Optical-resolution Photoacoustic Microscopy Research Articles

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).

Multiscale high-speed photoacoustic microscopy based on free-space light transmission and a MEMS scanning mirror.

The conventional photoacoustic microscopy (PAM) system allows trade-offs between lateral resolution and imaging depth, limiting its applications in biological imaging in vivo. Here we present an integrated optical-resolution (OR) and acoustic-resolution (AR) multiscale PAM based on free-space light transmission and fast microelectromechanical systems (MEMS) scanning. The lateral resolution for OR is 4.9 µm, and the lateral resolution for AR is 114.5 µm. The maximum imaging depth for OR is 0.7 mm, and the maximum imaging depth for AR is 4.1 mm. The imaging speed can reach 50 k Alines per second. The high signal-to-noise ratios and wavelength throughput are achieved by delivering light via free-space, and the high speed is achieved by a MEMS scanning mirror. The blood vasculature from superficial skin to the deep tissue of a mouse leg was imaged in vivo using two different resolutions to demonstrate the multiscale imaging capability.

A microvascular image analysis method for optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) has been shown to be an excellent tool for high-resolution imaging of microvasculature, and quantitative analysis of the microvasculature can provide valuable information for the early diagnosis and treatment of various vascular-related diseases. In order to address the characteristics of weak signals, discontinuity and small diameters in photoacoustic microvascular images, we propose a method adaptive to the microvascular segmentation in photoacoustic images, including Hessian matrix enhancement and the morphological connection operators. The accuracy of our vascular segmentation method is quantitatively evaluated by the multiple criteria. To obtain more precise and continuous microvascular skeletons, an improved skeleton extraction framework based on the multistencil fast marching (MSFM) method is developed. We carried out in vivo OR-PAM microvascular imaging in mouse ears and subcutaneous hepatoma tumor model to verify the correctness and superiority of our proposed method. Compared with the previous methods, our proposed method can extract the microvascular network more completely, continuously and accurately, and provide an effective solution for the quantitative analysis of photoacoustic microvascular images with many small branches.

Micro-rocket robot with all-optic actuating and tracking in blood

Micro/nanorobots have long been expected to reach all parts of the human body through blood vessels for medical treatment or surgery. However, in the current stage, it is still challenging to drive a microrobot in viscous media at high speed and difficult to observe the shape and position of a single microrobot once it enters the bloodstream. Here, we propose a new micro-rocket robot and an all-optic driving and imaging system that can actuate and track it in blood with microscale resolution. To achieve a high driving force, we engineer the microrobot to have a rocket-like triple-tube structure. Owing to the interface design, the 3D-printed micro-rocket can reach a moving speed of 2.8 mm/s (62 body lengths per second) under near-infrared light actuation in a blood-mimicking viscous glycerol solution. We also show that the micro-rocket robot is successfully tracked at a 3.2-µm resolution with an optical-resolution photoacoustic microscope in blood. This work paves the way for microrobot design, actuation, and tracking in the blood environment, which may broaden the scope of microrobotic applications in the biomedical field.

Development of optical resolution photoacoustic microscopy with sub-micron lateral resolution for visualization of cells and their structures

Photoacoustic (PA) imaging is a hybrid imaging technique with light-dependent contrast and ultrasound-dependent depth. In previous studies, the interest of PA imaging was mainly clinical research. However, in recent years, PA imaging that targets cells also has attracted attention. Cells are the smallest unit in our body, and cell abnormalities cause various diseases. Therefore, PA imaging of cells is important for elucidating the relationship between diseases and PA properties and linking the results to the clinical application of PA imaging. For PA imaging of cells and its internal structures, sub-micron lateral resolution is required. In this study, we have developed an optical resolution photoacoustic microscopy (OR-PAM) system using a high NA objective lens and a single-mode fiber to achieve sub-micron lateral resolution. With the OR-PAM, it was confirmed that the lateral resolution was better than 700 nm, and the characteristic shapes of red blood cells and melanoma cells could be visualized.

Photoacoustic imaging of microenvironmental changes in facial cupping therapy.

As a traditional medicine practice, cupping therapy has been widely used to relieve symptoms like fatigue, tension, and muscle pain. During the therapy, negative pressure is applied to the skin for a while with an intention to enhance blood circulation or induce micro-bleeding. The therapeutic effect, however, is not clear due to the lack of direct quantification. Aiming at a quantitative assessment of the treatment effect, we explore optical-resolution photoacoustic microscopy (OR-PAM) in monitoring the structural and functional changes after cupping. We find that, after 5-minutes of ∼ 20 kPa negative pressure cupping, more capillaries appear in the focus, and micro-blooding is observed from the capillaries. We quantify the images and find the blood vessel density is increased by 64%, and the total hemoglobin concentration in both the veins and the arteries exhibits 62% and 40% elevation, respectively. Oxygen saturation in the vein and artery decreased by 17% and 3% right after cupping, respectively. After two hours of recovery, the three blood-related parameters return to their original levels, indicating that the effects in the tissue last only a short period after cupping at the given pressure and time duration. Note that no significant cupping marks are induced with the treatment parameters in this study. This work proposes OR-PAM to quantitatively monitor and evaluate the effect of cupping therapy from the perspective of imaging. The method is also useful for accurate control of the therapeutic outcome.

