Photoacoustic Microscopy System Research Articles

Quad-mode functional and molecular photoacoustic microscopy

A conventional photoacoustic microscopy (PAM) system typically has to make tradeoffs between its spatial resolution and penetration depth, by choosing a fixed configuration of optical excitation and acoustic detection. The single-scale imaging capability of PAM may limit its applications in biomedical studies. Here, we report a quad-mode photoacoustic microscopy (QM-PAM) system with four complementary spatial resolutions and maximum penetration depths. For this we first developed a ring-shaped focused ultrasound transducer that has two independent elements with respective central frequencies at 20 MHz and 40 MHz, providing complementary acoustically-determined spatial resolutions and penetration depths. To accommodate the dual-element ultrasound transducer, we implemented two optical excitation modes to provide tightly- and weakly-focused light illumination. The dual-element acoustic detection combined with the two optical focusing modes can thus provide four imaging scales in a single imaging device, with consistent contrast mechanisms and co-registered field of views. We have demonstrated the multiscale morphological, functional, and molecular imaging capability of QM-PAM in the mouse head, leg and ear in vivo. We expect the high scale flexibility of QM-PAM will enable broad applications in preclinical studies.

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.

Miniaturized Optical Resolution Photoacoustic Microscope Based on a Microelectromechanical Systems Scanning Mirror.

In this paper, we report a miniaturized optical resolution photoacoustic microscopy system based on a microelectromechanical system (MEMS) scanning mirror. A two-dimensional MEMS scanning mirror was used to achieve raster scanning of the excitation optical focus. The wideband photoacoustic signals were detected by a flat ultrasound transducer with a center frequency of 10 MHz and an active area of 2 mm in diameter. The size and weight of this device were 60 mm × 30 mm × 20 mm and 40 g, respectively. We evaluated this system using sharp blades, carbon fibers, and a silver strip target. In vivo experiments of imaging vasculatures in the mouse ear, brain, and human lip were completed to demonstrate its potential for biological and clinical applications.

All-optical photoacoustic Doppler transverse blood flow imaging.

The method of measuring blood flow in photoacoustic microscopy usually relies on ultrasonic transducers in contact fashion, which is not favored in many applications, such as wound areas, burns, and anabrosis. Here we present a noncontact photoacoustic velocity measurement method to quantitatively map transverse blood flow based on the photoacoustic Doppler (PAD) bandwidth broadening method with an all-optical photoacoustic microscopy system. It is validated that the PAD bandwidth broadening is proportional to the transverse flow within a certain range. The transverse flow speed ranging from 0 to 5.5mm/s, as well as sectional flow images, was obtained in the blood-mimicking flow phantoms. Furthermore, the blood flow image of the mouse ear demonstrates that the all-optical photoacoustic Doppler method can acquire the information of blood flow in vivo, which could significantly broaden the scope of applications for obtaining the blood flow velocity of the microvasculature in biomedicine.

In vivo study of rat cortical hemodynamics using a stereotaxic-apparatus-compatible photoacoustic microscope.

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic-apparatus-compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2-dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm2 . Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.

Photoacoustic microscopy for evaluating combretastatin A4 phosphate induced vascular disruption in orthotopic glioma.

The use of an optical resolution photoacoustic microscopy (OR-PAM) system to evaluate the vascular disruptive effect of combretastatin A4 Phosphate (CA4P) on a murine orthotopic glioma with intact skull is described here. Second generation optical-resolution photoacoustic microscopy scanner with a 532 nm pulsed diode-pumped solid-state laser that specifically matches the absorption maximum of hemoglobin in tissues was used to image orthotopic glioma inoculated in mouse brain. Two-dimensional maps of brain vasculature with a lateral resolution of 5 μm and a depth of 700 μm at a field of view 5 × 4 mm were acquired on normal brain and glioma brain. Longitudinal imaging of the brain pre- and post-administration of CA4P, a FDA approved drug for solid tumors, enabled the monitoring of hemodynamic changes in tumor vasculature revealing the well documented vascular shutdown and recovery associated with this drug. Our study marks the beginning of potential prospects of this technology as an imaging tool for preclinical and clinical study of pathologies characterized by changes in the vasculature.

