Imaging and sensing based on dual-pulse nonlinear photoacoustic contrast: a preliminary study on fatty liver.

The feasibility of diagnostic imaging and tissue characterization based on a new contrast realized by dual-pulse photoacoustic measurement was studied. Unlike current photoacoustic methods which are mostly focused on the measurement of tissue optical absorption, this contrast revealed by a dual-pulse laser excitation process takes advantage of the temperature dependence of the Grüneisen parameter of tissue. The first laser pulse heats the sample and causes a temperature rise in the target tissue, which leads to a change of the Grüneisen parameter and the amplitude of the photoacoustic signal from the second laser pulse. This new contrast is then quantified by percentile change in the second pulse signal as a result of the first laser pulse. Since the temperature-dependent Grüneisen parameter is tissue specific and closely relevant to chemical and molecular properties of the sample, the dual-pulse photoacoustic measurement can differentiate various tissue types and conditions. The preliminary study on phantoms and a mouse model has suggested the capability of the proposed contrast in the characterization of fatty livers and the potential for future clinical diagnosis of liver conditions.

Highly stable polymer coated nano-clustered silver plates: a multimodal optical contrast agent for biomedical imaging.

Here, we present a new optical contrast agent based on silver nanoplate clusters embedded inside of a polymer nano matrix. Unlike nanosphere clusters, which have been well studied, nanoplate clusters have unique properties due to the different possible orientations of interaction between the individual plates, resulting in a significant broadening of the absorption spectra. These nanoclusters were immobilized inside of a polymer cladding so as to maintain their stability and optical properties under in vivo conditions. The polymer-coated silver nanoplate clusters show a lower toxicity compared to the uncoated nanoparticles. At high nanoparticle concentrations, cell death occurs mostly due to apoptosis. These nanoparticles were used for targeted fluorescence imaging in a rat glioma cell line by incorporating a fluorescent dye into the matrix, followed by conjugation of a tumor targeting an F3 peptide. We further used these nanoparticles as photoacoustic contrast agents in vivo to enhance the contrast of the vasculature structures in a rat ear model. We observed a contrast enhancement of over 90% following the nanoparticle injection. It is also shown that these NPs can serve as efficient contrast agents, with specific targeting abilities for broadband multimodal imaging that are usable for diagnostic applications and that extend into use as therapeutic agents as well.

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.

Antiosteoporosis effect and possible mechanisms of the ingredients of Radix Achyranthis Bidentatae in animal models of osteoporosis: systematic review and meta-analysis of in vivo studies.

BACKGROUND: The effect and mechanisms of the ingredients (IRAB) of Radix Achyranthis Bidentatae (RAB) on treating osteoporosis (OP) remains debated. We aimed to summary the evidence to evaluate the efficacy of IRAB for animal model OP and elucidate the potential mechanism of IRAB in the treatment of OP. METHODS: In this review and meta-analysis, we searched PubMed, EMBASE, Web of Science, Cochrane Library, Chinese National Knowledge Infrastructure, Wanfang, Chinese Biomedical Literature Database, as well as Chinese VIP databases for targeting articles published from inception to March 2023 in English or Chinese. All randomized controlled animal trials that assessed the efficacy and safety of IRAB for OP were included. We excluded trials according to exclusion criteria. The CAMARADES 10-item quality checklist was utilized to test the risk of potential bias for each including study and modifications were performed accordingly. The primary outcome measures were bone mineral density of the femoral neck (F-BMD), serum calcium (Ca), serum phosphorus (P), serum alkaline phosphatase (ALP), bone gla protein (BGP), bone maximum stress (M-STRESS). The secondary outcome measure was the antiosteoporosis mechanisms of IRAB. RESULTS: Data from nine articles were included in the systematic review and meta-analysis, which focused on 196 animals. Egger's test revealed the presence of publication bias in various studies regarding the primary outcome. Administration of IRAB or RAB could significantly increases the F-BMD (SMD = 2.09; 95% CI = 1.29 to 2.89; P < 0.001, I2 = 76%), Ca (SMD = 0.86; 95% CI = 0.39to1.34; P = 0.07, I2 = 49%); P (SMD = 1.01; 95% CI = 0.45-4.57; P = 0.08, I2 = 50%), BGP (SMD = 2.13; 95% CI = 1.48 to 2.78; I2 = 46%, P = 0.10), while the ALP (SMD = - 0.85; 95% CI = - 1.38 to - 0.31; I2 = 46%, P = 0.10) was remarkably decreased in OP model animals. Moreover, the bone biomechanical indicator M-STRESS (SMD = 2.39; 95% CI = 1.74-3.04; I2 = 32%, P = 0.21) was significantly improved. CONCLUSION: Collectively, the findings suggest that the RAB or IRAB could be an effective drug or an ingredient in diet for the clinical treatment of OP in future.

