Biodistributions and Imaging of Poly(ethylene glycol)-Conjugated Silicon Quantum Dot Nanoparticles in Osteosarcoma Models via Intravenous and Intratumoral Injections.

Osteosarcoma is a malignant tumor with relatively high mortality rates in children and adolescents. While nanoparticles have been widely used in assisting the diagnosis and treatment of cancers, the biodistributions of nanoparticles in osteosarcoma models have not been well studied. Herein, we synthesize biocompatible and highly photoluminescent silicon quantum dot nanoparticles (SiQDNPs) and investigate their biodistributions in osteosarcoma mouse models after intravenous and intratumoral injections by fluorescence imaging. The bovine serum albumin (BSA)-coated and poly(ethylene glycol) (PEG)-conjugated SiQDNPs, when dispersed in phosphate-buffered saline (PBS), can emit red photoluminescence with the photoluminescence quantum yield more than 30% and have very low in vitro and in vivo toxicity. The biodistributions after intravenous injections reveal that the SiQDNPs are mainly metabolized through the livers in mice, while only slight accumulation in the osteosarcoma tumor is observed. Furthermore, the PEG conjugation can effectively extend the circulation time. Finally, a mixture of SiQDNPs and indocyanine green (ICG), which complement each other in the spectral range and diffusion length, is directly injected into the tumor for imaging. After the injection, the SiQDNPs with relatively large particle sizes stay around the injection site, while the ICG molecules diffuse over a broad range, especially in the muscular tissue. By taking advantage of this property, the difference between the osteosarcoma tumor and normal muscular tissue is demonstrated.

Sensing of triglyceride concentration in blood solution using photoacoustic microscopy.

The level of triglyceride (TG) in blood is essential to human health, and hypertriglyceridemia (TG level > 150 mg/dL) would lead to cardiovascular disease and acute pancreatitis that threaten human life. Routine methods for measuring the TG level in blood depend on a lipid panel blood test, which is invasive and not convenient. Here, we use photoacoustic (PA) microscopy to test the PA amplitude of blood solutions (based on hemoglobin powder as well as flowing sheep blood) with different TG concentrations. Interestingly, we observe that the PA amplitude increases with increasing TG concentration in blood solutions, which is attributed to the increase of the Grüneisen coefficient. The preliminary in vitro study shows that the PA methodology is able to detect the TG level down to 450 mg/dL. This finding provides an opportunity for using photoacoustics to noninvasively diagnose hypertriglyceridemia.

Biodegradable germanium nanoparticles as contrast agents for near-infrared-II photoacoustic imaging.

Photoacoustic (PA) imaging using contrast agents with strong near-infrared-II (NIR-II, 1000-1700 nm) absorption enables deep penetration into biological tissue. Besides, biocompatibility and biodegradability are essential for clinical translation. Herein, we developed biocompatible and biodegradable germanium nanoparticles (GeNPs) with high photothermal stability as well as strong and broad absorption for NIR-II PA imaging. We first demonstrate the excellent biocompatibility of the GeNPs through experiments, including the zebrafish embryo survival rates, nude mouse body weight curves, and histological images of the major organs. Then, comprehensive PA imaging demonstrations are presented to showcase the versatile imaging capabilities and excellent biodegradability, including in vitro PA imaging which can bypass blood absorption, in vivo dual-wavelength PA imaging which can clearly distinguish the injected GeNPs from the background blood vessels, in vivo and ex vivo PA imaging with deep penetration, in vivo time-lapse PA imaging of a mouse ear for observing biodegradation, ex vivo time-lapse PA imaging of the major organs of a mouse model for observing the biodistribution after intravenous injection, and notably in vivo dual-modality fluorescence and PA imaging of osteosarcoma tumors. The in vivo biodegradation of GeNPs is observed not only in the normal tissue but also in the tumor, making the GeNPs a promising candidate for clinical NIR-II PA imaging applications.

Implantable QR code subcutaneous microchip using photoacoustic and ultrasound microscopy for secure and convenient individual identification and authentication.

