Optical-resolution Photoacoustic Imaging Research Articles

In-vivo assessment of a rat rectal tumor using optical-resolution photoacoustic endoscopy.

Optical-resolution photoacoustic endoscopy (OR-PAE) has been proven to realize imaging on the vascular network in the gastrointestinal (GI) tract with high sensitivity and spatial resolution, providing morphological information. Various photoacoustic endoscopic catheters were developed to improve the resolution and adaptivity of in-vivo imaging. However, this technology has not yet been validated on in-vivo GI tumors, which generally feature angiogenesis. The tumor causes thickened mucosa and neoplasia, requiring large depth-of-field (DOF) in imaging, which contradicts to high-resolution imaging. In this work, a novel catheter was developed with a high resolution of ∼27 µm, providing a matched DOF of ∼400 µm to cover the vessels up to the submucosa layer. Optical-resolution photoacoustic endoscopic imaging was first performed on in-vivo rat rectal tumors. In addition, to further characterize the vessel morphology, tumor-suspected regions and normal regions were selected for quantification and analysis of vessel dimension distribution and tortuosity. All the results suggest that the OR-PAE has great application potential in tumor diagnosis, evaluation, and monitoring of therapeutic efficacy.

Super-resolution reconstruction algorithm for optical-resolution photoacoustic microscopy images based on sparsity and deconvolution.

The lateral resolution of the optical-resolution photoacoustic microscopy (OR-PAM) system depends on the focusing diameter of the probe beam. By increasing the numerical aperture (NA) of optical focusing, the lateral resolution of OR-PAM can be improved. However, the increase in NA results in smaller working distances, and the entire imaging system becomes very sensitive to small optical imperfections. The existing deconvolution-based algorithms are limited by the image signal-to-noise ratio when improving the resolution of OR-PAM images. In this paper, a super-resolution reconstruction algorithm for OR-PAM images based on sparsity and deconvolution is proposed. The OR-PAM image is sparsely reconstructed according to the constructed loss function, which utilizes the sparsity of the image to combat the decrease in the resolution. The gradient accelerated Landweber iterative algorithm is used to deconvolve to obtain high-resolution OR-PAM images. Experimental results show that the proposed algorithm can improve the resolution of mouse retinal images by approximately 1.7 times without increasing the NA of the imaging system. In addition, compared to the Richardson-Lucy algorithm, the proposed algorithm can further improve the image resolution and maintain better imaging quality, which provides a foundation for the development of OR-PAM in clinical research.

High-fluence relay-based disposable photoacoustic-ultrasonic endoscopy for in vivo anatomical imaging of gastrointestinal tract

Photoacoustic endomicroscopy combined with ultrasound (PAEM-US) has been a long-standing expectation for gastrointestinal tumor examination. Here, we introduce a prototype disposable PAEM-US catheter and corresponding power interface unit, featuring catheter switchability, self-internal three-dimensional scanning, and system repeatability for gastrointestinal endoscopy. By utilizing high-fluence relays, cascade insertion loss of the optical waveguide is minimized to 0.6 dB with a high performance of power resistance, and a focus-customizable acousto-optic coaxial probe is designed for high-sensitivity optical-resolution photoacoustic imaging. Imaging capability was demonstrated with in vivo anatomical imaging at 30 frames per second. Imaging results showed co-registered microscopic visualization of the microvascular and stratification of the rat colorectum with lateral resolution of 18 μm and axial resolution of 63 μm, holding great potential in the clinical detection of gastrointestinal diseases.

Quickly Alternating Green and Red Laser Source for Real-time Multispectral Photoacoustic Microscopy

Multispectral photoacoustic microscopy uses a wavelength-dependent absorption difference as a contrast mechanism to image the target molecule. In this paper, we present a novel multispectral pulsed fiber laser source, which selectively alternates the excitation wavelengths between green and red colors based on the stimulated Raman scattering (SRS) effect for imaging. This laser has a high pulse repetition rate (PRR) of 300 kHz and high pulse energy of more than 200 nJ meeting the real-time requirements of optical-resolution photoacoustic microscopy imaging. By switching the polarization state of the pump light and optical paths of the pump light, the operating wavelengths of the light source can be selectively alternated at the same fast PRR for any two SRS peak wavelengths between 545 and 655 nm. At 545 nm excitation wavelength, molecular photoacoustic signals from both blood vessels and gold nanorods were obtained simultaneously. However, at 655 nm, the photoacoustic signals of gold nanorods were dominant because the absorption of light by the blood vessels decreased drastically in the spectral region over 600 nm. Thus the multispectral photoacoustic system designed using the novel laser source implemented here could simultaneously monitor the time-dependent fast movement of two molecules independently, having different wavelength-dependent absorption properties at a high repetition rate of 0.49 frames per second (fps).

High-Resolution 3D NIR-II Photoacoustic Imaging of Cerebral and Tumor Vasculatures Using Conjugated Polymer Nanoparticles as Contrast Agent.