Miniaturized all-optical photoacoustic microscopy based on microelectromechanical systems mirror scanning.

Achieving photoacoustic microscopic imaging through a miniaturized scanning head is a crucial step toward high-resolution photoacoustic endoscopy. In this work, we have developed a miniaturized probe head using a microelectromechanical systems (MEMS) based mirror for raster scan of the laser beam and our newly developed super broad bandwidth microring resonator based ultrasound detector for photoacoustic signal detection. Through this all-optical design, which offers unique advantages for endoscopic applications, this system is capable of three-dimensional (3D) imaging with high resolution of 17.5 µm in lateral direction and 20 µm in axial direction at a distance of 3.7 mm. After the performance of this system was validated through the experiments on printed grids and a resolution test target, microscopic imaging of the 3D microvasculatures in canine bladders was also conducted successfully, demonstrating the potential of novel photoacoustic endoscopic in future clinical management of bladder cancer.

Pure optical photoacoustic microscopy.

The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After the refinements of the microring's working wavelength and in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×10(5) was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29 Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM with high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5 µm and an axial resolution of 8 µm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at depths in unfrozen tissue specimens or thicker tissue sections, which is not now imageable with current optical or acoustic microscopes of comparable resolution.

In vivo flow speed measurement of capillaries by photoacoustic correlation spectroscopy.

We recently proposed photoacoustic correlation spectroscopy (PACS) and demonstrated a proof-of-concept experiment. Here we use the technique for in vivo flow speed measurement in capillaries in a chick embryo model. The photoacoustic microscopy system is used to render high spatial resolution and high sensitivity, enabling sufficient signals from single red blood cells. The probe beam size is calibrated by a blood-mimicking phantom. The results indicate the feasibility of using PACS to study flow speeds in capillaries.

Evaluation of bladder microvasculature with high-resolution photoacoustic imaging.

We explored the potential of an emerging laser-based technology, photoacoustic imaging (PAI), for bladder cancer diagnosis through high-resolution imaging of microvasculature in the bladder tissues. Imaging results from ex vivo canine bladders demonstrated the excellent ability of PAI in mapping three-dimensional microvasculature in optically scattering bladder tissues. By comparing the results from human bladder specimens affected by cancer to those from the normal control, the feasibility of PAI to differentiate malignant from benign bladder tissues was also explored. The distinctive morphometric characteristics of tumor microvasculature can be seen in the images from cancer samples, suggesting that PAI may allow in vivo assessment of neoangiogenesis that is closely associated with bladder cancer generation and progression. By presenting subsurface morphological and physiological information in bladder tissues, PAI, when performed in a similar way as in conventional endoscopy, provides an opportunity for improved diagnosis, staging, and treatment guidance of bladder cancer.

Simultaneous multimodal imaging with integrated photoacoustic microscopy and optical coherence tomography.

We have developed a multimodal imaging technique by integrating photoacoustic microscopy and spectral-domain optical coherence tomography to provide simultaneous volumetric microscopic imaging of both optical absorption and scattering contrasts in biological tissues. In the integrated system, the two imaging modalities share the same optical scanning and delivery mechanisms after their probing and illumination light beams are combined. By further synchronizing the image acquisitions, the images from the two modalities are intrinsically registered. The capabilities of this novel technique were demonstrated by imaging both the microanatomy and microvasculature in mouse ears in vivo.

Optical fluence distribution study in tissue in dark-field confocal photoacoustic microscopy using a modified Monte Carlo convolution method.