Acoustic-spectrum-compensated photoacoustic microscopy.

Photoacoustic microscopy (PAM) can label-free image oxy- and deoxy-hemoglobin (${{\rm HbO}_2}$HbO2 and Hb) concentrations in vivo, providing useful information for metabolic researches and diagnostic applications. Conventional PAM assumes a linear relationship between the photoacoustic amplitude and the absorption coefficient. However, many factors, including absorber size, laser pulse width, and frequency response of the ultrasound transducer, may affect the measured acoustic spectrum and the shape of the temporal photoacoustic signal. The ultrasound transducer may weigh the blood vessels differently according to their diameters. In addition, the pulse width also affects the photoacoustic signal amplitude. These factors may cause inaccurate measurement of Hb and ${{\rm HbO}_2}$HbO2 concentrations. To address this issue, we develop an acoustic-spectrum-compensated optical-resolution PAM (OR-PAM) that corrects the nonuniform acoustic spectrum and makes the quantitative results to be independent of the vessel diameter and pulse width. In dual-wavelength OR-PAM, we demonstrate that the acoustic spectrum compensation can improve the accuracy of oxygen saturation imaging by $\sim{15}\% $∼15%.

Optical-resolution photoacoustic microscopy with ultrafast dual-wavelength excitation.

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength-switching time. Recent development of stimulated Raman scattering in optical fiber offers a low-cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A-line rate. In addition, a long-time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual-wavelength excitation, we present optical-resolution photoacoustic microscopy with ultrafast dual-wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50-ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual-wavelength excitation technique to in vivo OR-PAM. Results demonstrate that A-lines at two wavelengths can be successfully separated, and sO2 values can be reliably computed from the separated data. The ultrafast dual-wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.

Vascular bifurcation mapping with photoacoustic microscopy

The early detection of microvascular changes in cancer diagnosis is needed in the clinic. A change in the vascular bifurcation density is a biomarker for the sprouting activity. Here, Optical-Resolution PhotoAcoustic Microscopy is used for quantitative vascular bifurcation mapping in 2D after the creation of Virtual Tubes out of Bifurcations. In stacks of OR-PAM images of the hemoglobin distribution, bifurcations become tubes and are selected by the 3D tubeness filter. These fast analyses will be compared to a classical approach and are easier to implement for functional analysis of the vascular bifurcation density in healthy and diseased tissues.

Fully waterproof two-axis galvanometer scanner for enhanced wide-field optical-resolution photoacoustic microscopy.

A large field-of-view and fast scanning of photoacoustic microscopy (PAM) relatively have been difficult to obtain due to the water-drowned structure of the system for the transmission of ultrasonic signals. Researchers have widely studied the achievement of a waterproof scanner for dynamic biological applications with a high-resolution and high signal-to-noise ratio. This Letter reports a novel, to the best of our knowledge, waterproof galvanometer scanner-based PAM system with a successfully attainable ${9.0}\;{\rm mm} \times {14.5}\;{\rm mm}$9.0mm×14.5mm scan region, amplitude scan rate of 40 kHz, and spatial resolution of 4.9 µm. The in vivo characterization of a mouse brain in intact-skull microvascular visualization demonstrated its capability in biomedical imaging and is anticipated to be an effective technique for various preclinical and clinical studies.

High acoustic numerical aperture photoacoustic microscopy with improved sensitivity

Limited by the numerical aperture of ultrasonic detection, optical resolution photoacoustic microscopy (OR-PAM) has not achieved optimal sensitivity. To address this problem, we have developed a high acoustic numerical aperture ($ {\sim} 0.74 $∼0.74) OR-PAM (HNA-OR-PAM). Via engineering the acoustic lens, we implement the highest acoustic numerical aperture that a spherical concave lens can achieve. The sensitivity of HNA-OR-PAM is improved to around 160%-the state-of-the-art OR-PAM. Without averaging, the new system can image oxygen saturation in vivo with only 10-nJ pulse energy. The improved sensitivity allows us to image weaker absorbers, penetrate deeper, and reduce nonlinear effects induced by high pulse energy. Moreover, the photoacoustic view angle is augmented to 51.8 deg and makes tilted features more visible. We validate the improved view angle in both a phantom study and brain imaging.

