Optical-resolution Photoacoustic Microscopy Imaging Research Articles

Reconstructing Cancellous Bone From Down-Sampled Optical-Resolution Photoacoustic Microscopy Images With Deep Learning

ObjectiveBone diseases deteriorate the microstructure of bone tissue. Optical-resolution photoacoustic microscopy (OR-PAM) enables high spatial resolution of imaging bone tissues. However, the spatiotemporal trade-off limits the application of OR-PAM. The purpose of this study was to improve the quality of OR-PAM images without sacrificing temporal resolution. MethodsIn this study, we proposed the Photoacoustic Dense Attention U-Net (PADA U-Net) model, which was used for reconstructing full-scanning images from under-sampled images. Thereby, this approach breaks the trade-off between imaging speed and spatial resolution. ResultsThe proposed method was validated on resolution test targets and bovine cancellous bone samples to demonstrate the capability of PADA U-Net in recovering full-scanning images from under-sampled OR-PAM images. With a down-sampling ratio of [4, 1], compared to bilinear interpolation, the Peak Signal-to-Noise Ratio and Structural Similarity Index Measure values (averaged over the test set of bovine cancellous bone) of the PADA U-Net were improved by 2.325 dB and 0.117, respectively. ConclusionThe results demonstrate that the PADA U-Net model reconstructed the OR-PAM images well with different levels of sparsity. Our proposed method can further facilitate early diagnosis and treatment of bone diseases using OR-PAM.

WSA-MP-Net: Weak-signal-attention and multi-scale perception network for microvascular extraction in optical-resolution photoacoustic microcopy

The unique advantage of optical-resolution photoacoustic microscopy (OR-PAM) is its ability to achieve high-resolution microvascular imaging without exogenous agents. This ability has excellent potential in the study of tissue microcirculation. However, tracing and monitoring microvascular morphology and hemodynamics in tissues is challenging because the segmentation of microvascular in OR-PAM images is complex due to the high density, structure complexity, and low contrast of vascular structures. Various microvasculature extraction techniques have been developed over the years but have many limitations: they cannot consider both thick and thin blood vessel segmentation simultaneously, they cannot address incompleteness and discontinuity in microvasculature, there is a lack of open-access datasets for DL-based algorithms. We have developed a novel segmentation approach to extract vascularity in OR-PAM images using a deep learning network incorporating a weak signal attention mechanism and multi-scale perception (WSA-MP-Net) model. The proposed WSA network focuses on weak and tiny vessels, while the MP module extracts features from different vessel sizes. In addition, Hessian-matrix enhancement is incorporated into the pre-and post-processing of the input and output data of the network to enhance vessel continuity. We constructed normal vessel (NV-ORPAM, 660 data pairs) and tumor vessel (TV-ORPAM, 1168 data pairs) datasets to verify the performance of the proposed method. We developed a semi-automatic annotation algorithm to obtain the ground truth for our network optimization. We applied our optimized model successfully to monitor glioma angiogenesis in mouse brains, thus demonstrating the feasibility and excellent generalization ability of our model. Compared to previous works, our proposed WSA-MP-Net extracts a significant number of microvascular while maintaining vessel continuity and signal fidelity. In quantitative analysis, the indicator values of our method improved by about 1.3% to 25.9%. We believe our proposed approach provides a promising way to extract a complete and continuous microvascular network of OR-PAM and enables its use in many microvascular-related biological studies and medical diagnoses.

Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Super-resolution reconstruction algorithm for optical-resolution photoacoustic microscopy images based on sparsity and deconvolution.

The lateral resolution of the optical-resolution photoacoustic microscopy (OR-PAM) system depends on the focusing diameter of the probe beam. By increasing the numerical aperture (NA) of optical focusing, the lateral resolution of OR-PAM can be improved. However, the increase in NA results in smaller working distances, and the entire imaging system becomes very sensitive to small optical imperfections. The existing deconvolution-based algorithms are limited by the image signal-to-noise ratio when improving the resolution of OR-PAM images. In this paper, a super-resolution reconstruction algorithm for OR-PAM images based on sparsity and deconvolution is proposed. The OR-PAM image is sparsely reconstructed according to the constructed loss function, which utilizes the sparsity of the image to combat the decrease in the resolution. The gradient accelerated Landweber iterative algorithm is used to deconvolve to obtain high-resolution OR-PAM images. Experimental results show that the proposed algorithm can improve the resolution of mouse retinal images by approximately 1.7 times without increasing the NA of the imaging system. In addition, compared to the Richardson-Lucy algorithm, the proposed algorithm can further improve the image resolution and maintain better imaging quality, which provides a foundation for the development of OR-PAM in clinical research.

