptured atherosclerotic plaque has been achieved, which offers an important opportunity to recognize patients with moderate lumen stenosis but high risk of stroke. With the use of second-generation smaller GNBs, the potential to improve sentinel lymph node assessment and biopsy was advanced with respect to rapidity and sensitivity of detection in mice. Finally, for angiogenesis, an essential microanatomical biomarker of tumor and cardiovascular disease progression, integrin-targeted GNBs allowed visualization of numerous angiogenic sprouts and bridges that were otherwise undetectable from inherent blood signal alone, offering sensitive and specific discrimination and quantification of angiogenesis in vivo.
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Gold nanocages as contrast agents for photoacoustic imaging.
Contrast Media Mol Imaging
PUBLISHED: 10-26-2011
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Gold nanoparticles with tunable absorption and scattering properties have been developed as contrast agents for various optical imaging techniques. As a hybrid modality that combines the merits of both optical and ultrasonic imaging, photoacoustic (PA) imaging also benefits from the use of these nanoparticles to greatly enhance the contrast for visualization of structures and biomarkers in biological tissues. Gold nanocages characterized by hollow interiors, ultrathin and porous walls are of particular interest for in vivo PA imaging because of their compact sizes, bio-inertness and well-defined surface chemistry, as well as their strong and highly wavelength-tunable optical absorption in the near-infrared (NIR) optical window of soft tissues. This review discusses the application of gold nanocages as a new class of contrast agents for PA imaging in the context of cancer diagnosis.
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Photoacoustic tomography: fundamentals, advances and prospects.
Contrast Media Mol Imaging
PUBLISHED: 10-26-2011
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Optical microscopy has been contributing to the development of life science for more than three centuries. However, due to strong optical scattering in tissue, its in vivo imaging ability has been restricted to studies at superficial depths. Advances in photoacoustic tomography (PAT) now allow multiscale imaging at depths from sub-millimeter to several centimeters, with spatial resolutions from sub-micrometer to sub-millimeter. Because of this high scalability and its unique optical absorption contrast, PAT is capable of performing anatomical, functional, molecular and fluid-dynamic imaging at various system levels, and is playing an increasingly important role in fundamental biological research and clinical practice. This review discusses recent technical progress in PAT and presents corresponding applications. It ends with a discussion of several prospects and their technical challenges.
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Performance benchmarks of an array-based hand-held photoacoustic probe adapted from a clinical ultrasound system for non-invasive sentinel lymph node imaging.
Philos Trans A Math Phys Eng Sci
PUBLISHED: 10-19-2011
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Clinical translation of photoacoustic (PA) imaging can be facilitated by integration with commercial ultrasound (US) scanners to enable dual-modality imaging. An array-based US scanner was modified for hand-held PA imaging. The performance was benchmarked in terms of signal-to-noise ratio (SNR), axial spatial resolution and sensitivity. PA images of a tube, filled with methylene blue (MB; approx. 30?mM) and placed at various depths in chicken tissue, were acquired. A 5?cm penetration depth was achieved with an 18.6?dB SNR using a laser fluence of 3?mJ?cm(-2), only one-seventh of the safety limit (20?mJ?cm(-2)). An axial resolution of approximately 400??m was maintained at all imaging depths. The PA sensitivity to MB placed 2.3?cm deep in chicken tissue was less than 100??M. Further, after intradermal injection of MB (approx. 30?mM), a rat sentinel lymph node was clearly identified in vivo, beneath a 3.8?cm thick layer of chicken breast. The accumulated concentration of MB in the node was estimated to be approximately 7?mM. The noise-equivalent sensitivities (approx. 2?cm depth) were 17 and 85??M, ex vivo and in vivo, respectively. These results support the use of this PA system for non-invasive mapping and image-guided needle biopsy of sentinel nodes in breast cancer patients.
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Quantitative photoacoustic imaging: correcting for heterogeneous light fluence distributions using diffuse optical tomography.
J Biomed Opt
PUBLISHED: 09-29-2011
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The specificity of molecular and functional photoacoustic (PA) images depends on the accuracy of the photoacoustic absorption spectroscopy. The PA signal is proportional to the product of the optical absorption coefficient and local light fluence; quantitative PA measurements of the optical absorption coefficient therefore require an accurate estimation of optical fluence. Light-modeling aided by diffuse optical tomography (DOT) can be used to map the required fluence and to reduce errors in traditional PA spectroscopic analysis. As a proof-of-concept, we designed a tissue-mimicking phantom to demonstrate how fluence-related artifacts in PA images can lead to misrepresentations of tissue properties. To correct for these inaccuracies, the internal fluence in the tissue phantom was estimated by using DOT to reconstruct spatial distributions of the absorption and reduced scattering coefficients of multiple targets within the phantom. The derived fluence map, which only consisted of low spatial frequency components, was used to correct PA images of the phantom. Once calibrated to a known absorber, this method reduced errors in estimated absorption coefficients from 33% to 6%. These results experimentally demonstrate that combining DOT with PA imaging can significantly reduce fluence-related errors in PA images, while producing quantitatively accurate, high-resolution images of the optical absorption coefficient.
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Three-dimensional photoacoustic tomography based on the focal-line concept.
J Biomed Opt
PUBLISHED: 09-29-2011
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A full ring ultrasonic array-based photoacoustic tomography system was recently developed for small animal brain imaging. The 512-element array is cylindrically focused in the elevational direction, and can acquire a two-dimensional (2D) image in 1.6 s. In this letter, we demonstrate the three-dimensional (3D) imaging capability of this system. A novel 3D reconstruction algorithm was developed based on the focal-line concept. Compared to 3D images acquired simply by stacking a series of 2D images, the 3D focal-line reconstruction method renders images with much less artifacts, and improves the elevational resolution by 30% and the signal-to-noise ratio by two times. The effectiveness of the proposed algorithm was first validated by numerical simulations and then demonstrated with a hair phantom experiment and an ex vivo mouse embryo experiment.
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Nonionizing photoacoustic cystography in vivo.
Opt Lett
PUBLISHED: 09-21-2011
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We demonstrate the feasibility of a novel and nonionizing process for bladder imaging in vivo, called photoacoustic cystography (PAC). Using a photoacoustic imaging system, we have successfully imaged a rat bladder filled with clinically used Methylene Blue (MB) dye. An image contrast of ~8 was achieved. Further, spectroscopic PAC confirmed the accumulation of MB in the bladder. Using a laser pulse energy of less than 1 mJ/cm² (1/20 of the ANSI safety limit), a deeply (1.2 cm) positioned bladder in biological tissues was clearly visible in the PA image. Our results suggest that PAC can potentially provide a nonionizing, relatively cheap, and portable tool for bladder mapping. Among our clinical interests, nonionizing PAC with an injection of MB can potentially monitor vesicoureteral reflux in children.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.