Integrating photoacoustic microscopy, optical coherence tomography, OCT angiography, and fluorescence microscopy for multimodal imaging.

We have developed a multimodal imaging system, which integrated optical resolution photoacoustic microscopy, optical coherence tomography, optical coherence tomography angiography, and confocal fluorescence microscopy in one platform. The system is able to image complementary features of a biological sample by combining different contrast mechanisms. We achieved fast imaging and large field of view by combining optical scanning with mechanical scanning, similar to our previous publication. We have demonstrated the capability of the multimodal imaging system by imaging a mouse ear in vivo. Impact statement Photoacoustic microscopy-based multimodal imaging technology can provide high-resolution complementary information for biological tissues in vivo. It will potentially bring significant impact on the research and diagnosis of diseases by providing combined structural and functional information.

Optical-resolution photoacoustic microscopy continually monitors macrophages activities of acute inflammation in vivo

Photoacoustic imaging has been developed to image the immune study at the macro scale. Macrophages play diverse roles in the acute response to infection and tissue repair. However, macrophages activities in acute inflammation at the microscopic level still remain challenging. In this work, we proposed optical-resolution photoacoustic microscopy to promptly monitor the labeled macrophages activities in normal and inflammatory groups. The result showed that many labeled macrophages emerged around the vessels firstly, then exuded into tissues, and finally disappeared in the inflammatory group injected with labeled macrophages. In summary, our method allows us to exactly image and track the immune cells of inflammatory diseases.

Single-shot photoacoustic microscopy of hemoglobin concentration, oxygen saturation, and blood flow in sub-microseconds

We present fast functional optical-resolution photoacoustic microscopy (OR-PAM) that can simultaneously image hemoglobin concentration, blood flow speed, and oxygen saturation with three-pulse excitation. To instantaneously determine the blood flow speed, dual-pulse photoacoustic flowmetry is developed to determine the blood flow speed from photoacoustic signal decay in sub-microseconds. Grueneisen relaxation effect is compensated for in the oxygen saturation calculation. The blood flow imaging is validated in phantom and in vivo experiments. The results show that the flow speed can be measured accurately in sub-microseconds by comparing the dual-pulse flowmetric method with photoacoustic Doppler flowmetry. Wide-field OR-PAM of hemoglobin concentration, blood flow speed, and oxygen saturation are demonstrated in the mouse ear. This technical advance enables more biomedical applications for fast functional OR-PAM.

Three-dimensional label-free imaging of mammalian yolk sac vascular remodeling with optical resolution photoacoustic microscopy

Vessel development in the yolk sac is important for the embryo development and the malfunction of which can lead to cardiac dysfunction, embryonic malformation and miscarriage. Although substantial emphasis has been placed on the yolk sac vascular remodeling, no detailed three-dimensional (3D) imaging and quantitative analysis of this process has been described. Herein, we explored the development of the vascular system in the visceral yolk sac (VYS) on E11.5, E12.5 and E13.5 mouse embryos using a home-built large field-of-view (FOV) optical-resolution photoacoustic microscopy (OR-PAM). The results showed that OR-PAM can be used as a label-free imaging tool for studying the 2D/3D morphology changes of the vascular system during organogenesis. In addition, after a quantitative analysis the results showed that the microvascular density in the VYS gradually reduced along with the embryo growth. Vascular density in the VYS of E11.5 mouse embryos was almost 6-fold than that of E13.5. Hovever, the averaged vessel diameter of the entire VYS membrane increased gradually with the development of embryos. This study suggests that OR-PAM is a potential tool for acquiring the hemodynamic parameters of mammalian embryos, which could be further used for studying diseases related with the vascular remodeling such as vascular malformations and heart defects.

Fabrication of Coaxial and Confocal Transducer Based on Sol-Gel Composite Material for Optical Resolution Photoacoustic Microscopy.

We have newly developed coaxial and confocal optical-resolution photoacoustic microscopy based on sol-gel composite materials. This transducer contains a concave-shaped piezoelectric layer with a focus depth of 5 mm and a hole with a diameter of 3 mm at the center to pass a laser beam into a phantom. Therefore, this system can directly detect an excited photoacoustic signal without prisms or acoustic lenses. We demonstrate the capability of the system through pulse-echo and photoacoustic imaging experiments. The center frequency of the fabricated transducer is approximately 7 MHz, and its relative bandwidth is 86%. An ex-vivo experiment is conducted, and photoacoustic signals are clearly obtained. As a result, 2- and 3-dimensional maximum amplitude projection images are reconstructed.

