Label-free imaging of zebrafish larvae in vivo by photoacoustic microscopy.
ABSTRACT: Zebrafish play an important role in biological and biomedical research. Traditional in vivo imaging methods for studying zebrafish larvae primarily require fluorescence labeling. In this work, relying on tissue intrinsic optical absorption contrast, we acquired high resolution label-free 3D images of zebrafish larvae by using photoacoustic microscopy (PAM) in vivo. The spatial resolution reaches several microns, allowing the study of microstructures in various living organs. We demonstrated that our method has the potential to be a powerful non-invasive imaging method for studying various small animal models, including zebrafish larvae, Caenorhabditis elegans, frogs and drosophila larvae.
Project description:With their optically transparent appearance, zebrafish larvae are readily imaged with optical-resolution photoacoustic (PA) microscopy (OR-PAM). Previous OR-PAM studies have mapped endogenous chromophores (e.g. melanin and hemoglobin) within larvae; however, anatomical features cannot be imaged with OR-PAM alone due to insufficient optical absorption. We have previously reported on the photoacoustic radiometry (PAR) technique, which can be used simultaneously with OR-PAM to generate images dependent upon the optical attenuation properties of a sample. Here we demonstrate application of the duplex PAR/PA technique for label-free imaging of the anatomy and vasculature of zebrafish larvae in vivo at 200 and 400?MHz ultrasound detection frequencies. We then use the technique to assess the effects of anti-angiogenic drugs on the development of the larval vasculature. Our results demonstrate the effectiveness of simultaneous PAR/PA for acquiring anatomical images of optically transparent samples in vivo, and its potential applications in assessing drug efficacy and embryonic development.
Project description:A dual modality microscopy with the highest imaging resolution reported so far based on reflection-mode photoacoustic and confocal fluorescence is presented in this study. The unique design of the imaging head of the microscope makes it highly convenient for scalable high-resolution imaging by simply switching the optical objectives. The submicron resolution performance of the system is demonstrated via in vivo imaging of zebrafish, normal mouse ear, and a xenograft tumor model inoculated in the mouse ear. The imaging results confirm that the presented dual-modality microscopy imaging system could play a vital role in observing model organism, studying tumor angiogenesis and assessment of antineoplastic drugs.
Project description:The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290?nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling.
Project description:Optical-resolution photoacoustic microscopy (OR-PAM) has become a major experimental tool of photoacoustic tomography, with unique imaging capabilities for various biological applications. However, conventional imaging systems are all table-top embodiments, which preclude their use in internal organs. In this study, by applying the OR-PAM concept to our recently developed endoscopic technique, called photoacoustic endoscopy (PAE), we created an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system, which enables internal organ imaging with a much finer resolution than conventional acoustic-resolution PAE systems. OR-PAEM has potential preclinical and clinical applications using either endogenous or exogenous contrast agents.
Project description:We propose to enhance the axial resolution of photoacoustic microscopy (PAM) by reducing the speed of sound within the imaging region of interest. With silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. When tissue-compatible low-speed liquid becomes available, this approach may find broad applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging.
Project description:The temperature-dependent property of the Grueneisen parameter has been employed in photoacoustic imaging mainly to measure tissue temperature. Here we explore this property using a different approach and develop Grueneisen relaxation photoacoustic microscopy (GR-PAM), a technique that images nonradiative absorption with confocal optical resolution. GR-PAM sequentially delivers two identical laser pulses with a microsecond-scale time delay. The first laser pulse generates a photoacoustic signal and thermally tags the in-focus absorbers. When the second laser pulse excites the tagged absorbers within the thermal relaxation time, a photoacoustic signal stronger than the first one is produced, owing to the temperature dependence of the Grueneisen parameter. GR-PAM detects the amplitude difference between the two colocated photoacoustic signals, confocally imaging the nonradiative absorption. We greatly improved axial resolution from 45???m to 2.3???m and, at the same time, slightly improved lateral resolution from 0.63???m to 0.41???m. In addition, the optical sectioning capability facilitates the measurement of the absolute absorption coefficient without fluence calibration.
Project description:Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using lacZ, one of the most widely used reporter genes in biology. We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ?1.0 mm and ?0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 µm and an axial resolution of 15 µm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.
Project description:This study investigates the feasibility of characterizing the microstructures within a biological tissue by analyzing the frequency spectrum of the photoacoustic signal from the tissue. Hypotheses are derived from theoretical analyses on the relationships between the dimensions/concentrations of the photoacoustic sources within the region-of-interest and the linear model fitted to the power spectra of photoacoustic signals. The hypotheses are validated, following the procedures of ultrasound spectrum analysis, by simulations and experiments with phantoms fabricated by embedding the polyethylene microspheres in porcine gelatin, indicating that photoacoustic spectrum analysis could be a potential tool for characterizing microstructures in biological samples.
Project description:We report the development of functional photoacoustic microscopy capable of video-rate high-resolution in vivo imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light delivery system. Focused broadband ultrasound detection provides a 44-?m lateral resolution and a 28-?m axial resolution based on the envelope (a 15-?m axial resolution based on the raw RF signal). Due to the efficient bright-field light delivery, the system can image as deep as 4.8 mm in vivo using low excitation pulse energy (28 ?J per pulse, 0.35??mJ/cm² on the skin surface). The photoacoustic probe is mounted on a fast-scanning voice-coil scanner to acquire 40 two-dimensional (2-D) B-scan images per second over a 9-mm range. High-resolution anatomical imaging is demonstrated in the mouse ear and brain. Via fast dual-wavelength switching, oxygen dynamics of mouse cardio-vasculature is imaged in realtime as well.
Project description:Photoacoustic imaging is a novel hybrid imaging modality combining the high spatial resolution of optical imaging with the high penetration depth of ultrasound imaging. Here, for the first time, we evaluate the efficacy of various photosensitizers that are widely used as photodynamic therapeutic (PDT) agents as photoacoustic contrast agents. Photoacoustic imaging of photosensitizers exhibits advantages over fluorescence imaging, which is prone to photobleaching and autofluorescence interference. In this work, we examined the photoacoustic activity of 5 photosensitizers: zinc phthalocyanine, protoporphyrin IX, 2,4-bis [4-(N,N-dibenzylamino)-2,6-dihydroxyphenyl] squaraine, chlorin e6 and methylene blue in phantoms, among which zinc phthalocyanine showed the highest photoacoustic activity. Subsequently, we evaluated its tumor localization efficiency and biodistribution at multiple time points in a murine model using photoacoustic imaging. We observed that the probe localized at the tumor within 10 minutes post injection, reaching peak accumulation around 1 hour and was cleared within 24 hours, thus, demonstrating the potential of photosensitizers as photoacoustic imaging contrast agents in vivo. This means that the known advantages of photosensitizers such as preferential tumor uptake and PDT efficacy can be combined with photoacoustic imaging capabilities to achieve longitudinal monitoring of cancer progression and therapy in vivo.