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.

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.

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.

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.

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.

Optomechanical Time-Gated Fluorescence Imaging Using Long-Lived Silicon Quantum Dot Nanoparticles.

We demonstrate a novel optomechanical synchronization method to achieve ultrahigh-contrast time-gated fluorescence imaging using live zebrafish as models. Silicon quantum dot nanoparticles (SiQDNPs) with photoluminescence lifetime of about 16 µs were used as the long-lived probes to enable background autofluorescence removal and multiplexing through time-gating. A continuous-wave 405 nm laser as the excitation source was focused on a rotating optical chopper on which the emission light beam obtained from an inverted fluorescence microscope was also focused but with a phase difference such that in a short delay after the excitation laser is blocked, the emission light beam passes through the optical chopper, initiating the image acquisition by a conventional sensor. Both excitation and detection time windows were synchronized by one optical chopper, eliminating the need for pulsed light source and image intensifier which is often used as ultrafast optical shutter. Through use of the cost-effective time-gating method, nearly all background autofluorescence emitted from the yolk sac of a zebrafish embryo microinjected with the SiQDNPs was removed, leading to a 45-fold increase in signal-to-background ratio. Furthermore, two kinds of fluorescence signals emitted from the microinjected SiQDNPs and the intrinsic green fluorescent protein of transgenic zebrafish larvae can be clearly separated through time-gating.

Miniature fiber-optic high-intensity focused ultrasound device using a candle soot nanoparticles-polydimethylsiloxane composites-coated photoacoustic lens.

We present a miniature fiber-optic ultrasound transmitter for generating high-intensity focused ultrasound (HIFU) based on photoacoustic transduction. The HIFU device consists of a fiber and a photoacoustic lens. We develop a simple fabrication procedure for making the photoacoustic lens, which is coated with candle soot nanoparticles-polydimethylsiloxane composites. The fiber is used to deliver pulsed laser for photoacoustic excitation, which facilitates the use of the HIFU device by eliminating the need of free-space optical alignment. The HIFU device (6.5 mm in diameter) produces focused acoustic pressures up to >30 MPa in peak positive with a tight -6-dB focal volume of ~100 µm and ~500 µm in the lateral and axial directions, respectively. Acoustic cavitation induced by the HIFU device is demonstrated. The miniature HIFU device facilitates handheld operation. It holds promise for clinical applications in intraoperative high-precision HIFU therapy. It can even be used for intracavitary therapy with further miniaturization.

Miniature probe for in vivo optical- and acoustic-resolution photoacoustic microscopy.

We present a miniature probe capable of both optical-resolution (OR) and acoustic-resolution (AR) photoacoustic microscopy. A gradient-index-lens fiber and a multimode fiber are used to deliver light for OR and AR illumination, respectively. The probe achieves lateral resolution of 3.1 µm for OR mode and 46-249 µm (at depth of 1.2-4.3 mm) for AR mode, respectively. The size of the probe attains 3.7 mm in diameter, which can be used for endoscopic applications. In vivo imaging of several different parts of a mouse demonstrates the excellent imaging ability of the probe.

Ultrasound detection at fiber end-facets with surface plasmon resonance cavities.

Ultrasound detection is performed by measuring laser reflection off a surface plasmon resonance cavity which is integrated at a single-mode fiber end facet. It shows a total noise (or laser RIN) equivalent pressure of 9.7 KPa (or 5.2 KPa) over 0-20 MHz, a 6-dB angular detection range as large as 70° near 10 MHz, a detection bandwidth larger than 125 MHz, and a stable performance for over 20 min without feedback in ambient conditions. Its small form factor, fiber-optic integration, almost omnidirectional responsivity and large bandwidth are favorable for in vivo applications and high resolution imaging.

Dual-view photoacoustic microscopy for quantitative cell nuclear imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging imaging modality for studying biological tissues. However, in conventional single-view OR-PAM, the lateral and axial resolutions-determined optically and acoustically, respectively-are highly anisotropic. In this Letter, we introduce dual-view OR-PAM to improve axial resolution, achieving three-dimensional (3D) resolution isotropy. We first use 0.5 µm polystyrene beads and carbon fibers to validate the resolution isotropy improvement. Imaging of mouse brain slices further demonstrates the improved resolution isotropy, revealing the 3D structure of cell nuclei in detail, which facilitates quantitative cell nuclear analysis.

Miniature all-optical probe for large synthetic aperture photoacoustic-ultrasound imaging.

