Optical-resolution Photoacoustic Microscopy Research Articles

Deep-learning-based motion-correction algorithm in optical resolution photoacoustic microscopy

In this study, we propose a deep-learning-based method to correct motion artifacts in optical resolution photoacoustic microscopy (OR-PAM). The method is a convolutional neural network that establishes an end-to-end map from input raw data with motion artifacts to output corrected images. First, we performed simulation studies to evaluate the feasibility and effectiveness of the proposed method. Second, we employed this method to process images of rat brain vessels with multiple motion artifacts to evaluate its performance for in vivo applications. The results demonstrate that this method works well for both large blood vessels and capillary networks. In comparison with traditional methods, the proposed method in this study can be easily modified to satisfy different scenarios of motion corrections in OR-PAM by revising the training sets.

Dual-Polarized Fiber Laser Sensor for Photoacoustic Microscopy.

Optical resolution photoacoustic microscopy (OR-PAM) provides high-resolution, label-free and non-invasive functional imaging for broad biomedical applications. Dual-polarized fiber laser sensors have high sensitivity, low noise, a miniature size, and excellent stability; thus, they have been used in acoustic detection in OR-PAM. Here, we review recent progress in fiber-laser-based ultrasound sensors for photoacoustic microscopy, especially the dual-polarized fiber laser sensor with high sensitivity. The principle, characterization and sensitivity optimization of this type of sensor are presented. In vivo experiments demonstrate its excellent performance in the detection of photoacoustic (PA) signals in OR-PAM. This review summarizes representative applications of fiber laser sensors in OR-PAM and discusses their further improvements.

Optical Resolution Photoacoustic Microscopy of Ovary and Fallopian Tube

Ovarian cancer is the leading cause of death among gynecological cancers, but is poorly amenable to preoperative diagnosis. In this study, we investigate the feasibility of “optical biopsy,” using high-optical-resolution photoacoustic microscopy (OR-PAM) to quantify the microvasculature of ovarian and fallopian tube tissue. The technique is demonstrated using excised human ovary and fallopian tube specimens imaged immediately after surgery. Quantitative parameters are derived using Amira software. The parameters include three-dimensional vascular segment count, total volume and length, which are associated with tumor angiogenesis. Qualitative results of OR-PAM demonstrate that malignant ovarian tissue has larger and more tortuous blood vessels as well as smaller vessels of different sizes, while benign and normal ovarian tissue has smaller vessels of uniform size. Quantitative analysis shows that malignant ovaries have greater tumor vessel volume, length and number of segments, as compared with benign and normal ovaries. The vascular pattern of benign fallopian tube is different than that of benign ovarian tissue. Our initial results demonstrate the potential of OR-PAM as an imaging tool for fast assessment of ovarian tissue and fallopian tube and could avoid unnecessary surgery if the risk of the examined ovary is extremely low.

In Vivo Reflection-Mode Photoacoustic Microscopy Enhanced by Plasmonic Sensing with an Acoustic Cavity.

Relying on high-sensitivity refractive index sensing and a highly constrained evanescent field of surface plasmon resonance (SPR), broadband photoacoustic (PA) pressure transients were measured using an SPR sensor instead of routinely used piezoelectric ultrasonic transducers. An acoustic cavity made from stainless steel and having a designed ellipsoidal inner surface redirected laser-induced PA waves from the PA excitation spot to the SPR sensor. By incorporating the SPR sensor with the acoustic cavity, we developed optical-resolution photoacoustic microscopy (OR-PAM) with multiple advantages, including reflection-mode signal capture, improved PA detection sensitivity, increased PA spectral bandwidth as broad as ∼98 MHz, and micrometer-scale lateral resolution. This allowed label-free volumetric PA imaging of vasculature in not only the thin ear but also the thick forelimb of living mice. With these combined advantages, our OR-PAM system potentially offers more opportunities for biomedical investigation, for example, when studying microcirculations in the eye and cortex.

Quantitative investigation of vascular response to mesenteric venous thrombosis using large-field-of-view photoacoustic microscopy.

Mesenteric venous thrombosis (MVT) is one of major causes leading to severe mesenteric ischemia. Vascular network plays an important role during the occurrence and development of MVT. However, there lacks an appropriate imaging method, which features advanced volumetric resolving capability, superior sensitivity to hemoglobin, and ultra-large field-of-view (FOV), to investigate vascular response of MVT. In this study, we developed and applied a large-FOV optical resolution photoacoustic microscopy to quantify the vascular response during the entire course of two different MVT models in which we ligated the superior mesenteric vein and inferior mesenteric vein, respectively. Furthermore, we developed a quantitative algorithm to derive total vascular length, relative concentration of total hemoglobin and vascular density over the FOV to reveal different vascular responses in different MVT models.

