Photoacoustic Microscopy Imaging Research Articles

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging.

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.

Retinal safety evaluation of photoacoustic microscopy

Photoacoustic microscopy (PAM) has significant potential as a promising diagnostic method for eye diseases and can provide anatomic and functional information of the retinal and choroidal vasculature. However, there are no FDA-approved PAM systems for ophthalmic imaging. In this study, a comprehensive safety evaluation was performed to evaluate the safety of PAM retinal imaging and whether PAM causes damage to retinal structure or function in rabbit eyes. 12 Dutch-Belted pigmented rabbits received photoacoustic imaging to 57% of the retinal surface area with a laser energy of 5% of the ANSI safety limit for five consecutive days and followed before imaging and 3 days, 1, 2, 3, and 4 weeks post imaging. Retinal morphologic analyses using slit lamp examination, fundus photography, red free, FA, FAF, ICGA, and OCT showed no retinal hemorrhage, edema, detachment, vascular abnormalities, or pigmentary abnormalities in the retina or choroid after PAM imaging. Full-field ERG analysis showed no significant difference in scotopic or photopic a- and b-wave amplitudes or implicit times between the control and experimental eyes over time (n = 6, P values > 0.05). Retinal ultrastructural evaluation using TEM showed normal structure of organelles and nuclei, and no significant loss of cells after PAM. TUNEL assay showed no evidence of cells apoptosis in retina. Retinal histopathology indicated that the architecture and thickness of the retinal layers was well preserved in all experimental eyes. A positive control at 500% of the ANSI limit demonstrated significant damage. The comprehensive retinal safety evaluation demonstrated no damage to retinal structure or function for 4 weeks after PAM imaging in rabbits.

Feasibility Study of Precise Balloon Catheter Tracking and Visualization with Fast Photoacoustic Microscopy

Correct guiding of the catheter is a critical issue in almost all balloon catheter applications, including arterial stenosis expansion, coronary arterial diseases, and gastrointestinal tracking. To achieve safe and precise guiding of the balloon catheter, a novel imaging method with high-resolution, sufficient depth of penetration, and real-time display is required. Here, we present a new balloon catheter guiding method using fast photoacoustic microscopy (PAM) technique for precise balloon catheter tracking and visualization as a feasibility study. We implemented ex vivo and in vivo experiments with three different medium conditions of balloon catheter: no air, air, and water. Acquired cross-sectional, maximum amplitude projection (MAP), and volumetric 3D PAM images demonstrated its capability as a new imaging guiding tool for balloon catheter tracking and visualization.

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 Multimodal Photoacoustic Microscopy and Optical Coherence Tomography Visualization of Choroidal Vascular Occlusion

Photoacoustic microscopy is a novel, non-ionizing, non-invasive imaging technology that evaluates tissue absorption of short-pulsed light through the sound waves emitted by the tissue and has numerous biomedical applications. In this study, a custom-built multimodal imaging system, including photoacoustic microscopy (PAM) and optical coherence tomography (OCT), has been developed to evaluate choroidal vascular occlusion (CVO). CVO was performed on three living rabbits using laser photocoagulation. Longitudinal imaging of CVO was obtained using multiple imaging tools such as color fundus photography, fluorescein angiography, indocyanine green angiography (ICGA), OCT, and PAM. PAM images were acquired at different wavelengths, ranging from 532 to 700 nm. The results demonstrate that the CVO was clearly observed on PAM in both two dimensions (2D) and 3D with high resolution longitudinally over 28 days. In addition, the location and margin of the CVO were distinguished from the surrounding choroidal vasculature after the injection of ICG contrast agent. PAM imaging was achieved using a laser energy of approximately 80 nJ, which is about half of the American National Standards Institute safety limit. The proposed imaging technique may provide a potential tool for the evaluation of different chorioretinal vascular disease pathogeneses and other biological studies.

Improved Depth-of-Field Photoacoustic Microscopy with a Multifocal Point Transducer for Biomedical Imaging.