Exogenous contrast-agent-assisted NIR-II optical-resolution photoacoustic microscopy imaging (ORPAMI) holds promise to decipher wide-field 3D biological structures with deep penetration, large signal-to-background ratio (SBR), and high maximum imaging depth to depth resolution ratio. Herein, NIR-II conjugated polymer nanoparticle (CP NP) assisted ORPAMI is reported for pinpointing cerebral and tumor vasculatures. The CP NPs exhibit a large extinction coefficient of 48.1 L g-1 at the absorption maximum of 1161 nm, with an ultrahigh PA sensitivity up to 2 µg mL-1 . 3D ORPAMI of wide-field mice ear allows clear visualization of regular vasculatures with a resolution of 19.2 µm and an SBR of 29.3 dB at the maximal imaging depth of 539 µm. The margin of ear tumor composed of torsional dense vessels among surrounding normal regular vessels can be clearly delineated via 3D angiography. In addition, 3D whole-cortex cerebral vasculatures with large imaging area (48 mm2 ), good resolution (25.4 µm), and high SBR (22.3 dB) at a depth up to 1001 µm are clearly resolved through the intact skull. These results are superior to the recently reported 3D NIR-II fluorescence confocal vascular imaging, which opens up new opportunities for NIR-II CP-NP-assisted ORPAMI in various biomedical applications.

Deep non-contact photoacoustic initial pressure imaging

Photoacoustic imaging techniques have been extensively developed for biomedical applications, including functional and molecular imaging, due in part to their high optical contrast, high spatial resolution, and non-ionizing imaging properties. However, there are currently depth limitations in cellular-resolution, optically focused photoacoustic microscopy systems. In addition, most common photoacoustic systems need to be in contact with the sample through an ultrasound medium. In this work, by taking advantage of large photoacoustic initial pressures, all-optical non-contact optical resolution photoacoustic imaging is reported at depths beyond the optical transport mean-free path of the excitation wavelength. The proposed technique is called deep photoacoustic remote sensing (dPARS) microscopy. Visible pulsed excitation wavelengths are used to produce large initial-pressure-induced refractive index modulations in absorbing targets. These localized pressure rises create transient variations to the local scattering properties, which are detected as back-reflected intensity modulations from a deep-penetrating interrogation beam and do not require an interferometric detection pathway. Experiments demonstrate that dPARS is capable of providing optical resolution images to depths of 2.5 mm in tissue-mimicking scattering media. Signal-to-noise ratio ∼50 dB is reported for in vivo imaging of microvascular networks. Also, imaging of single red blood cells, oxygen saturation mapping, and deep-vascular imaging applications are demonstrated. dPARS’s capabilities such as remote sensing, deep optical resolution imaging, and high signal-to-noise ratio, may yield new opportunities for several pre-clinical and clinical applications.

Three-dimensional Hessian matrix-based quantitative vascular imaging of rat iris with optical-resolution photoacoustic microscopy in vivo.

For the diagnosis and evaluation of ophthalmic diseases, imaging and quantitative characterization of vasculature in the iris are very important. The recently developed photoacoustic imaging, which is ultrasensitive in imaging endogenous hemoglobin molecules, provides a highly efficient label-free method for imaging blood vasculature in the iris. However, the development of advanced vascular quantification algorithms is still needed to enable accurate characterization of the underlying vasculature. We have developed a vascular information quantification algorithm by adopting a three-dimensional (3-D) Hessian matrix and applied for processing iris vasculature images obtained with a custom-built optical-resolution photoacoustic imaging system (OR-PAM). For the first time, we demonstrate in vivo 3-D vascular structures of a rat iris with a the label-free imaging method and also accurately extract quantitative vascular information, such as vessel diameter, vascular density, and vascular tortuosity. Our results indicate that the developed algorithm is capable of quantifying the vasculature in the 3-D photoacoustic images of the iris in-vivo, thus enhancing the diagnostic capability of the OR-PAM system for vascular-related ophthalmic diseases in vivo.

Optical-resolution photoacoustic imaging with speckle illumination

Conventional approaches for optical-resolution photoacoustic microscopy generally involves raster scanning a focused spot over the sample. Here, we show that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of ghost imaging [1], and is based here on solving a linear inverse problem under sparsity assumptions: the object is reconstructed through a pseudo-inverse computation of a reference matrix made of speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti et al. [2]: in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. Because speckle patterns naturally appear in many vari...

2 MHz multi-wavelength pulsed laser for functional photoacoustic microscopy.

Fast functional photoacoustic microscopy requires multi-wavelength pulsed laser sources with high pulse repetition rates, short wavelength switching time, and sufficient pulse energies. Here, we report the development of a stimulated-Raman-scattering-based multi-wavelength pulsed laser source for fast functional photoacoustic imaging. The new laser source is pumped with a 532 nm 1 MHz pulsed laser. The 532 nm laser beam is split into two: one pumps a 5 m optical fiber to excite a 558 nm wavelength via stimulated Raman scattering; the other goes through a 50 m optical fiber to delay the 532 nm pulse by 220 ns. The two beams are combined and coupled into an optical fiber for photoacoustic excitation. As a result, the new laser source can generate 2 million pulses per second, switch wavelengths in 220 ns, and provide hundreds of nanojoules pulse energy for each wavelength. Using this laser source, we demonstrate optical-resolution photoacoustic imaging of microvascular structures and oxygen saturation in the mouse ear. The ultrashort wavelength switching time enables oxygen saturation imaging of flowing red blood cells, which is valuable for high-resolution functional imaging.