Individual identification and authentication techniques are merged into many aspects of human life with various applications, including access control, payment or banking transfer, and healthcare. Yet conventional identification and authentication methods such as passwords, biometrics, tokens, and smart cards suffer from inconvenience and/or insecurity. Here, inspired by quick response (QR) code and implantable microdevices, implantable and minimally-invasive QR code subcutaneous microchips (QRC-SMs) are proposed to be an effective approach to carry useful and private information, thus enabling individual identification and authentication. Two types of QRC-SMs, QRC-SMs with "hole" and "flat" elements and QRC-SMs with "titanium-coated" and "non-coated" elements, are designed and fabricated to store personal information. Corresponding ultrasound microscopy and photoacoustic microscopy are used for imaging the QR code pattern underneath skin, and open-source artificial intelligence algorithm is applied for QR code detection and recognition. Ex vivo experiments under tissue and in vivo experiments with QRC-SMs implanted in live mice have been performed, demonstrating successful information retrieval from implanted QRC-SMs. QRC-SMs are hidden subcutaneously and invisible to the eyes. They cannot be forgotten, misplaced or lost, and can always be ready for timely medical identification, access control, and payment or banking transfer. Hence, QRC-SMs provide promising routes towards private, secure, and convenient individual identification and authentication.

De-Noising of Photoacoustic Microscopy Images by Attentive Generative Adversarial Network.

As a hybrid imaging technology, photoacoustic microscopy (PAM) imaging suffers from noise due to the maximum permissible exposure of laser intensity, attenuation of ultrasound in the tissue, and the inherent noise of the transducer. De-noising is an image processing method to reduce noise, and PAM image quality can be recovered. However, previous de-noising techniques usually heavily rely on manually selected parameters, resulting in unsatisfactory and slow de-noising performance for different noisy images, which greatly hinders practical and clinical applications. In this work, we propose a deep learning-based method to remove noise from PAM images without manual selection of settings for different noisy images. An attention enhanced generative adversarial network is used to extract image features and adaptively remove various levels of Gaussian, Poisson, and Rayleigh noise. The proposed method is demonstrated on both synthetic and real datasets, including phantom (leaf veins) and in vivo (mouse ear blood vessels and zebrafish pigment) experiments. In the in vivo experiments using synthetic datasets, our method achieves the improvement of 6.53 dB and 0.26 in peak signal-to-noise ratio and structural similarity metrics, respectively. The results show that compared with previous PAM de-noising methods, our method exhibits good performance in recovering images qualitatively and quantitatively. In addition, the de-noising processing speed of 0.016 s is achieved for an image with 256×256 pixels, which has the potential for real-time applications. Our approach is effective and practical for the de-noising of PAM images.

Miniature Probe for Optomechanical Focus-Adjustable Optical-Resolution Photoacoustic Endoscopy.

Photoacoustic microscopy (PAM) is a promising imaging modality because it is able to reveal optical absorption contrast in high resolution on the order of a micrometer. It can be applied in an endoscopic approach by implementing PAM into a miniature probe, termed photoacoustic endoscopy (PAE). Here we develop a miniature focus-adjustable PAE (FA-PAE) probe characterized by both high resolution (in micrometers) and large depth of focus (DOF) via a novel optomechanical design for focus adjustment. To realize high resolution and large DOF in a miniature probe, a 2-mm plano-convex lens is specially adopted, and the mechanical translation of a single-mode fiber is meticulously designed to allow the use of multi-focus image fusion (MIF) for extended DOF. Compared with existing PAE probes, our FA-PAE probe achieves high resolution of [Formula: see text] within unprecedentedly large DOF of 3.2 mm, more than 27 times the DOF of the probe without performing focus adjustment for MIF. The superior performance is first demonstrated by imaging both phantoms and animals including mice and zebrafish in vivo by linear scanning. Further, in vivo endoscopic imaging of a rat's rectum by rotary scanning of the probe is conducted to showcase the capability of adjustable focus. Our work opens new perspectives for PAE biomedical applications.

In vivo evaluation of a lipopolysaccharide-induced ear vascular leakage model in mice using photoacoustic microscopy.