In this study, a photoacoustic microscopy (PAM) system based on a multifocal point (MFP) transducer was fabricated to produce a large depth-of-field tissue image. The customized MFP transducer has seven focal points, distributed along with the transducer’s axis, fabricated by separate spherically-focused surfaces. These surfaces generate distinct focal zones that are overlapped to extend the depth-of-field. This design allows extending the focal zone of 10 mm for the 11 MHz MFP transducer, which is a great improvement over the 0.48 mm focal zone of the 11 MHz single focal point (SFP) transducer. The PAM image penetration depths of a chicken-hemoglobin phantom using SFP and MFP transducers were measured as 5 mm and 8 mm, respectively. The significant increase in the PAM image-based penetration depth of the chicken-hemoglobin phantom was a result of using the customized MFP transducer.

Functional optical coherence tomography and photoacoustic microscopy imaging for zebrafish larvae.

We present a dual modality functional optical coherence tomography and photoacoustic microscopy (OCT-PAM) system. The photoacoustic modality employs an akinetic optical sensor with a large imaging window. This imaging window enables direct reflection mode operation, and a seamless integration of optical coherence tomography (OCT) as a second imaging modality. Functional extensions to the OCT-PAM system include Doppler OCT (DOCT) and spectroscopic PAM (sPAM). This functional and non-invasive imaging system is applied to image zebrafish larvae, demonstrating its capability to extract both morphological and hemodynamic parameters in vivo in small animals, which are essential and critical in preclinical imaging for physiological, pathophysiological and drug response studies.

The Biointegration of a Porcine Acellular Dermal Matrix in a Novel Radiated Breast Reconstruction Model.

Ideal acellular dermal matrices (ADM) for breast reconstruction exhibit native extracellular matrix (ECM) structure to allow rapid biointegration and appropriate mechanical properties for desired clinical outcomes. In a novel in vivo model of irradiated breast reconstruction, we describe the cellular and vascular ingrowth of Artia, a porcine product chemically prepared to mimic the biomechanics of human ADM, with retained natural ECM structure to encourage cellular ingrowth. Utilizing the murine dorsal skinfold model, Artia was implanted into 16 C57bl/6 mice. Eight of the mice received a single dose 35 Gy radiation to the skin, followed by 12 weeks to produce radiation fibrosis and 8 mice served as nonradiated controls. Real-time photoacoustic microscopy of vascular integration and oxygen saturation within the ADM were made over 14 days. At 21 days, vascular ingrowth (CD31), fibroblast scar tissue formation (alpha smooth-muscle actin α-SMA, vimentin), and macrophage function (M2/M1 ratio) were evaluated. Scanning electron microscopy images of Artia were produced to help interpret the potential orientation of cellular and vascular ingrowth. Repeated photoacoustic microscopy imaging demonstrated vascular ingrowth increasing over 14 days, with a commensurate increase in oxygen saturation within both radiated and nonradiated ADM-albeit at an insignificantly lower rate in the radiated group. By day 21, robust CD31 staining was seen that was insignificantly greater in the nonradiated group. Of the fibroblast markers, vimentin expression was significantly greater in the radiated group (P < 0.05). Macrophage lineage phenotype was consistent with remodeling physiology in both radiated and nonradiated groups. Scanning electron microscopy demonstrated transversely organized collagen fibrils with natural porous ECM structure to allow cellular ingrowth. Artia demonstrates appropriate biointegration, with increased oxygen saturation by 14 days, consistent with the performance of other collagen substrates in this model. Radiation fibrosis resulted in higher vimentin expression yet did not impact macrophage phenotype while only modestly decreasing Artia biointegration suggesting that ADM may have a role in reconstructive efforts in a radiated setting. Taken together with its enhanced biomechanics, this porcine ADM product is well poised to be clinically applicable to breast reconstruction.