High-speed simultaneous multiscale photoacoustic microscopy

.Photoacoustic microscopy (PAM) is a fast-growing biomedical imaging technique that provides high-resolution in vivo imaging beyond the optical diffusion limit. Depending on the scalable lateral resolution and achievable penetration depth, PAM can be classified into optical resolution PAM (OR-PAM) and acoustic resolution PAM (AR-PAM). The use of a microelectromechanical systems (MEMS) scanner has improved OR-PAM imaging speed significantly and is highly beneficial in the development of miniaturized handheld devices. The shallow penetration depth of OR-PAM limits the use of such devices for a wide range of clinical applications. We report the use of a high-speed MEMS scanner for both OR-PAM and AR-PAM. A high-speed, wide-area scanning integrated OR-AR-PAM system combining MEMS scanner and raster mechanical movement was developed. A lateral resolution of and penetration depth in vivo was achieved using OR-PAM at 586 nm, whereas a lateral resolution of and penetration depth of in vivo was achieved using AR-PAM at 532 nm.

Optical coherence tomography-guided dynamic focusing for combined optical and mechanical scanning multimodal photoacoustic microscopy.

.To achieve fast imaging and large field of view (FOV), we improved our multimodal imaging system, which integrated optical resolution photoacoustic microscopy, optical coherence tomography (OCT), and confocal fluorescence microscopy in one platform, by combining optical scanning with mechanical scanning. To ensure good focusing of the objective lens over all the imaged area, we employed OCT-guided dynamic focusing. Different from our previous point-by-point dynamic focusing, we employed an area-by-area focusing adjustment strategy, in which each fast optical scanning area has a fixed focusing depth. We have demonstrated the performance of the system by imaging biological samples ex vivo (plant leaf) and in vivo (mouse ear). The system has achieved uniform resolution in an FOV of with an imaging time of about 5 min.

Optical-resolution photoacoustic microscopy for monitoring vascular normalization during anti-angiogenic therapy

Monitoring the changes in tumor vascularity is important for anti-angiogenic therapy assessment with therapeutic implications. However, monitoring vascularity is quite challenging due to the lack of appropriate imaging techniques. Here, we describe a non-invasive imaging technique using optical-resolution photoacoustic microscopy (OR-PAM) to track vascular changes in prostate cancer treated with an anti-angiogenic agent, DC101, on a mouse ear xenograft model. Approximately 1–3 days after the initial therapy, OR-PAM imaging detected tumor vascular changes such as reduced vessel tortuosity, decreased vessel diameter and homogenized intratumoral vessel distribution. These observations indicated vessel normalization, which was pathologically validated as increased fractional pericyte coverage, functional perfusion and drug delivery of the vessels. After four DC101 interventions, OR-PAM imaging eventually revealed intratumoral vessel regression. Therefore, OR-PAM imaging of the vasculature offers a promising method to study anti-angiogenic drug mechanisms of action in vivo and holds potential in monitoring and guiding anti-angiogenic therapy.

Isometrically Resolved Photoacoustic Microscopy Based on Broadband Surface Plasmon Resonance Ultrasound Sensing.

Photoacoustic microscopy (PAM) can measure optical absorption-based molecular specificities within tissues. Despite the diffraction-limited lateral resolution in optical-resolution photoacoustic microscopy (OR-PAM), the ongoing challenge is poor axial resolution because of an insufficient ultrasound detection bandwidth, which hampers PAM volumetric imaging. We propose polarization-differential surface plasmon resonance (SPR) sensing for broadband and high-sensitivity photoacoustic (PA) detection, allowing OR-PAM with comparable resolution along lateral and axial directions. This sensor possesses an estimated noise-equivalent-pressure sensitivity of ∼477 Pa over an approximately linear pressure response up to 107 kPa. Moreover, an improved PA detection bandwidth of ∼173 MHz permits an axial resolution (∼7.6 μm) that approaches the lateral resolution (∼4.5 μm) of our OR-PAM system. The capability in spatially isometric micrometer-scale resolution enables in vivo volumetric label-free imaging of the microvasculature of a mouse ear. The SPR sensing technology promises broader applications of PAM in biomedical studies such as microcirculation.