Sepsis is caused by dysregulated host inflammatory response to infection. During sepsis, early identification and monitoring of vascular leakage are pivotal for improved diagnosis, treatment, and prognosis. However, there is a lack of research on noninvasive observation of inflammation-related vascular leakage. Here, we investigate the use of photoacoustic microscopy (PAM) for in vivo visualization of lipopolysaccharide (LPS)-induced ear vascular leakage in mice using Evans blue (EB) as an indicator. A model combining needle pricking on the mouse ear, topical smearing of LPS on the mouse ear, and intravenous tail injection of EB is developed. Topical application of LPS is expected to induce local vascular leakage in skin. Inflammatory response is first validated by ex vivo histology and enzyme-linked immunosorbent assay. Then, local ear vascular leakage is confirmed by ex vivo measurement of swelling, thickening, and EB leakage. Finally, PAM for in vivo identification and evaluation of early vascular leakage using the model is demonstrated. For PAM, common excitation wavelength of 532 nm is used, and an algorithm is developed to extract quantitative metrics for EB leakage. The results show potential of PAM for noninvasive longitudinal monitoring of peripheral skin vascular leakage, which holds promise for clinical sepsis diagnosis and management.

High-fidelity deconvolution for acoustic-resolution photoacoustic microscopy enabled by convolutional neural networks.

Acoustic-resolution photoacoustic microscopy (AR-PAM) image resolution is determined by the point spread function (PSF) of the imaging system. Previous algorithms, including Richardson-Lucy (R-L) deconvolution and model-based (MB) deconvolution, improve spatial resolution by taking advantage of the PSF as prior knowledge. However, these methods encounter the problems of inaccurate deconvolution, meaning the deconvolved feature size and the original one are not consistent (e.g., the former can be smaller than the latter). We present a novel deep convolution neural network (CNN)-based algorithm featuring high-fidelity recovery of multiscale feature size to improve lateral resolution of AR-PAM. The CNN is trained with simulated image pairs of line patterns, which is to mimic blood vessels. To investigate the suitable CNN model structure and elaborate on the effectiveness of CNN methods compared with non-learning methods, we select five different CNN models, while R-L and directional MB methods are also applied for comparison. Besides simulated data, experimental data including tungsten wires, leaf veins, and in vivo blood vessels are also evaluated. A custom-defined metric of relative size error (RSE) is used to quantify the multiscale feature recovery ability of different methods. Compared to other methods, enhanced deep super resolution (EDSR) network and residual in residual dense block network (RRDBNet) model show better recovery in terms of RSE for tungsten wires with diameters ranging from 30 µ m to 120 µ m . Moreover, AR-PAM images of leaf veins are tested to demonstrate the effectiveness of the optimized CNN methods (by EDSR and RRDBNet) for complex patterns. Finally, in vivo images of mouse ear blood vessels and rat ear blood vessels are acquired and then deconvolved, and the results show that the proposed CNN method (notably RRDBNet) enables accurate deconvolution of multiscale feature size and thus good fidelity.

Image enhancement in acoustic-resolution photoacoustic microscopy enabled by a novel directional algorithm.

By considering the line pattern of acoustic-resolution photoacoustic microscopy (AR-PAM) vessel images, we develop modified algorithms for synthetic aperture focusing technique (SAFT) and deconvolution based on a directional approach to enhance images. The modified algorithms consist of Fourier accumulation SAFT (FA-SAFT) and directional model-based (D-MB) deconvolution. To evaluate the performance of our algorithms, we conduct a series of imaging experiments and apply our algorithms, and existing SAFT and deconvolution algorithms are also applied for side-by-side comparison. By imaging tungsten wire phantom, our algorithms enable full width at half maximum of 26 - 31 µm over depth of focus of 1.8 mm and minimum resolvable distance of 46 - 49 µm, besting existing SAFT and deconvolution algorithms. Imaging of leaf skeleton phantom and in vivo imaging of mouse blood vessels also prove that our algorithm is capable of providing high-resolution, high-signal-to-noise ratio, and good-fidelity results for complex structures and for in vivo applications, especially for the images with the line pattern. The proposed directional approach can not only be used in AR-PAM but also in other imaging modalities to deal with the line pattern, such as FA-SAFT for ultrasound imaging and D-MB deconvolution for optical coherence tomography angiography.

Investigation of nonlinear photoacoustic microscopy using a low-cost infrared lamp.