All-fibre supercontinuum laser for in vivo multispectral photoacoustic microscopy of lipids in the extended near-infrared region

Among the numerous endogenous biological molecules, information on lipids is highly coveted for understanding both aspects of developmental biology and research in fatal chronic diseases. Due to the pronounced absorption features of lipids in the extended near-infrared region (1650−1850 nm), visualisation and identification of lipids become possible using multi-spectral photoacoustic (optoacoustic) microscopy. However, the spectroscopic studies in this spectral region require lasers that can produce high pulse energies over a broad spectral bandwidth to efficiently excite strong photoacoustic signals. The most well-known laser sources capable of satisfying the multi-spectral photoacoustic microscopy requirements (tunability and pulse energy) are tunable nanosecond optical parametric oscillators. However, these lasers have an inherently large footprint, thus preventing their use in compact microscopy systems. Besides, they exhibit low-repetition rates. Here, we demonstrate a compact all-fibre, high pulse energy supercontinuum laser that covers a spectral range from 1440 to 1870 nm with a 7 ns pulse duration and total energy of 18.3 μJ at a repetition rate of 100 kHz. Using the developed high-pulse energy source, we perform multi-spectral photoacoustic microscopy imaging of lipids, both ex vivo on adipose tissue and in vivo to study the development of Xenopus laevis tadpoles, using six different excitation bands over the first overtone transition of C–H vibration bonds (1650−1850 nm).

Miniature probe for dual‐modality photoacoustic microscopy and white‐light microscopy for image guidance: A prototype toward an endoscope

In this study, a novel photoacoustic microscopy (PAM) probe integrating white-light microscopy (WLM) modality that provides guidance for PAM imaging and complementary information is implemented. One single core of an imaging fiber bundle is employed to deliver a pulsed laser for photoacoustic excitation for PAM mode, which provides high resolution with deep penetration. Meanwhile, for WLM mode, the imaging fiber bundle is used to transmit two-dimensional superficial images. Lateral resolution of 7.2 μm for PAM is achieved. Since miniature components are used, the probe diameter is only 1.7 mm. Imaging of phantom and animals in vivo is conducted to show the imaging capability of the probe. The probe has several advantages by introducing the WLM mode, such as being able to conveniently identify regions of interest and align the focus for PAM mode. The prototype of an endoscope shows potential to facilitate clinical photoacoustic endoscopic applications.

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...

Photoacoustic Imaging of Lithium Metal Batteries

We demonstrate that photoacoustic microscopy (PAM) can be a potential novel imaging tool to investigate the Li metal dendrite growth, a critical issue leading to short circuit and even explosion of Li metal batteries. Our results suggest several advantages of PAM imaging of Li metal batteries: high resolution (micrometers), 3D imaging capability, deep penetration in a separator, and high contrast from bulk Li metal. Further, PAM has potential for in situ real-time imaging of Li metal batteries.

Evaluation of Retinal Pigment Epithelium and Choroidal Neovascularization in Rats Using Laser-Scanning Optical-Resolution Photoacoustic Microscopy

Purpose: To demonstrate the value of the laser-scanning optical-resolution (LSOR)-photoacoustic (PA) microscopy (PAM) system and the conventional multimodal imaging techniques in the evaluation of laser-induced retinal injury and choroidal neovascularization (CNV) in rats. Methods: Different degrees of retinal injury were induced using laser photocoagulation. We compared the LSOR-PAM system with conventional imaging techniques in evaluating retinal injury with or without CNV. Six additional rats, treated with an anti-VEGF antibody or immunoglobulin G immediately after photocoagulation, were imaged 7 and 14 days after injection, and CNV lesion areas were compared. Results: In the retinal injury model, fundus autofluorescence showed well-defined hyperreflection, while the lesion displayed abundant PA signals demonstrating nonuniform melanin distribution in retinal pigment epithelium (RPE). RPE was detected with higher contrast in the PAM B-scan image than optical coherence tomography (OCT). Additionally, the CNV lesion was present with multiple PA signal intensities which distinctly characterized the location and area of CNV as found in fundus fluorescein angiography. Furthermore, the decreased PA signals extending from the CNV lesion were similar to those of the vascular bud in ex vivo imaging, which was invisible in other in vivo images. When treated with anti-VEGF agents, statistically significant differences can be demonstrated by PAM similar to other modalities. Conclusions: LSOR-PAM can detect the melanin distribution of RPE in laser-induced retinal injury and CNV in rats. PAM imaging provides a potential new tool to evaluate the vitality and functionality of RPE in vivo as well as to monitor the development and treatment of CNV.