Label-free photoacoustic microscopy for in-vivo tendon imaging using a fiber-based pulse laser

Tendons are tough, flexible, and ubiquitous tissues that connect muscle to bone. Tendon injuries are a common musculoskeletal injury, which affect 7% of all patients and are involved in up to 50% of sports-related injuries in the United States. Various imaging modalities are used to evaluate tendons, and both magnetic resonance imaging and sonography are used clinically to evaluate tendons with non-invasive and non-ionizing radiation. However, these modalities cannot provide 3-dimensional (3D) structural images and are limited by angle dependency. In addition, anisotropy is an artifact that is unique to the musculoskeletal system. Thus, great care should be taken during tendon imaging. The present study evaluated a functional photoacoustic microscopy system for in-vivo tendon imaging without labeling. Tendons have a higher density of type 1 collagen in a cross-linked triple-helical formation (65–80% dry-weight collagen and 1–2% elastin in a proteoglycan-water matrix) than other tissues, which provides clear endogenous absorption contrast in the near-infrared spectrum. Therefore, photoacoustic imaging with a high sensitivity to absorption contrast is a powerful tool for label-free imaging of tendons. A pulsed near-infrared fiber-based laser with a centered wavelength of 780 nm was used for the imaging, and this system successfully provided a 3D image of mouse tendons with a wide field of view (5 × 5 mm2).

Retinal and choroidal imaging in vivo using integrated photoacoustic microscopy and optical coherence tomography.

Most reported photoacoustic ocular imaging work to date uses small animals, such as mice and rats, the eyes of which are small and less than one-third the size of a human eye, which poses a challenge for clinical translation. Here we achieved chorioretinal imaging of larger animals, i.e. rabbits, using a dual-modality photoacoustic microscopy (PAM) and optical coherence tomography (OCT) system. Preliminary experimental results in living rabbits demonstrate that the PAM can noninvasively visualize depth-resolved retinal and choroidal vessels using a safe laser exposure dose; and the OCT can finely distinguish different retinal layers, the choroid, and the sclera. This reported work might be a major step forward in clinical translation of photoacoustic microscopy.

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

Large area laser scanning optical resolution photoacoustic microscopy using a fibre optic sensor.

A laser scanning optical resolution photoacoustic microscopy (LS OR-PAM) system based on a stationary fibre optic sensor is described. The sensor comprises an optically resonant interferometric polymer cavity formed on the tip of a rounded single mode optical fibre. It provides low noise equivalent pressure (NEP = 68.7 Pa over a 20 MHz measurement bandwidth), a broad bandwidth that extends to 80 MHz and a near omnidirectional response. The latter is a significant advantage, as it allows large areas (>1cm2) to be imaged without the need for translational mechanical scanning offering the potential for fast image acquisition. The system provides a lateral resolution of 8 µm, an axial resolution of 21 µm, and a field of view up to 10 mm × 10 mm. To demonstrate the system, in vivo 3D structural images of the microvasculature of a mouse ear were obtained, showing single capillaries overlaying larger vessels as well as functional images revealing blood oxygen saturation.

In vivo oral imaging with integrated portable photoacoustic microscopy and optical coherence tomography

Oral diseases, especially oral cancers, are becoming serious health problems in humans. To image vasculatures and structures simultaneously in the human oral cavity which are tightly associated with various oral diseases, we develop a dual-modality portable optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) system. This system utilizes a new rotary scanning mechanism and a compact design of the imaging head, making it portable and free of translation of the imaging interface or samples. Through the phantom experiments, both modalities yield high lateral resolutions of 8.1 μm (ORPAM) and 8.56 μm (OCT), respectively. The axial resolutions are measured to be 116.5 μm for ORPAM and 6.1 μm for OCT. In vivo imaging of a mouse ear was carried out to evaluate the performance of the system in biological tissues. In addition, in vivo oral imaging of a healthy human lip and monitoring recovery progress of a lip ulcer demonstrate the clinical potential of this system.

Portable optical-resolution photoacoustic microscopy for volumetric imaging of multiscale organisms.

Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical-resolution photoacoustic microscopy (pORPAM) system for 3-dimensional (3D) imaging of small-to-large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer-associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases.

Laser scanning laser diode photoacoustic microscopy system

The development of low-cost and fast photoacoustic microscopy systems enhances the clinical applicability of photoacoustic imaging systems. To this end, we present a laser scanning laser diode-based photoacoustic microscopy system. In this system, a 905nm, 325W maximum output peak power pulsed laser diode with 50ns pulsewidth is utilized as the light source. A combination of aspheric and cylindrical lenses is used for collimation of the laser diode beam. Two galvanometer scanning mirrors steer the beam across a focusing aspheric lens. The lateral resolution of the system was measured to be ∼21μm using edge spread function estimation. No averaging was performed during data acquisition. The imaging speed is ∼370 A-lines per second. Photoacoustic microscopy images of human hairs, ex vivo mouse ear, and ex vivo porcine ovary are presented to demonstrate the feasibility and potentials of the proposed system.