Towards new applications using capillary waveguides.

In this paper we demonstrate the enhancement of the sensing capabilities of glass capillaries. We exploit their properties as optical and acoustic waveguides to transform them potentially into high resolution minimally invasive endoscopic devices. We show two possible applications of silica capillary waveguides demonstrating fluorescence and optical-resolution photoacoustic imaging using a single 330 μm-thick silica capillary. A nanosecond pulsed laser is focused and scanned in front of a capillary by digital phase conjugation through the silica annular ring of the capillary, used as an optical waveguide. We demonstrate optical-resolution photoacoustic images of a 30 μm-thick nylon thread using the water-filled core of the same capillary as an acoustic waveguide, resulting in a fully passive endoscopic device. Moreover, fluorescence images of 1.5 μm beads are obtained collecting the fluorescence signal through the optical waveguide. This kind of silica-capillary waveguide together with wavefront shaping techniques such as digital phase conjugation, paves the way to minimally invasive multi-modal endoscopy.

In vivo optical resolution photoacoustic microscopy using glancing angle-deposited nanostructured Fabry-Perot etalons.

In this Letter, reflection-mode optical resolution photoacoustic microscopy (OR-PAM) using glancing angle-deposited (GLAD) nanostructured Fabry-Perot interferometers (FPI) for in vivo applications is reported. GLAD is a single-step physical vapor deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide, a transparent semiconductor with a high refractive index (n=2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The OR-PAM in vivo images of chorioallantoic membrane (CAM) of 5-day chicken embryo model are demonstrated. The phantom study shows lateral resolution and signal-to-noise ratio better than 7μm and 35dB, respectively. The sensitive GLAD FPI allows photoacoustic imaging down to a few-nJ pulse energy. To the best of our knowledge, this is the first time that a FPI-based reflection mode optical resolution photoacoustic imaging technique is demonstrated for in vivo applications.

Optical-resolution photoacoustic imaging through thick tissue with a thin capillary as a dual optical-in acoustic-out waveguide

We demonstrate the ability to guide high-frequency photoacoustic waves through thick tissue with a water-filled silica-capillary (150 μm inner diameter and 30 mm long). An optical-resolution photoacoustic image of a 30 μm diameter absorbing nylon thread was obtained by guiding the acoustic waves in the capillary through a 3 cm thick fat layer. The transmission loss through the capillary was about −20 dB, much lower than the −120 dB acoustic attenuation through the fat layer. The overwhelming acoustic attenuation of high-frequency acoustic waves by biological tissue can therefore be avoided by the use of a small footprint capillary acoustic waveguide for remote detection. We finally demonstrate that the capillary can be used as a dual optical-in acoustic-out waveguide, paving the way for the development of minimally invasive optical-resolution photoacoustic endoscopes free of any acoustic or optical elements at their imaging tip.

Concentration dependence of optical clearing on the enhancement of laser-scanning optical-resolution photoacoustic microscopy imaging

Quantitative analysis of optical clearing effects (OCE) induced by hyperosmotic agents is very important to optical tissue clearing applications in biomedical diagnostic imaging and therapeutics. This study aims at investigating the effect of glycerol concentration on the laser-scanning optical-resolution photoacoustic microscopy (LSOR-PAM) imaging contrast and light penetration depth. The photoacoustic (PA) signal amplitude changes are evaluated as a function of the concentration of glycerol. The results reveal that the PA signal amplitudes are enhanced with the glycerol concentration increasing, and also show that higher concentration of glycerol produces better light penetration and OCE on a phantom. The PA signal amplitude increases only 8.1% for 20% glycerol, but for higher concentrations, the increases are 76% and 165% for 40% and 60% glycerol, respectively. This preliminary study demonstrates that application of glycerol as an optical contrast agent reduces the tissue scattering and is beneficial to PAM imaging and optical diagnosis in clinical dermatology.

Wide-field two-dimensional multifocal optical-resolution photoacoustic-computed microscopy.

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 μm 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.

Optical-resolution photoacoustic microscopy by use of a multimode fiber

We demonstrate Optical-Resolution Photoacoustic Microscopy (OR-PAM), where the optical field is focused and scanned using Digital Phase Conjugation through a multimode fiber. The focus is scanned across the field of view using digital means, and the acoustic signal induced is collected by a transducer. Optical-resolution photoacoustic images of a knot made by two absorptive wires are obtained and we report on resolution smaller than 1.5 μm across a 201 μm × 201 μm field of view. The use of a multimode optical fiber for the optical excitation part can pave the way for miniature endoscopes that can provide optical-resolution photoacoustic images at large optical depth.

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ∼7 μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.