A miniature all-optical probe for high-resolution photoacoustic (PA)-ultrasound (US) imaging using a large synthetic aperture is developed. The probe consists of three optical fibers for PA excitation, US generation, and detection of acoustic waves, respectively. The fiber for PA excitation has a large numerical aperture (NA) for wide-angle laser illumination. On the other hand, the fiber with a carbon black-polydimethylsiloxane composite coated on the end face of the optical fiber is used for wide-angle US transmission through laser-US conversion. Both the excited PA and backscattered US signals are detected by a fiber-tip Fabry-Perot cavity for wide-angle acoustic detection. The probe outer diameter is only ~2 mm. The synergy of the three optical fibers makes a large-NA synthetic aperture focusing technique for high-resolution PA and US imaging possible. High PA lateral resolutions of 104-154 µm and high US lateral resolutions of 64-112 µm over a depth range of > 4 mm are obtained. Compared with other existing miniature PA-US probes, to our knowledge, our probe achieves by far the best performance in terms of lateral resolutions and imaging depth range. The constructed probe has potential for endoscopic and intravascular imaging applications that require PA and US contrasts with high resolutions over a large depth range.

Photoacoustic microscopy in vivo using synthetic-aperture focusing technique combined with three-dimensional deconvolution.

Acoustic-resolution photoacoustic microscopy (ARPAM) plays an important role in studying the microcirculation system of biological tissues with deep penetration. High lateral resolution of ARPAM is achieved by using a high numerical aperture acoustic transducer. The deteriorated lateral resolution in the out-of-focus region can be alleviated by synthetic aperture focusing technique (SAFT). Previously, we reported a three-dimensional (3D) deconvolution ARPAM to improve both lateral and axial resolutions in the focus region. In this study, we present our extension of resolution enhancement to the out-of-focus region based on two-dimensional SAFT combined with the 3D deconvolution (SAFT+Deconv). In both the focus and out-of-focus regions, depth-independent lateral resolution provided by SAFT, together with inherently depth-independent axial resolution, ensures a depth-independent point spread function for 3D deconvolution algorithm. Imaging of 10 µm polymer beads shows that SAFT+Deconv ARPAM improves the -6 dB lateral resolutions from 65-700 µm to 20-29 µm, and the -6 dB axial resolutions from 35-42 µm to 12-19 µm in an extended depth of focus (DOF) of ∼2 mm. The signal-to-noise ratio is also increased by 6-30 dB. The resolution enhancement in three dimensions is validated by in vivo imaging of a mouse's dorsal subcutaneous microvasculature. Our results suggest that SAFT+Deconv ARPAM may allow fine spatial resolution with deep penetration and extended DOF for biomedical photoacoustic applications.

Synthetic aperture focusing technique for photoacoustic endoscopy.

Photoacoustic endoscopy (PAE) is a promising tool for the detection of atherosclerotic plaque. In this work, we propose a novel design of a side-viewing PAE probe based on a synthetic aperture focusing technique (SAFT) to enable high transverse resolution over large depth of focus (DOF) along the radial direction. A point-like ultrasonic detector is used to ensure a wide detection angle and thus a large synthetic aperture for SAFT. We first perform numerical simulation to optimize the PAE probe design, which involves the placement of the point-like detector and the diameter of a reflection rod mirror. Then, experiments are conducted based on the optimized probe design. High transverse resolution of 115-190 µm over large DOF of 3.5 mm along the radial direction is experimentally obtained. The SAFT-based PAE holds promise for endoscopic imaging with a high transverse resolution for both the surface and deep regions of tissue.

Simultaneous viewing of individual cells and ambient microvasculature using optical absorption and fluorescence contrasts.

Viewing individual cells and ambient microvasculature simultaneously is crucial for understanding tumor angiogenesis and microenvironments. We developed a confocal fluorescence microscopy (CFM) and photoacoustic microscopy (PAM) dual-modality imaging system that can assess fluorescent contrast and optical absorption contrast in biologic samples simultaneously. After staining tissues with fluorescent dye at an appropriate concentration, each laser pulse can generate not only sufficient fluorescent signals from cells for CFM but also sufficient photoacoustic signals from microvessels for PAM. To explore the potential of this system for diagnosis of bladder cancer, experiments were conducted on a rat bladder model. The CFM image depicts the morphology of individual cells, showing not only large polygonal umbrella cells but also intracellular components. The PAM image acquired at the same time provides complementary information on the microvascular distribution in the bladder wall, ranging from large vessels to capillaries. This device provides an opportunity to realize both histologic assay and microvascular characterization simultaneously. The combination of the information of individual cells and local microvasculature in the bladder offers the capability of envisioning the viability and activeness of these cells and holds promise for more comprehensive study of bladder cancer in vivo.

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.

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.

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.

All-optical scanhead for ultrasound and photoacoustic dual-modality imaging.

We propose a new scanhead design for combined ultrasound (US)/photoacoustic (PA) imaging that can be applied to dual-modality microscopy and biomedical imaging. Both imaging modalities employ the optical generation and detection of acoustic waves. The scanhead consists of an optical fiber with an axicon tip for excitation, and a microring for acoustic detection. No conventional piezoelectric device is needed, and the cost of the design makes it suitable for one-time, disposable use. Furthermore, a single laser pulse is employed to generate both US and PA signals. A subband imaging method can be applied to the receiver to enhance the contrast between the US and PA signals. Phantom data demonstrate the feasibility of this approach.