Front Cover: In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion (J. Biophotonics 7/2019)

How does the ischemic tissue re‐vascularize? Now we can visualize the reperfusion process at high spatial resolution by using a dual‐wavelength MEMS scanning based optical resolution photoacoustic microscopy (OR‐PAM) system. The fast imaging capability enables continuous monitoring of skin reperfusion in a mouse model. It's also found that the ischemic tissue has a significantly higher oxygen consumption rate in the reperfusion stage comparing to the normal tissue.Further details can be found in the article by Renzhe Bi, U.S. Dinish, Chi Ching Goh, et al. (e201800454). image

Optical-resolution photoacoustic microscopy of oxygen saturation with nonlinear compensation.

Optical-resolution photoacoustic microscopy (OR-PAM) of oxygen saturation (sO2) offers high-resolution functional information on living tissue. Limited by the availability of high-speed multi-wavelength lasers, most OR-PAM systems use wavelengths around 532nm. Blood has high absorption coefficients in this spectrum, which may cause absorption saturation and induce systematic errors in sO2 imaging. Here, we present nonlinear OR-PAM that compensates for the absorption saturation in sO2 imaging. We model the absorption saturation at different absorption coefficients and ultrasonic bandwidths. To compensate for the absorption saturation, we develop an OR-PAM system with three optical wavelengths and implement a nonlinear algorithm to compute sO2. Phantom experiments on bovine blood validate that the nonlinear OR-PAM can improve the sO2 accuracy by up to 0.13 for fully oxygenated blood. In vivo sO2 imaging has been conducted in the mouse ear. The nonlinear sO2 results agree with the normal physiological values. These results show that the absorption saturation effect can be compensated for in nonlinear OR-PAM, which improves the accuracy of functional photoacoustic imaging.

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.

Wearable optical resolution photoacoustic microscopy.

Optical resolution photoacoustic microscopy (ORPAM) is an emerging imaging technique, which has been extensively used to study various brain activities and disorders of the anesthetized/restricted rodents with a special focus on the morphological and functional visualization of cerebral cortex. However, it is challenging to develop a wearable photoacoustic microscope, which enables the investigation of brain activities/disorders on freely moving rodents. Here, we report a wearable and robust optical resolution photoacoustic microscope (W-ORPAM), which utilizes a small, light, stable and fast optical scanner. This wearable imaging probe features high spatiotemporal resolution, large field of view (FOV) and easy assembly as well as adjustable optical focus during the in vivo experiment, which makes it accessible to image cerebral cortex activities of freely moving rodents. To demonstrate the advantages of this technique, we used W-ORPAM to monitor both morphological and functional variations of vasculature in cerebral cortex during the induction of ischemia and reperfusion of a freely moving rat.

In vivo label-free functional photoacoustic monitoring of ischemic reperfusion.

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell-specific, fluorescent reporter mouse skin with induced ischemia-reperfusion (I-R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual-wavelength micro electro mechanical system (MEMS) scanning-based optical resolution photoacoustic microscopy (OR-PAM) system for continuous label-free functional imaging of vascular reperfusion in an IR mouse model. This MEMS-OR-PAM system provides fast scanning speed for concurrent dual-wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO2 ) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS-OR-PAM system provides novel perspectives to understand the I-R injuries. It solves the problem of dynamic label-free functional monitoring of the vascular reperfusion at high spatial resolution.

ADMM based low-rank and sparse matrix recovery method for sparse photoacoustic microscopy

Photoacoustic microscopy (PAM) has evolved into a new promising medical imaging tool available for both in vivo surficial and deep-tissue imaging with a high spatial resolution. However, the long data acquisition time has made real-time imaging highly challenging. This paper presents an Alternating Direction Method of Multipliers (ADMM) based low-rank and sparse matrix recovery method for a sparse optical-scanning PAM system to realize fast PAM vascular imaging.For our system, an x-y galvanometer scanner is used to achieve compressive sampling, and the associated image recovery process is formulated as a matrix completion problem. The sparse scanning scheme might be easy to integrate with several other optical-resolution PAM modalities. Further, the sparse constraint (the total variation norm) and the low-rank constraint (nuclear norm) are combined for solving the optimization program under ADMM framework for matrix recovery in order to achieve better PAM image recovery even for images that are not well-approximated by their low-rank components.A prototype PAM system has been implemented and the recovery method has been validated. From both visual effects and quantitative parameters, such as PSNR, SSIM, Rerr and MSE, comparable image qualities with conventional full sampling optical resolution PAM could be acquired by just half of the data acquisition time. Besides, the ADMM based low-rank and sparse matrix recovery method could show better results than GoDec. The obtained results demonstrate the preclinical and clinical potential of sparse PAM system in investigating vascular diseases.

