Conventional Optical Resolution Photoacoustic Microscopy Research Articles

Multi-focus image fusion with enhancement filtering for robust vascular quantification using photoacoustic microscopy.

Accurate identification and quantification of microvascular patterns are important for clinical diagnosis and therapeutic monitoring using optical-resolution photoacoustic microscopy (OR-PAM). Due to its limited depth of field, conventional OR-PAM may not fully reveal microvascular patterns with enough details in depth range, which affects the segmentation and quantification. Here, we propose a robust vascular quantification approach via combining multi-focus image fusion with enhancement filtering (MIFEF). The multi-focus image fusion is constructed based on multi-scale gradients and image matting to improve image fusion quality by considerably achieving accurate focus measurement for initial segmentation as well as decision map refinement. The enhancement filtering identifies the vessels and handles noise without deforming microvasculature. The performance of the MIFEF were evaluated employing a leaf phantom, mouse livers and brains. The proposed method for OR-PAM can significantly facilitate the clinical provision of optical biopsy of vascular-related diseases.

Video-rate high-resolution single-pixel nonscanning photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) is widely utilized in biomedical applications because of its ability to noninvasively image biological tissues in vivo while providing high-resolution morphological and functional information. However, one drawback of conventional OR-PAM is its imaging speed, which is restricted by the scanning technique employed. To achieve a higher imaging frame rate, we present video-rate high-resolution single-pixel nonscanning photoacoustic microscopy (SPN-PAM), which utilizes Fourier orthogonal basis structured planar illumination to overcome the above-mentioned limitations. A 473 × 473 µm2 imaging field of view (FOV) with 3.73 µm lateral resolution and video-rate imaging of 30 Hz were achieved. In addition, in both in vitro cell and in vivo mouse vascular hemodynamic imaging experiments, high-quality images were obtained at ultralow sampling rates. Thus, the proposed high-resolution SPN-PAM with video-rate imaging speed provides new insights into high-speed PA imaging and could be a powerful tool for rapid biological imaging.

Optical resolution photoacoustic computed microscopy

Optical resolution photoacoustic microscopy (ORPAM) has demonstrated both high resolution and rich contrast imaging of optical chromophores in biologic tissues. To date, sensitivity remains a major challenge for ORPAM, which limits the capability of resolving biologic microvascular networks. In this study, we propose and evaluate a new ORPAM modality termed as optical resolution photoacoustic computed microscopy (ORPACM), through the combination of a two-dimensional laser-scanning system with a medical ultrasonographic platform. Apart from conventional ORPAMs, we record multiple photoacoustic (PA) signals using a 128-element ultrasonic transducer array for each pulse excitation. Then, we apply a reconstruction algorithm to recover one depth-resolved PA signal referred to as an A-line, which reveals more detailed information compared with conventional single-element transducer-based ORPAMs. In addition, we carried out both in vitro and in vivo experiments as well as quantitative analyses to show the advanced features of ORPACM.

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.

Subdiffraction-limited second harmonic photoacoustic microscopy based on nonlinear thermal diffusion.

We have developed a second harmonic photoacoustic microscopy (SH-PAM) for subdiffraction-limited imaging based on nonlinear thermal diffusion. When a sine-modulated Gaussian temperature field is introduced by a laser beam, the temperature dependence of the thermal diffusivity induces a nonlinear photoacoustic (PA) effect and thus results in the production of second harmonic PA signals. We demonstrate through both simulation and experiment that the second harmonic PA images can be reconstructed with a lateral resolution exceeding that of conventional optical resolution PA microscopy. The feasibility of SH-PAM was verified on phantom samples. Amphioxus zygotes and germinated pollens have been studied by SH-PAM to demonstrate its biomedical imaging capability. This method expands the scope of conventional PA imaging and opens up new possibilities for super-resolution imaging, prefiguring great potential for biological imaging and material inspection.

Portable optical resolution photoacoustic microscopy (pORPAM) for human oral imaging.

Optical resolution photoacoustic microscopy (ORPAM) represents one of the fastest evolving optical microscopic techniques. However, due to the bulky size and complicated system configuration of conventional ORPAM, it is largely limited to small animal experiments. In this Letter, we present the design and evaluation of a portable ORPAM with a high spatiotemporal resolution and a large field of view. In this system, we utilize a rotatory scanning mechanism instead of the conventional raster scanning to achieve translationless imaging of the probe/samples, making it accessible to the human oral lip and tongue. After phantom evaluation, we applied this system to monitor longitudinal neo-angiogenesis of tumor growth and, for the first time, to the best of our knowledge, image the oral vascular network of humans to show its potential in clinical detection of early-stage oral cancer.

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.

Microsphere enabled subdiffraction-limited optical-resolution photoacoustic microscopy: a simulation study.

Optical resolution photoacoustic microscopy (ORPAM) is a high-resolution hybrid imaging modality having potential for microscale in vivo imaging. Optical diffraction limits the lateral resolution of ORPAM. A photonic nanojet (PNJ) was used to break this diffraction limit. A single round microsphere can generate a PNJ with subwavelength waist, but its short axial length limits its applications to surface imaging only. We investigate different sphere designs to achieve ultralong nanojets that will make the nanojet more viable in far-field applications, such as photoacoustic imaging. The PNJ properties, including effective length, waist size, working distance, and peak intensity, can be tuned and controlled by changing the sphere design and its refractive index. A truncated multilayer microsphere design could generate an ultraelongated PNJ with length larger than ∼172λ (∼138 μm) while retaining a large working distance ∼32λ (∼26 μm). Through simulation study, we observed ∼11-fold enhancement in lateral resolution with 5 μm round sphere (refractive index 2.2) when used in a conventional ORPAM setup with NA=0.1 and λ=800 nm.

Super-resolution photoacoustic microscopy using photonic nanojets: a simulation study.

Optical resolution photoacoustic microscopy (ORPAM) is important for various biomedical applications, such as the study of cellular structures, microcirculation systems, and tumor angiogenesis. However, the lateral resolution of a conventional ORPAM is limited by optical diffraction. In this work, we report a simulation study to achieve subdiffraction-limited super-resolution in ORPAM using microspheres. Laser radiation is focused through a microsphere to generate a photonic nanojet, which provides the possibility to break the diffraction limit in ORPAM by reducing the size of the excitation volume. In our simulations using microspheres, we observed improvement in the lateral resolution up to compared to conventional ORPAM. The method is simple, cost effective, and can provide far-field resolution. This approach may provide new opportunities for many biomedical imaging applications that require finer resolution.

Microvascular quantification based on contour-scanning photoacoustic microscopy.

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.

Blind-deconvolution optical-resolution photoacoustic microscopy in vivo

Optical-resolution photoacoustic microscopy (OR-PAM) is becoming a vital tool for studying the microcirculation system in vivo. By increasing the numerical aperture of optical focusing, the lateral resolution of OR-PAM can be improved; however, the depth of focus and thus the imaging range will be sacrificed correspondingly. In this work, we report our development of blind-deconvolution optical-resolution photoacoustic microscopy (BD-PAM) that can provide a lateral resolution ~2-fold finer than that of conventional OR-PAM (3.04 vs. 5.78μm), without physically increasing the system's numerical aperture. The improvement achieved with BD-PAM is demonstrated by imaging graphene nanoparticles and the microvasculature of mice ears in vivo. Our results suggest that BD-PAM may become a valuable tool for many biomedical applications that require both fine spatial resolution and extended depth of focus.

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.