Nonlinear photoacoustic microscopy (PAM) is a novel approach to enhance contrast and resolution. In this study, a low-cost infrared (IR) lamp as a simple approach for nonlinear PAM is demonstrated. Numerical simulations are first performed to verify the nonlinear photoacoustic effect under steady heating for two cases: (a) Differentiation of absorbers with different Grüneisen coefficients; (b) enhancement of photoacoustic amplitude. Then, sets of experiments are conducted to experimentally demonstrate our proposed approach: (a) Longitudinal monitoring of photoacoustic A-line signals from two samples, porcine tissue ex vivo and hemoglobin and indocyanine green (ICG) solutions in tubes in vitro for demonstrating the above-mentioned two cases; (b) PAM imaging of hemoglobin and ICG solutions in tubes before and after IR lamp heating. Different signal change and amplitude enhancement are observed in different demonstrations, showing the efficacy of the proposed approach. By virtue of cost-effectiveness and decent performance, our work facilitates nonlinear PAM studies.

Cerebrovascular imaging in vivo by non-contact photoacoustic microscopy based on photoacoustic remote sensing with a laser diode for interrogation.

Photoacoustic microscopy (PAM) is a unique tool for biomedical applications because it can visualize optical absorption contrast in vivo. Recently, non-contact PAM based on non-interferometric photoacoustic remote sensing (PARS), termed PARS microscopy, has shown promise for selected imaging applications. A variety of superluminescent diodes (SLDs) have been employed in the PARS microscopy system as the interrogation light source. Here, we investigate the use of a low-cost laser diode (LD) as the interrogation light source in PARS microscopy, termed PARS-LD. A side-by-side comparison of PARS-LD and a PARS microscopy system using an SLD was conducted that showed comparable performance in terms of resolution and signal-to-noise ratio. More importantly, for the first time to our knowledge, in vivo PAM imaging of mouse brain vessels was conducted in a non-contact manner, and the results show that PARS-LD provides great performance.

Miniature non-contact photoacoustic probe based on fiber-optic photoacoustic remote sensing microscopy.

Photoacoustic (PA) remote sensing (PARS) microscopy, featured by non-contact operation, has shown great potential for PA microscopy (PAM) imaging applications. However, current PARS microscopy systems are mainly based on free-space light, making the imaging head bulky and inconvenient to use. These issues hinder selected applications such as PA endoscopy and handheld PAM. Here, we report a miniature probe capable of non-contact PAM based on PARS microscopy. By utilizing fiber-optic components including a wavelength division multiplexer and an optical circulator, the imaging head can be highly miniaturized with a diameter of ∼3.0mm. Also, since all light is transmitted via fibers, the fiber-optic PARS microscopy system is relatively easy to build and facilitates scanning of the probe. In vivo imaging of a zebrafish larva and imaging of lithium metal batteries are conducted using the probe, showing its good imaging capability.

Label-free photoacoustic microscopy: a potential tool for the live imaging of blood disorders in zebrafish.

The zebrafish has emerged as a useful model for human hematological disorders. Transgenic zebrafish that express green fluorescence protein (GFP) in red blood cells (RBCs) visualized by fluorescence microscopy (FLM) is a fundamental approach in such studies to understand the cellular processes and biological functions. However, additional and cumbersome efforts are required to breed a transgenic zebrafish line with reliable GFP expression. Further, the yolk autofluorescence and finite GFP fluorescence lifetimes also have an adverse impact on the observation of target signals. Here, we investigate the identification of intracerebral hemorrhage (ICH) and hemolytic anemia (HA) in zebrafish embryos using label-free photoacoustic microscopy (PAM) for imaging. First, ICH and HA in transgenic LCR-EGFP zebrafish are mainly studied by PAM and FLM. The results show that PAM is comparable to FLM in good identification of ICH and HA. Besides, PAM is more advantageous in circumventing the issue of autofluorescence. Secondly, ICH and HA in the transparent casper zebrafish without fluorescent labeling are imaged by PAM and bright-field microscopy (BFM). Because of the high contrast to reveal RBCs, PAM obviously outperforms BFM in the identification of both ICH and HA. Note that FLM cannot observe casper zebrafish due to its lack of fluorescent labeling. Our work proves that PAM can be a useful tool to study blood disorders in zebrafish, which has advantages: (i) Reliable results enabled by intrinsic absorption of RBCs; (ii) wide applicability to zebrafish strains (no requirement of a transgene); (iii) high sensitivity in identification of ICH and HA compared with BFM.

Accelerated Growth of Electrically Isolated Lithium Metal during Battery Cycling.