We have modified the existing convolution method of the Monte Carlo simulation for finite photon beams with both translational and rotational invariance. The modified convolution method was applied to simulate the optical fluence distribution in tissue in dark-field confocal photoacoustic microscopy. We studied the influence of the size of the dark field and the illumination incident angle on the depth position of the effective optical focus (the region with the highest fluence) and the fluence ratio (the ratio of the optical fluence at the effective optical focus inside the tissue to the optical fluence on the tissue surface along the ultrasonic axis). Within the reach of diffuse photons, the depth position of the effective optical focus increases with the size of the dark field and is much less sensitive to the incident angle. The findings show that, while the fluence at the effective optical focus decreases, the fluence ratio increases with the size of the dark field. The incident angle has a weaker influence on the fluence ratio than the size of the dark field does. An incident angle between 30 and 50 degrees gives the highest fluence at the effective optical focus.

Plasmonic Nanoparticles with Quantitatively Controlled Bioconjugation for Photoacoustic Imaging of Live Cancer Cells.

Detection and imaging of single cancer cells is critical for cancer diagnosis and understanding of cellular dynamics. Photoacoustic imaging (PAI) provides a potential tool for the study of cancer cell dynamics, but faces the challenge that most cancer cells lack sufficient endogenous contrast. Here, a type of colloidal gold nanoparticles (AuNPs) are physically fabricated and are precisely functionalized with quantitative amounts of functional ligands (i.e., polyethyleneglycol (PEG) and (Arginine(R)-Glycine(G)-Aspartic(D))4 (RGD) peptides) to serve as an exogenous contrast agent for PAI of single cells. The functionalized AuNPs, with a fixed number of PEG but different RGD densities, are delivered into human prostate cancer cells. Radioactivity and photoacoustic analyses show that, although cellular uptake efficiency of the AuNPs linearly increases along with RGD density, photoacoustic signal generation efficiency does not and only maximize at a moderate RGD density. The functionalization of the AuNPs is in turn optimized based on the experimental finding, and single cancer cells are imaged using a custom photoacoustic microscopy with high-resolution. The quantitatively functionalized AuNPs together with the high-resolution PAI system provide a unique platform for the detection and imaging of single cancer cells, and may impact not only basic science but also clinical diagnostics on a range of cancers.

Dual-pulse nonlinear photoacoustic technique: a practical investigation.

The dual-pulse nonlinear photoacoustic technique is a recently developed technology based on temperature dependence of the Grüneisen parameter and involves consecutive excitations of biological tissue using two laser pulses with a short time delay. Here we review the principle of the technique and give a discussion about its technical aspects, including selection and combination of excitation laser wavelengths, determination of laser fluence, estimation of thermal relaxation function and probability of photoablation or cavitation. Comparisons between the dual-pulse technique and conventional photoacoustics as well as thermal photoacoustics are also presented. These investigations are supported by experimental results and will give a practical reference and guide for further developments of the technique.

A fiber-optic system for dual-modality photoacoustic microscopy and confocal fluorescence microscopy using miniature components.

Imaging of the cells and microvasculature simultaneously is beneficial to the study of tumor angiogenesis and microenvironments. We designed and built a fiber-optic based photoacoustic microscopy (PAM) and confocal fluorescence microscopy (CFM) dual-modality imaging system. To explore the feasibility of this all-optical device for future endoscopic applications, a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector were employed trying to meet the requirements of miniaturization. Both the lateral resolutions of PAM and CFM were quantified to be 8.8 µm. Axial resolutions of PAM and CFM were experimentally measured to be 19 µm and 53 µm, respectively. The experiments on ex vivo animal bladder tissues demonstrate the good performance of this system in imaging not only microvasculature but also cellular structure, suggesting that this novel imaging technique holds potential for improved diagnosis and guided treatment of bladder cancer.

Tissue-compliant neural implants from microfabricated carbon nanotube multilayer composite.

