Optical-resolution Photoacoustic Microscopy System Research Articles

In vivo photoacoustic monitoring using 700-nm region Raman source for targeting Prussian blue nanoparticles in mouse tumor model

Photoacoustic imaging (PAI) is a noninvasive imaging tool to visualize optical absorbing contrast agents. Due to high ultrasonic resolution and superior optical sensitivity, PAI can be used to monitor nanoparticle-mediated cancer therapy. The current study synthesized Food and Drug Administration-approved Prussian blue (PB) in the form of nanoparticles (NPs) with the peak absorption at 712 nm for photoacoustically imaging tumor-bearing mouse models. To monitor PB NPs from the background tissue in vivo, we also developed a new 700-nm-region stimulated Raman scattering (SRS) source (pulse energy up to 200 nJ and repetition rate up to 50 kHz) and implemented optical-resolution photoacoustic microscopy (OR-PAM). The SRS-assisted OR-PAM system was able to monitor PB NPs in the tumor model with micrometer resolution. Due to strong light absorption at 712 nm, the developed SRS light yielded a two-fold higher contrast from PB NPs, in comparison with a 532-nm pumping source. The proposed laser source involved cost-effective and simple system implementation along with high compatibility with the fiber-based OR-PAM system. The study highlights the OR-PAM system in conjunction with the tunable-color SRS light source as a feasible tool to assist NP-mediated cancer therapy.

Correcting the limited view in optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) has proven useful for anatomical and functional imaging with high spatial resolutions. However, the coherent signal generation and the desired reflection-mode detection in OR-PAM can result in a limited detectability of features aligned with the acoustic axis (ie, vertical structures). Here, we investigated the limited-view phenomenon in OR-PAM by simulating the generation and propagation of the acoustic pressure waves and determined the key optical parameters affecting the visibility of vertical structures. Proof-of-concept numerical experiments were performed with different illumination angles, optical foci and numerical apertures (NA) of the objective lens. The results collectively show that an NA of 0.3 can readily improve the visibility of vertical structures in a typical reflection-mode OR-PAM system. This conclusion was confirmed by numerical simulations on the cortical blood vessels in a mouse brain and by experiments in a suture-cross phantom and in a mouse brain in vivo.

Handheld Photoacoustic Microscopy Probe

Optical resolution photoacoustic microscopy (OR-PAM) is a non-invasive, label-free method of in vivo imaging with microscopic resolution and high optical contrast. Based on intrinsic contrasts, OR-PAM has expanded to include in vivo vessel imaging, flow cytometry, physiological parameter analysis, and single-cell characterization. However, since conventional OR-PAM systems have a fixed tabletop configuration, a large system size, and slow imaging speed, their use in preclinical and clinical studies remains limited. In this study, using microelectromechanical systems (MEMS) technology, we developed a handheld PAM probe with a high signal-to-noise ratio and image rate. To enable broader application of the OR-PAM system, we reduced its size and combined its fast scanning capabilities into a small handheld probe that uses a 2-axis waterproof MEMS scanner (2A-WP-MEMS scanner). All acoustical, optical, and mechanical components are integrated into a single probe with a diameter of 17 mm and a weight of 162 g. This study shows phantom and in vivo images of various samples acquired with the probe, including carbon fibers, electrospun microfibers, and the ear, iris, and brain of a living mouse. In particular, this study investigated the possibility of clinical applications for melanoma diagnosis by imaging the boundaries and morphology of a human mole.

Development of a Fiber Laser with Independently Adjustable Properties for Optical Resolution Photoacoustic Microscopy.