Severe capacity loss during cycling of lithium-metal batteries is one of the most concerning obstacles hindering their practical application. As this capacity loss is related to the variety of side reactions occurring to lithium metal, identification and quantification of these lithium-loss processes are extremely important. In this work, we systematically distinguish and quantify the different rates of lithium loss associated with galvanic corrosion, the formation of a solid-electrolyte interphase, and the formation of electrically isolated lithium metal (i.e., "dead" lithium). We show that the formation of "dead" Li is accelerated upon cycling, dominating the total lithium loss, with much slower rates of lithium loss associated with galvanic corrosion and formation of the solid-electrolyte interphase. Furthermore, photoacoustic imaging reveals that the three-dimensional spatial distribution of "dead" Li is distinctly different from that of freshly deposited lithium. This quantification is further extended to a solid-state Li/Cu cell based on a Li10GeP2S12 solid-state electrolyte. The lithium loss in the solid-state cell is much severer than that of a conventional lithium-metal battery based on a liquid electrolyte. Our work highlights the importance of quantitative studies on conventional and solid-state lithium-metal batteries and provides a strong basis for the optimization of lithium-metal electrochemistry.

Photoacoustic-fluorescence microendoscopy in vivo.

A miniature endoscope capable of imaging multiple tissue contrasts in high resolution is highly attractive, because it can provide complementary and detailed tissue information of internal organs. Here we present a photoacoustic (PA)-fluorescence (FL) endoscope for optical-resolution PA microscopy (PAM) and FL microscopy (FLM). The endoscope with a diameter of 2.8 mm achieves high lateral resolutions of 5.5 and 6.3 µm for PAM and FLM modes, respectively. In vivo imaging of zebrafish larvae and a mouse ear is conducted, and high-quality images are obtained. Additionally, in vivo endoscopic imaging of a rat rectum is demonstrated, showing the endoscopic imaging capability of our endoscope. By providing dual contrasts with high resolution, the endoscope may open up new opportunities for clinical endoscopic imaging applications.

Photoacoustic microscopy with sparse data by convolutional neural networks.

The point-by-point scanning mechanism of photoacoustic microscopy (PAM) results in low-speed imaging, limiting the application of PAM. In this work, we propose a method to improve the quality of sparse PAM images using convolutional neural networks (CNNs), thereby speeding up image acquisition while maintaining good image quality. The CNN model utilizes attention modules, residual blocks, and perceptual losses to reconstruct the sparse PAM image, which is a mapping from a 1/4 or 1/16 low-sampling sparse PAM image to a latent fully-sampled one. The model is trained and validated mainly on PAM images of leaf veins, showing effective improvements quantitatively and qualitatively. Our model is also tested using in vivo PAM images of blood vessels of mouse ears and eyes. The results suggest that the model can enhance the quality of the sparse PAM image of blood vessels in several aspects, which facilitates fast PAM and its clinical applications.

Review recent developments in photoacoustic imaging and sensing for nondestructive testing and evaluation.

Photoacoustic (PA) imaging has been widely used in biomedical research and preclinical studies during the past two decades. It has also been explored for nondestructive testing and evaluation (NDT/E) and for industrial applications. This paper describes the basic principles of PA technology for NDT/E and its applications in recent years. PA technology for NDT/E includes the use of a modulated continuous-wave laser and a pulsed laser for PA wave excitation, PA-generated ultrasonic waves, and all-optical PA wave excitation and detection. PA technology for NDT/E has demonstrated broad applications, including the imaging of railway cracks and defects, the imaging of Li metal batteries, the measurements of the porosity and Young's modulus, the detection of defects and damage in silicon wafers, and a visualization of underdrawings in paintings.

Dual-modal imaging with non-contact photoacoustic microscopy and fluorescence microscopy.

Simultaneous imaging of complementary absorption and fluorescence contrasts with high spatial resolution is useful for biomedical studies. However, conventional dual-modal photoacoustic (PA) and fluorescence imaging systems require the use of acoustic coupling media due to the contact operation of PA imaging, which causes issues and complicates the procedure in certain applications such as cell imaging and ophthalmic imaging. We present a novel dual-modal imaging system which combines non-contact PA microscopy (PAM) based on PA remote sensing and fluorescence microscopy (FLM) into one platform. The system enables high lateral resolution of 2 and 2.7 µm for PAM and FLM modes, respectively. In vivo imaging of a zebrafish larva injected with a rhodamine B solution is demonstrated, with PAM visualizing the pigment and FLM revealing the injected rhodamine B.