Parallel Computing for Quantitative Blood Flow Imaging in Photoacoustic Microscopy

Photoacoustic microscopy (PAM) is an emerging biomedical imaging technology capable of quantitative measurement of the microvascular blood flow by correlation analysis. However, the computational cost is high, limiting its applications. Here, we report a parallel computation design based on graphics processing unit (GPU) for high-speed quantification of blood flow in PAM. Two strategies were utilized to improve the computational efficiency. First, the correlation method in the algorithm was optimized to avoid redundant computation and a parallel computing structure was designed. Second, the parallel design was realized on GPU and optimized by maximizing the utilization of computing resource in GPU. The detailed timings and speedup for each calculation step were given and the MATLAB and C/C++ code versions based on CPU were presented as a comparison. Full performance test shows that a stable speedup of ~80-fold could be achieved with the same calculation accuracy and the computation time could be reduced from minutes to just several seconds with the imaging size ranging from 1 × 1 mm2 to 2 × 2 mm2. Our design accelerates PAM-based blood flow measurement and paves the way for real-time PAM imaging and processing by significantly improving the computational efficiency.

Co-impulse multispectral photoacoustic microscopy and optical coherence tomography system using a single supercontinuum laser.

A combination of multispectral photoacoustic microscopy (PAM) and optical coherence tomography (OCT) by a single light source was previously realized discretely; however, this is unfavorable for visualizing vital physiological and pathological activities in vivo. Here, a co-impulse dual-mode imaging system that simultaneously enables multispectral PAM and OCT using a megahertz supercontinuum pulse laser in vivo is presented. The 500-600nm band is used for functional PAM imaging, which can flexibly switch between different wavelengths, while the 600-840nm band is selected for OCT imaging. A mimicking phantom experiment and in vivo imaging of normal and melanoma mouse ears demonstrate that the co-impulse multispectral PAM-OCT system can simultaneously provide structural and functional information for bioimaging.

Towards a Low-Cost and Portable Photoacoustic Microscope for Point-of-Care and Wearable Applications.

Several breakthrough applications in biomedical imaging have been reported in the recent years using advanced photoacoustic microscopy imaging systems. While two photon and other optical microscopy systems have recently emerged in portable and wearable form, there is much less work reported on the portable and wearable photoacoustic microscopy (PAM) systems. Working towards this goal, we report our studies on a low-cost and portable photoacoustic microscopy system that uses a custom fabricated 2.5 mm diameter ring ultrasound transducer integrated with a fiber-coupled laser diode. The ultrasound transducer is centered at 17.25 MHz, and shows ~ 45% and ~ 100% fractional bandwidths for ultrasound pulse-echo and photoacoustic A-line signals respectively. To achieve overall system portability, besides the imaging head, other backend imaging system components need to be readily portable as well. In this direction, we have studied the potential use of compact pre-amplifiers, scanning stages and microcontroller based data acquisition and reconstruction for photoacoustic imaging. The portable PAM system is validated by imaging phantoms embedded with light absorbing targets. Future directions that will likely help achieve a completely portable and wearable photoacoustic microscopy system are discussed.

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.

Contrast Agent Enhanced Multimodal Photoacoustic Microscopy and Optical Coherence Tomography for Imaging of Rabbit Choroidal and Retinal Vessels in vivo