Current neural prosthetic devices (NPDs) induce chronic inflammation due to complex mechanical and biological reactions related, in part, to staggering discrepancies of mechanical properties with neural tissue. Relatively large size of the implants and traumas to blood-brain barrier contribute to inflammation reactions, as well. Mitigation of these problems and the realization of long-term brain interface require a new generation of NPDs fabricated from flexible materials compliant with the brain tissue. However, such materials will need to display hard-to-combine mechanical and electrical properties which are not available in the toolbox of classical neurotechnology. Moreover, these new materials will concomitantly demand different methods of (a) device micromanufacturing and (b) surgical implantation in brains because currently used processes take advantage of high stiffness of the devices. Carbon nanotubes (CNTs) serve as a promising foundation for such materials because of their record mechanical and electrical properties, but CNT-based tissue-compliant devices have not been realized yet. In this study, we formalize the mechanical requirements to tissue-compliant implants based on critical rupture strength of brain tissue and demonstrate that miniature CNT-based devices can satisfy these requirements. We fabricated them using MEMS-like technology and miniaturized them so that at least two dimensions of the electrodes would be comparable to brain tissue cells. The nanocomposite-based flexible neural electrodes were implanted into the rat motor cortex using a surgical procedure specifically designed for soft tissue-compliant implants. The post-surgery implant localization in the motor cortex was successfully visualized with magnetic resonance and photoacoustic imaging. In vivo functionality was demonstrated by successful registration of the low-frequency neural recording in the live brain of anesthetized rats. Investigation of inflammation processes around these electrodes will be required to establish their prospects as long-term neural electrodes.

Photoacoustic microscopy: a potential new tool for evaluation of angiogenesis inhibitor.

The feasibility of photoacoustic microscopy (PAM) for evaluation of angiogenesis inhibitor was investigated on a chick embryo model in vivo. Different concentrations of the angiogenesis inhibitor, Sunitinib, were applied to the chorioallantoic membrane (CAM) of the chick embryos. Imaging of microvasculature in embryo CAMs was acquired using a laser-scanning PAM system; while the optical microscopy (OM) capturing the microvascular images of the same set of CAMs for comparison served as a gold standard for validating the results from PAM. The microvascular density as a function of applied Sunitinib concentration has been quantified in both PAM and OM images. The results from these two modalities have a good agreement, suggesting that PAM could provide an unbiased quantification of microvascular density for objective evaluation of anti-angiogenesis medication. In comparison with conventional OM which enables only two-dimensional enface imaging, PAM is capable of three-dimensional analysis of microvessels, including not only morphology but also functions, as demonstrated in part by the imaging result on a canine bladder model. The emerging PAM technique shows promise to be used in clinical and preclinical settings for comprehensive and objective evaluation of anti-angiogenesis medications.

Combined photoacoustic and acoustic imaging of human breast specimens in the mammographic geometry.

A photoacoustic volume imaging (PAVI) system was designed to study breast cancer detection and diagnosis in the mammographic geometry in combination with automated 3-D ultrasound (AUS). The goal of the work described here was to validate the design and evaluate its performance in human breast tissues for non-invasive imaging of deeply positioned structures covering such geometry. The good penetration of near-infrared light and high receiving sensitivity of a broad-bandwidth, 572-element, 2-D polyvinylidene fluoride (PVDF) array at a low center frequency of 1 MHz were used with 20 channel simultaneous acquisition. Pseudo-lesions filled with dilute blood were imaged in three human breast specimens at various depths up to 49 mm. With near-infrared light illumination and 256-sample averaging, the extrapolated maximum depth in imaging a 2.4-mm blood-rich lesion with a 3-dB contrast-to-noise ratio in a compressed breast was 54 mm. Three-dimensional photoacoustic volume image stacks of the breasts were co-registered with 3-D ultrasound image stacks, suggesting for the first time that PAVI, based on the intrinsic tissue contrast, can visualize tissue interfaces other than those with blood, including the inner skin surface and connective tissue sheets. With the designed system, PAVI revealed satisfactory imaging depth and sensitivity for coverage of the entire breast when imaged from both sides in the mammographic geometry with mild compression.

In vitro testing of a protease-sensitive contrast agent for optoacoustic imaging.

We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers, causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules deaggregate, giving rise to a spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is detection of the cleaved probe that is of the greatest import, since it reports on the protease activity of interest.

Laser-scanning optical-resolution photoacoustic microscopy.

We have developed a laser-scanning optical-resolution photoacoustic microscopy method that can potentially fuse with existing optical microscopic imaging modalities. To acquire an image, the ultrasonic transducer is kept stationary during data acquisition, and only the laser light is raster scanned by an x-y galvanometer scanner. A lateral resolution of 7.8 microm and a circular field of view with a diameter of 6 mm were achieved in an optically clear medium. Using a laser system working at a pulse repetition rate of 1,024 Hz, the data acquisition time for an image consisting of 256 x 256 pixels was less than 2 min.