Quantitative analysis on in vivo tumor-microvascular images from optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) has been shown to be an excellent imaging modality for monitoring and study of tumor microvasculature. However, previous studies focused mainly on the normal tissues and did not quantify the tumor microvasculature. In this study, we present an in vivo OR-PAM imaging of the melanomas and hepatoma implanted in the mouse ear. We quantify the vessel growth by extracting the skeletons of both dense and thin branches of the tumor microvasculature obtained by Hessian matrix enhancement followed by improved two-step multistencils fast marching method. Compared with the previous methods of using OR-PAM for normal tissues, our method was more effective in extracting the binary vascular network in the tumor images and in obtaining the complete and continuous microvascular skeleton maps. Our demonstration of using OR-PAM in improving microvasculature of tumors and quantification of tumor growth would push deep this technology for the early diagnosis and treatment of cancers.

Multi-wavelength photo-acoustic microscopy in the frequency domain for simultaneous excitation and detection of dyes.

An optical-resolution photoacoustic microscope with modulated CW laser diodes allowing multi-channel imaging is presented that can be used for both imaging biological tissues and for targeted photo-dynamic therapy (PDT) varying the optical power and exposure time. The effects of this therapy are immediately monitored in order to optimize the time of irradiation. After the description of the experimental setup, in vitro and in vivo applications are presented on a synthetic sample and on the mouse ear using hemoglobin as endogenous and methylene blue as exogenous dye for imaging and PDT, respectively.

Photoacoustic microscopy for cells in a 3D microenvironment: Systems and applications

Imaging of living cells in a three-dimensional (3D) culture system is of critical importance in order to understand the interplay between cells and the surrounding matrix components. However, the use of conventional imaging methods, such as optical microscopy, is limited due to the lack of penetration. To overcome the limitation, optical-resolution photoacoustic microscopy (OR-PAM) has been proposed and widely investigated. The OR-PAM is based on the photoacoustic effect with which high frequency acoustic waves are generated when the medium absorbs light energy within a short pulse duration. It, therefore, takes advantages of the high optical absorption contrast with low acoustic scattering, allowing non-invasive visualization the cells at depths with optical resolution. With the advance of targeted contrast agents, the OR-PAM is also capable of facilitating molecular characterization for pre-clinical research on personalized diagnostics or guided therapeutics. In this presentation, the applications and challenges of photoacoustic microscopy in a 3D cellular microenvironment are covered. Potential future development of photoacoustic microscopy in pre-clinical applications is also discussed.

Optimal Generation of Ten Individual Green-to-Red Raman Source for Wavelength-Dependent Real-Time OR-PAM Images

In this study, ten individual green-to-red stimulated Raman scattering (SRS) peaks are optimally generated for wavelength-dependent real-time optical-resolution photoacoustic microscopy (OR-PAM) with contrast agents. Exogenous contrast agents, such as dyes, semiconducting polymers, and metallic nanoparticles, are frequently used in OR-PAM to provide structural, functional, and molecular images of biological samples with a high spatial resolution. For an effective use of various exogenous contrast agents for functional or molecular OR-PAM, selectable visible and near-infrared wavelengths are needed to select the absorption wavelengths of the contrast agents. Therefore, we optimized an SRS source, which can selectively generate ten narrow-band SRS peaks with a bandwidth smaller than 11 nm and individually produce sufficient threshold pulse energies (116–756 nJ) at a high pulse repetition rate (PRR) of 300 kHz. The wavelength-dependent photoacoustic signal was easily obtained in a methylene blue phantom and in vivo mouse studies using the optimal generation conditions of the ten-wavelength green-to-red SRS source, from 545 to 695 nm. In particular, for the first time, wavelength-dependent real-time in vivo OR-PAM images at a PRR of hundreds of kilohertz were obtained to distinguish gold nanoparticles from distributed blood vessels of a mouse ear.

2P5-14 Visualization of cell structure by optical resolution photoacoustic microscopy with sub-micron lateral resolution

2P5-14 Visualization of cell structure by optical resolution photoacoustic microscopy with sub-micron lateral resolution