Dual modality reflection mode optical coherence and photoacoustic microscopy using an akinetic sensor.

This Letter presents a novel dual modality reflection mode optical coherence and photoacoustic microscopy (OC-PAM) system. The optical coherence microscopy modality features a broadband source to accomplish 5 μm axial resolution. The photoacoustic microscopy modality uses a rigid akinetic Fabry-Perot etalon encapsulated in an optically transparent medium, which forms a 2 mm×11 mm translucent imaging window, permitting reflection mode dual modality imaging. After characterization, the OC-PAM system was applied to image zebrafish larvae in vivo, demonstrating its capability in biomedical imaging with complementary optical scattering and absorption contrasts by revealing morphology in the fish larvae.

High-speed dual-layer scanning photoacoustic microscopy using focus tunable lens modulation at resonant frequency.

The range of imaging depth in optical resolution photoacoustic microscopy (PAM) is limited by the short depth of focus of high-numerical aperture objective lenses. In this paper, focus tunable lens modulation has been employed at the resonant frequency of focus tunable lenses in order to enhance both the range of imaging depth and the scanning speed. By electrically controlling the focal length in the axial direction of the sample, the range of imaging depth was extended approximately 1.22 times and the scanning speed was enhanced by approximately 7.40 times, in comparison to corresponding values of conventional PAM systems.

Noninvasive chorioretinal imaging in living rabbits using integrated photoacoustic microscopy and optical coherence tomography.

Most reported photoacoustic ocular imaging work to date uses small animals, such as mice and rats, the eyeball sizes of which are less than one-third of those of humans, posing challenges for clinical translation. Here we developed a novel integrated photoacoustic microscopy (PAM) and optical coherence tomography (OCT) system for dual-modality chorioretinal imaging of larger animals, such as rabbits. The system has quantified lateral resolutions of 4.1 µm (PAM) and 3.8 µm (OCT), and axial resolutions of 37.0 µm (PAM) and 4.0 µm (OCT) at the focal plane of the objective. Experimental results in living rabbits demonstrate that the PAM can noninvasively visualize individual depth-resolved retinal and choroidal vessels using a laser exposure dose of ~80 nJ below the American National Standards Institute (ANSI) safety limit 160 nJ at 570 nm; and the OCT can finely distinguish different retinal layers, the choroid, and the sclera. This reported work may be a major step forward in clinical translation of the technology.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging.

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 µm with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 µm with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,in vivosmall-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy.

The goal of breast-conserving surgery is to completely remove all of the cancer. Currently, no intraoperative tools can microscopically analyze the entire lumpectomy specimen, which results in 20 to 60% of patients undergoing second surgeries to achieve clear margins. To address this critical need, we have laid the foundation for the development of a device that could allow accurate intraoperative margin assessment. We demonstrate that by taking advantage of the intrinsic optical contrast of breast tissue, photoacoustic microscopy (PAM) can achieve multilayered histology-like imaging of the tissue surface. The high correlation of the PAM images to the conventional histologic images allows rapid computations of diagnostic features such as nuclear size and packing density, potentially identifying small clusters of cancer cells. Because PAM does not require tissue processing or staining, it can be performed promptly and intraoperatively, enabling immediate directed re-excision and reducing the number of second surgeries.

Performance Characterization of a Switchable Acoustic Resolution and Optical Resolution Photoacoustic Microscopy System.

Photoacoustic microscopy (PAM) is a scalable bioimaging modality; one can choose low acoustic resolution with deep penetration depth or high optical resolution with shallow imaging depth. High spatial resolution and deep penetration depth is rather difficult to achieve using a single system. Here we report a switchable acoustic resolution and optical resolution photoacoustic microscopy (AR-OR-PAM) system in a single imaging system capable of both high resolution and low resolution on the same sample. Lateral resolution of 4.2 µm (with ~1.4 mm imaging depth) and lateral resolution of 45 μm (with ~7.6 mm imaging depth) was successfully demonstrated using a switchable system. In vivo blood vasculature imaging was also performed for its biological application.