Unveiling the improved targeting migration of mesenchymal stem cells with CXC chemokine receptor 3-modification using intravital NIR-II photoacoustic imaging

BackgroundTherapy with genetically modified mesenchymal stem cells (MSCs) has clinical translation promise. Optimizing the targeting migratory ability of MSCs relies on accurate imaging of the distribution and extravasation kinetics of MSCs, and the corresponding imaging results could be used to predict therapeutic outcomes and guide the optimization of the treatment program. Among the different imaging modalities, second near-infrared (NIR-II) optical-resolution photoacoustic microscopy (OR-PAM) has merits, including a fine resolution, a deep penetration, a high sensitivity, and a large signal-to-background ratio. It would be an ideal candidate for precise monitoring of MSCs, although it has not been tested for this purpose so far.ResultsPenetrating peptide-decorated conjugated polymer nanoparticles (TAT-CPNPs) with strong NIR-II absorbance were used to label chemokine-receptor genetically modified MSCs, which were subsequently evaluated under intravital NIR-II OR-PAM regarding their targeting migratory ability. Based on the upregulation of chemokine (C-X-C motif) ligand 10 in the inflamed ears of contact hypersensitivity mice, MSCs with overexpression of corresponding receptor, chemokine (C-X-C motif) receptor 3 (Cxcr3) were successfully generated (MSCCxcr3). TAT-CPNPs labeling enabled NIR-II photoacoustic imaging to discern MSCCxcr3 covered by 1.2 cm of chicken breast tissue. Longitudinal OR-PAM imaging revealed enhanced inflammation-targeting migration of MSCCxcr3 over time attributed to Cxcr3 gene modification, which was further validated by histological analysis.ConclusionsTAT-CPNPs-assisted NIR-II PA imaging is promising for monitoring distribution and extravasation kinetics of MSCs, which would greatly facilitate optimizing MSC-based therapy.Graphical

Optical Resolution Photoacoustic Microscopy Imaging in the Detection of Early Oral Cancer under Image Reconstruction Algorithm.

This research was intended to explore the application value of photoacoustic imaging technology based on image intelligent iterative reconstruction algorithm in the detection and diagnosis of early oral cancer. An iterative algorithm model was constructed and systematically analyzed. The algorithm was used to debug the detection of B-scan images on the diameter of the imaging area. The results showed that the sensitivity of line-focused ultrasound detector was 86.72% and the specificity was 80.79%, while the sensitivity of the flat-field ultrasound detector was 63.15% and the specificity was 71.79%. The photoacoustic microscopy imaging technology can clearly observe the rich capillary network on human lips. A part of the vascular network at the depth of 100 μm, 500 μm, and 1000 μm grew out of the reticular capillaries and extended out of the loop-like capillaries, and the diameter gradually expanded. The imaging experiment of the sublingual capillary network in the human body showed that loop-like capillaries were observed, but there were some large blood vessels, which corresponded to the densely distributed blood vessel network under the tongue. The morphological changes of loop-like capillaries can be well observed by photoacoustic microscopy. In conclusion, the reconstructed photoacoustic microscopy imaging technology can realize high-resolution imaging of human oral capillaries and observe the morphological changes of loop-like capillaries, which had a certain application value for the early detection of oral cancer.

Detachable head-mounted photoacoustic microscope in freely moving mice.

Optical resolution photoacoustic microscopy (ORPAM) is a promising tool for investigating anatomical and functional dynamics in the cerebral cortex. However, observation in freely moving mice has been a longstanding challenge for ORPAM. In this Letter, we extended ORPAM from anesthetized, head-restrained to awake, freely moving mice by using a detachable head-mounted ORPAM probe. We used a micro-electro-mechanical-system scanner and a miniaturized piezoelectric ultrasonic detector to scan the excitation laser beam and detect generated photoacoustic signals, respectively. The probe weighs 1.8 g and has a large field of view of ∼3mm×3mm. We evaluated the performance of the probe by carrying out phantom experiments and the imaging of vascular networks in a mouse cerebral cortex. The results suggest that the ORPAM probe is capable of providing stable and high-quality ORPAM images in freely moving mice.

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.

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...

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-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.

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.

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.

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.

A handheld microscope integrating photoacoustic microscopy and optical coherence tomography.

The combination of optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) is capable of providing complementary imaging contrasts. Unfortunately, the miniaturization of ORPAM remains a major challenge in the development of a handheld dual-modality imaging microscope with OCT. Here, we report the design and evaluation of an integrated ORPAM and OCT imaging probe using a two-dimensional MEMS (micro-electro-mechanical-system)-based optical scanner. This microscope, weighting 35.4 g, has an ultracompact size of 65×30×18 mm3, and an effective imaging area of 2×2 mm2. The experimental lateral resolutions are 3.7 μm (ORPAM) and 5.6 μm (OCT), and the axial resolutions are measured as 120 μm (ORPAM) and 7.3 μm (OCT). Besides phantom and animal experiments, we carried out oral imaging of a healthy volunteer to show the clinical feasibility of this technique.