Photoacoustic imaging is based on the detection of generated acoustic waves through thermal expansion of tissue illuminated by short laser pulses. Fiber lasers as an excitation source for photoacoustic imaging have recently been preferred for their high repetition frequencies. Here, we report a unique fiber laser developed specifically for multiwavelength photoacoustic microscopy system. The laser is custom-made for maximum flexibility in adjustment of its parameters; pulse duration (5–10 ns), pulse energy (up to 10 μJ) and repetition frequency (up to 1 MHz) independently from each other and covers a broad spectral region from 450 to 1100 nm and also can emit wavelengths of 532, 355, and 266 nm. The laser system consists of a master oscillator power amplifier, seeding two stages; supercontinuum and harmonic generation units. The laser is outstanding since the oscillator, amplifier and supercontinuum generation parts are all-fiber integrated with custom-developed electronics and software. To demonstrate the feasibility of the system, the images of several elements of standardized resolution test chart are acquired at multiple wavelengths. The lateral resolution of optical resolution photoacoustic microscopy system is determined as 2.68 μm. The developed system may pave the way for spectroscopic photoacoustic microscopy applications via widely tunable fiber laser technologies.

A PDMS-Based 2-Axis Waterproof Scanner for Photoacoustic Microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) is an imaging tool to provide in vivo optically sensitive images in biomedical research. To achieve a small size, fast imaging speed, wide scan range, and high signal-to-noise ratios (SNRs) in a water environment, we introduce a polydimethylsiloxane (PDMS)-based 2-axis scanner for a flexible and waterproof structure. The design, theoretical background, fabrication process and performance of the scanner are explained in details. The designed and fabricated scanner has dimensions of 15 × 15 × 15 mm along the X, Y and Z axes, respectively. The characteristics of the scanner are tested under DC and AC conditions. By pairing with electromagnetic forces, the maximum scanning angles in air and water are 18° and 13° along the X and Y axes, respectively. The measured resonance frequencies in air and water are 60 and 45 Hz along the X axis and 45 and 30 Hz along the Y axis, respectively. Finally, OR-PAM with high SNRs is demonstrated using the fabricated scanner, and the PA images of micro-patterned samples and microvasculatures of a mouse ear are successfully obtained with high-resolution and wide-field of view. OR-PAM equipped with the 2-axis PDMS based waterproof scanner has lateral and axial resolutions of 3.6 μm and 26 μm, respectively. This compact OR-PAM system could potentially and widely be used in preclinical and clinical applications.

Fast optical-resolution photoacoustic microscopy using a 2-axis water-proofing MEMS scanner.

Optical-resolution photoacoustic microscopy (OR-PAM) is a novel label-free microscopic imaging tool to provide in vivo optical absorbing contrasts. Specially, it is crucial to equip a real-time imaging capability without sacrificing high signal-to-noise ratios (SNRs) for identifying and tracking specific diseases in OR-PAM. Herein we demonstrate a 2-axis water-proofing MEMS scanner made of flexible PDMS. This flexible scanner results in a wide scanning range (9 × 4 mm2 in a transverse plane) and a fast imaging speed (5 B-scan images per second). Further, the MEMS scanner is fabricated in a compact footprint with a size of 15 × 15 × 15 mm3. More importantly, the scanning ability in water makes the MEMS scanner possible to confocally and simultaneously reflect both ultrasound and laser, and consequently we can maintain high SNRs. The lateral and axial resolutions of the OR-PAM system are 3.6 and 27.7 μm, respectively. We have successfully monitored the flow of carbon particles in vitro with a volumetric display frame rate of 0.14 Hz. Finally, we have successfully obtained in vivo PA images of microvasculatures in a mouse ear. It is expected that our compact and fast OR-PAM system can be significantly useful in both preclinical and clinical applications.

Confocal photoacoustic microscopy using a single multifunctional lens

Photoacoustic microscopy (PAM) has remained one of the fastest developing biomedical imaging modalities in the past decade. The confocal strategy of optical illumination and acoustic detection is a way to boost the sensitivity of PAM. To achieve confocal PAM, current PAM systems utilize separate acoustic and optical converging devices, making the systems bulky and complicated. In this Letter, we demonstrate the use of a single-liquid lens to successfully achieve acoustic and optical confocal configuration for optical-resolution PAM (ORPAM). Using the lens with a numerical aperture of 0.43, we show that the resolution of the ORPAM system is 4.8μm with a significantly improved sensitivity of acoustic detection. We also apply this compact ORPAM system to in vivo imaging of the vasculature of a rat ear.