Optical-Resolution Photoacoustic Microscopy Using Transparent Ultrasound Transducer.

The opacity of conventional ultrasound transducers can impede the miniaturization and workflow of current photoacoustic systems. In particular, optical-resolution photoacoustic microscopy (OR-PAM) requires the coaxial alignment of optical illumination and acoustic-detection paths through complex beam combiners and a thick coupling medium. To overcome these hurdles, we developed a novel OR-PAM method on the basis of our recently reported transparent lithium niobate (LiNbO3) ultrasound transducer (Dangi et al., Optics Letters, 2019), which was centered at 13 MHz ultrasound frequency with 60% photoacoustic bandwidth. To test the feasibility of wearable OR-PAM, optical-only raster scanning of focused light through a transducer was performed while the transducer was fixed above the imaging subject. Imaging experiments on resolution targets and carbon fibers demonstrated a lateral resolution of 8.5 µm. Further, we demonstrated vasculature mapping using chicken embryos and melanoma depth profiling using tissue phantoms. In conclusion, the proposed OR-PAM system using a low-cost transparent LiNbO3 window transducer has a promising future in wearable and high-throughput imaging applications, e.g., integration with conventional optical microscopy to enable a multimodal microscopy platform capable of ultrasound stimulation.

Multiscale Vascular Enhancement Filter Applied toIn VivoMorphologic and Functional Photoacoustic Imaging of Rat Ocular Vasculature

Optical-resolution photoacoustic microscopy (OR-PAM) is used for in vivo imaging of a variety of albino and pigmented eyes taking advantages of requiring no exogenous dye, performing high-resolution imaging, and achieving morphologic and functional imaging at the same time. However, to accurately diagnose the ophthalmic disease in the OR-PAM images, vascular enhancement algorithms are necessary for extracting vessels and quantifying them correctly. Vascular enhancement algorithms developed for other imaging technologies, are not suitable to be used for OR-PAM, because of the underlying differences in the physics of the formation of images. In this study, a new vascular enhancement algorithm called photoacoustic imaging vasculature enhancement filter (PAIVEF) is proposed, which not only enhances vasculature including micro-vessels signals, suppresses noise signals effectively, but also achieves highly sensitive and accurate enhancement of the vasculature within a large depth range in and out of the system's depth of focus (DOF). Using the PAIVEF, the morphologic and functional 3D images of the whole rat's ocular anterior vasculature segment was displayed simultaneously for a depth range of ~0.6 mm, which was ~7 times of the system's DOF. This study paves the way for the application of OR-PAM technology in ophthalmic disease research.

Single-shot linear dichroism optical-resolution photoacoustic microscopy

Dichroism is a material property that causes anisotropic light-matter interactions for different optical polarizations. Dichroism relates to molecular types and material morphology and thus can be used to distinguish different dichroic tissues. In this paper, we present single-shot dichroism photoacoustic microscopy that can image tissue structure, linear dichroism, and polarization angle with a single raster scanning. We develop a fiber-based laser system to split one laser pulse into three with different polarization angles, sub-microseconds time delay, and identical pulse energy. A dual-fiber optical-resolution photoacoustic microscopy system is developed to acquire three A-lines per scanning step. In such a way, dichroism imaging can achieve the same speed as single-wavelength photoacoustic microscopy. Moreover, the three polarized pulses originate from one laser pulse, which decreases pulse energy fluctuations and reduces dichroism measurement noise by ∼35 %. The new dichroism photoacoustic imaging technique can be used to image endogenous or exogenous polarization-dependent absorption contrasts, such as dichroic tumor or molecule-labeled tissue.

Surface-Functionalized Silicon Nanoparticles as Contrast Agents for Photoacoustic Microscopy Imaging

We demonstrate high contrast optical-resolution photoacoustic microscopy (OR-PAM) imaging by using biocompatible, indirect band gap silicon nanoparticles (SiNPs) as contrast agents. The SiNPs were synthesized by high energy ball milling of crystalline silicon wafers, followed by hydrosilylation with bifunctional olefinic ligands to enable water solubility and bioconjugation. When uniformly dispersed in water, the SiNPs with an average particle size about 114 nm showed remarkably high extinction coefficients, more than 2 × 1010 M−1 cm−1, from the visible to near-infrared spectral range. For the first OR-PAM imaging demonstration, the SiNPs were microinjected to live zebrafish embryonic cells. As the embryo developed, the distribution of the cells carrying the SiNPs can be clearly traced by the spread of the photoacoustic signals. Secondly, photoacoustic-fluorescence dual-modality imaging was demonstrated on live zebrafish larvae which were microinjected with a mixture of the SiNPs and dextran-rhodamine dye...