Optical resolution photoacoustic microscopy based on multimode fibers.

Photoacoustic microscopy (PAM) is a multiscale imaging technique. In optical-resolution photoacoustic microscopy (OR-PAM), a single mode (SM) fiber is normally used as the source of optical excitation to be focused into a diffraction-limited spot. Recent advances in OR-PAM have improved its imaging speed using microelectromechanical systems (MEMS). Here we report for the first time the use of a multimode (MM) fiber as the optical excitation source for high resolution OR-PAM in vivo imaging. A high-speed MEMS scanner based OR-PAM system combined with the mechanical movement to provide wide area imaging was used. The use of multimode fiber for achieving tight optical focus would make the optical alignment easier and high repetition rate light delivery possible for high-speed OR-PAM imaging. A lateral resolution of 3.5 µm and axial resolution of 27 µm with ~1.5 mm imaging depth was successfully demonstrated using the system. The efficacy of multimode fibers for achieving tight focus is beneficial for developing high-resolution photoacoustic endoscopy systems and can be combined with other optical endoscopic imaging modalities as well.

Real-time functional optical-resolution photoacoustic microscopy using high-speed alternating illumination at 532 and 1064 nm.

Optical-resolution photoacoustic microscopy (OR-PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high-resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR-PAM. This study demonstrates real-time functional OR-PAM images with high-speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro-optical modulator is used as an optical switch. Therefore, the A-line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real-time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR-PAM images of the distribution of blood vessels and gold nanorods in a BALB/c-nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real-time display at 0.49 fps.

Sparse-representation-based denoising of photoacoustic images

Optical resolution photoacoustic microscopy (OR-PAM) is an emerging hybrid technology that combines optical contrast and acoustic resolution. The quality of photoacoustic (PA) images is degraded due to different parameters such as frequency, the diameter of the transducer or external noise induced from the laser. The diameter of the transducer is proportional to its near field to focus or unfocus the transducer, which affects the image quality, so reconstruction and denoising of photoacoustic images is an important issue in medical imaging, especially when it comes to diagnosing diseases at an early stage. Visualization of different structures in the PA images requires filtering to suppress noise. This paper investigates the use of K-means singular value decomposition (K-SVD) to eliminate noise and enhance the effect of vasculature in the PA images. The algorithm is tested on PA images of blood-filled tubes of different diameters and in vivo mouse ear images acquired using OR-PAM imaging. The results reveal a better denoising capability of PA images when compared with standard Wiener and wavelet-based filtering.

In vivo corneal neovascularization imaging by optical-resolution photoacoustic microscopy

Corneal neovascularization leads to blurred vision, thus in vivo visualization is essential for pathological studies in animal models. Photoacoustic (PA) imaging can delineate microvasculature and hemodynamics noninvasively, which is suitable for investigating corneal neovascularization. In this study, we demonstrate in vivo imaging of corneal neovascularization in the mouse eye by optical-resolution photoacoustic microscopy (OR-PAM), where corneal neovascularization is induced by deliberate alkali burn injuries in C57BL6/J inbred mice corneas on the left eye. We used OR-PAM to image five mice with corneal alkali burn injuries; the uninjured eyes (right eye) in these mice are then used as the controls. Corneal images acquired by OR-PAM with and without alkali burn injury are compared, clear signs of corneal neovascularization are present in the OR-PAM images of injured eyes; the OR-PAM results are also confirmed by postmortem fluorescence-labeled confocal microscopy.

In-Vivo functional optical-resolution photoacoustic microscopy with stimulated Raman scattering fiber-laser source

In this paper a multi-wavelength optical-resolution photoacoustic microscopy (OR-PAM) system using stimulated Raman scattering is demonstrated for both phantom and in vivo imaging. A 1-ns pulse width ytterbium-doped fiber laser is coupled into a single-mode polarization maintaining fiber. Discrete Raman-shifted wavelength peaks extending to nearly 800 nm are generated with pulse energies sufficient for OR-PAM imaging. Bandpass filters are used to select imaging wavelengths. A dual-mirror galvanometer system was used to scan the focused outputs across samples of carbon fiber networks, 200μm dye-filled tubes, and Swiss Webster mouse ears. Photoacoustic signals were collected in transmission mode and used to create maximum amplitude projection C-scan images. Double dye experiments and in vivo oxygen saturation estimation confirmed functional imaging potential.