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.

A SIMULTANEOUS MULTI-PROBE DETECTION LABEL-FREE OPTICAL-RESOLUTION PHOTOACOUSTIC MICROSCOPY TECHNIQUE BASED ON MICROCAVITY TRANSDUCER

We demonstrate the feasibility of simultaneous multi-probe detection for an optical-resolution photoacoustic microscopy (OR-PAM) system. OR-PAM has elicited the attention of biomedical imaging researchers because of its optical absorption contrast and high spatial resolution with great imaging depth. OR-PAM allows label-free and noninvasive imaging by maximizing the optical absorption of endogenous biomolecules. However, given the inadequate absorption of some biomolecules, detection sensitivity at the same incident intensity requires improvement. In this study, a modulated continuous wave with power density less than 3 mW/cm2 (1/4 of the ANSI safety limit) excited the weak photoacoustic (PA) signals of biological cells. A microcavity transducer is developed based on the bulk modulus of gas five orders of magnitude lower than that of solid; air pressure variation is inversely proportional to cavity volume at the same temperature increase. Considering that a PA wave expands in various directions, detecting PA signals from different positions and adding them together can increase detection sensitivity and signal-to-noise ratio. Therefore, we employ four detectors to acquire tiny PA signals simultaneously. Experimental results show that the developed OR-PAM system allows the label-free imaging of cells with weak optical absorption.

Reflection-mode multifocal optical-resolution photoacoustic microscopy

Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. A reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci as well as an ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously is presented. Using a customized microprism to reflect the incident laser beam to the microlens array, the multiple optical foci are aligned confocally with the focal zone of the ultrasonic array transducer. Experiments show the reflection-mode multifocal OR-PAM is capable of imaging microvessels in vivo, and it can image a 6×5×2.5 mm³ volume at 16 μm lateral resolution in ∼2.5 min, which was limited by the signal multiplexing ratio and laser pulse repetition rate.

Portable optical-resolution photoacoustic microscopy with a pulsed laser diode excitation

Optical-resolution photoacoustic microscopy (OR-PAM) has been significantly improved in terms of spatial resolution, detection sensitivity, imaging speed, and penetration depth. However, the popular producibility of OR-PAM system is still limited by the size and cost of solid-state laser excitation. Here, we developed a portable laser-diode-based OR-PAM (LD-OR-PAM) system using a pulsed semiconductor laser source, which was operated at 905 ± 15 nm with a pulse energy as low as 4.9 μJ. The measured lateral resolution has been improved to ∼1.5 μm from hundreds of microns. The compact and inexpensive natures of LD-OR-PAM would promote the potential clinical applications such as in dermatology.

Objective-free optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) becomes a premier microscopic imaging tool in biomedicine because it provides agent-free optical absorption information in tissues. By tightly focusing light to a spot, a significantly improved lateral resolution can be achieved in OR-PAM. The focal spot size is typically determined by the numerical aperture of the used objective lens. Here, we demonstrate objective-free OR-PAM using a fiber optic Bessel beam generator. In this approach, no objective lens is required and, beneficially, the complexities of conventional OR-PAM systems can be greatly relieved. We have obtained photoacoustic images of a carbon fiber with a diameter of ∼6 μm, whose lateral resolution was measured to be better than 6 to 7 μm.