Multimodal imaging with photoacoustic microscopy (PAM) and optical coherence tomography (OCT) can be an effective method to evaluate the choroidal and retinal microvasculature. To improve the efficiency for visualizing capillaries, colloidal gold nanoparticles (AuNPs) have been applied as a multimodal contrast agent for both OCT and PAM imaging by taking advantage of the strong optical scattering and the strong optical absorption of AuNPs due to their surface plasmon resonance. Ultra-pure AuNPs were fabricated by femtosecond laser ablation, capped with polyethylene glycol (PEG), and administered to 13 New Zealand white rabbits and 3 Dutch Belted pigmented rabbits. The synthesized PEG-AuNPs (20.0 ± 1.5 nm) were demonstrated to be excellent contrast agents for PAM and OCT, and do not demonstrate cytotoxicity to bovine retinal endothelial cells in cell studies. The image signal from the retinal and choroidal vessels in living rabbits was enhanced by up to 82% for PAM and up to 45% for OCT, respectively, by the administered PEG-AuNPs, which enables detection of individual blood vessels by both imaging modalities. The biodistribution study demonstrated the AuNP accumulated primarily in the liver and spleen. Histology and TUNEL staining did not indicate cell injury or death in the lung, liver, kidney, spleen, heart, or eyes up to seven days after AuNP administration. PEG-AuNPs offer an efficient and safe contrast agent for multimodal ocular imaging to achieve improved characterization of microvasculature.

Fenestration Improves Acellular Dermal Matrix Biointegration: An Investigation of Revascularization with Photoacoustic Microscopy.

Acellular dermal matrices have revolutionized alloplastic breast reconstruction. Furthering our knowledge of their biointegration will allow for improved design of these biomaterials. The ideal acellular dermal matrix for breast reconstruction would provide durable soft-tissue augmentation while undergoing rapid biointegration to promote physiologic elasticity and reduced infectious complications. The inclusion of fenestrations in their design is thought to promote the process of biointegration; however, the mechanisms underlying this theory have not been evaluated. Biointegration of standard and fenestrated acellular dermal matrices was assessed with serial photoacoustic microscopic imaging, in a murine dorsal skinfold window chamber model specifically designed to recapitulate the microenvironment of acellular dermal matrix-assisted alloplastic breast reconstruction. Photoacoustic microscopy allows for a serial, real-time, noninvasive assessment of hemoglobin content and oxygen saturation in living tissues, generating high-resolution, three-dimensional maps of the nascent microvasculature within acellular dermal matrices. Confirmatory histologic and immunohistochemical assessments were performed at the terminal time point. Fenestrated acellular dermal matrices demonstrated increased fibroblast and macrophage lineage host cell infiltration, greater mean percentage surface area vascular penetration (21 percent versus 11 percent; p = 0.08), and greater mean oxygen saturation (13.5 percent versus 6.9 percent; p < 0.05) than nonfenestrated matrices by 2 weeks after implantation. By 21 days, host cells had progressed nearly 1 mm within the acellular dermal matrix fenestrations, resulting in significantly more vascularity across the top of the fenestrated matrix (3.8 vessels per high-power field versus 0.07 vessels per high-power field; p < 0.05). Inclusion of fenestrations in acellular dermal matrices improves the recellularization and revascularization that are crucial to biointegration of these materials. Future studies will investigate the optimal distance between fenestrations.

Impacts of the murine skull on high-frequency transcranial photoacoustic brain imaging.

Non-invasive photoacoustic tomography (PAT) of mouse brains with intact skulls has been a challenge due to the skull's strong acoustic attenuation, aberration, and reverberation, especially in the high-frequency range (>15 MHz). In this paper, we systematically investigated the impacts of the murine skull on the photoacoustic wave propagation and on the PAT image reconstruction. We studied the photoacoustic acoustic wave aberration due to the acoustic impedance mismatch at the skull boundaries and the mode conversion between the longitudinal wave and shear wave. The wave's reverberation within the skull was investigated for both longitudinal and shear modes. In the inverse process, we reconstructed the transcranial photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) images of a point target enclosed by the mouse skull, showing the skull's different impacts on both modalities. Finally, we experimentally validated the simulations by imaging an in vitro mouse skull phantom using representative transcranial PAM and PACT systems. The experimental results agreed well with the simulations and confirmed the accuracy of our forward and inverse models. We expect that our results will provide better understanding of the impacts of the murine skull on transcranial photoacoustic brain imaging and pave the ways for future technical improvements.