Mosaic acquisition and processing for optical-resolution photoacoustic microscopy

In optical-resolution photo-acoustic microscopy (OR-PAM), data acquisition time is limited by both laser pulse repetition rate (PRR) and scanning speed. Optical-scanning offers high speed, but limited, field of view determined by ultrasound transducer sensitivity. In this paper, we propose a hybrid optical and mechanical-scanning OR-PAM system with mosaic data acquisition and processing. The system employs fast-scanning mirrors and a diode-pumped, nanosecond-pulsed, Ytterbium-doped, 532-nm fiber laser with PRR up to 600 kHz. Data from a sequence of image mosaic patches is acquired systematically, at predetermined mechanical scanning locations, with optical scanning. After all imaging locations are covered, a large panoramic scene is generated by stitching the mosaic patches together. Our proposed system is proven to be at least 20 times faster than previous reported OR-PAM systems.

Optical-resolution photoacoustic microscopy based on two-dimensional scanning galvanometer

An optical-resolution photoacoustic microscopy system was designed and fabricated by integration of a two-dimensional scanning galvanometer, an objective lens, an unfocused ultrasound transducer, and a sample stage. The lateral resolution of the system was measured to be ∼500 nm. Ex vivo erythrocytes were used to test the imaging capability of the system, and a single erythrocyte was mapped with high contrast. Furthermore, invivo blood vessels of a mouse ear were clearly shown, and the injured blood vessels were also monitored. The experimental results demonstrate that galvanometer-based photoacoustic microscopy holds clinical potential in detecting lesion of erythrocyte and blood vessel.

Real-time handheld optical-resolution photoacoustic microscopy

In this paper a new generation of optical-resolution photoacoustic microscopy (OR-PAM) with a wide range of potential clinical applications is demonstrated. Using fast scanning mirrors, an image guide with 30,000 fiber pixels, a refocusing lens and a unique probe we managed to reduce the footprint of an OR-PAM system from a stationary table-top system to a portable, 4 cm by 6 cm, probe weighing ~500 g tethered to a scanning unit. The phantom studies show that the handheld optical-resolution photoacoustic microscope is able to image with ~7 μm resolution. For in vivo studies images of the microvasculature in a Swiss Webster mouse ear are shown. The compact, flexible nature of the proposed design and the small footprint of the apparatus increase the usability of OR-PAM for potential clinical applications such as in dermatology.

In vivo near-realtime volumetric optical-resolution photoacoustic microscopy using a high-repetition-rate nanosecond fiber-laser

Optical-resolution photoacoustic microscopy (OR-PAM) is capable of achieving optical-absorption-contrast images with micron-scale spatial resolution. Previous OR-PAM systems have been frame-rate limited by mechanical scanning speeds and laser pulse repetition rate (PRR). We demonstrate OR-PAM imaging using a diode-pumped nanosecond-pulsed Ytterbium-doped 532-nm fiber laser with PRR up to 600 kHz. Combined with fast-scanning mirrors, our proposed system provides C-scan and 3D images with acquisition frame rate of 4 frames per second (fps) or higher, two orders of magnitude faster than previously published systems. High-contrast images of capillary-scale microvasculature in a live Swiss Webster mouse ear with ~6-µm optical lateral spatial resolution are demonstrated.

In vivo functional chronic imaging of a small animal model using optical‐resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) has been validated as a valuable tool for label-free volumetric microvascular imaging. More importantly, the advantages of noninvasiveness and measurement consistency suggest the use of OR-PAM for chronic imaging of intact microcirculation. Here, such chronic imaging is demonstrated for the first time by monitoring the healing process of laser-induced microvascular lesions in a small animal model in vivo. The central part of a 1 mm by 1 mm region in a nude mouse ear was treated under a continuous-wave laser to create a microvascular lesion for chronic study. The region of interest was imaged before the laser treatment, immediately after the treatment, and throughout the healing process using both the authors' OR-PAM system and a commercial transmission-mode optical microscope. Three-dimensional microvascular morphology and blood oxygenation information were imaged simultaneously at capillary-level resolution. Transmission-mode optical microscopic images were acquired for comparison. OR-PAM has potential important applications in microcirculatory physiology or pathophysiology, tumor angiogenesis, laser microsurgery, and neuroscience.