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Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.
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Optoacoustic (OA) imaging offers powerful capabilities for interrogating biological tissues with rich optical absorption contrast while maintaining high spatial resolution for deep tissue observations. The spectrally distinct absorption of visible and near-infrared photons by endogenous tissue chromophores facilitates extraction of diverse anatomic, functional, molecular, and metabolic information from living tissues across various scales, from organelles and cells to whole organs and organisms. The primarily blood-related contrast and limited penetration depth of OA imaging have fostered the development of multimodal approaches to fully exploit the unique advantages and complementarity of the method. We review the recent hybridization efforts, including multimodal combinations of OA with ultrasound, fluorescence, optical coherence tomography, Raman scattering microscopy and magnetic resonance imaging as well as ionizing methods, such as X-ray computed tomography, single-photon-emission computed tomography and positron emission tomography. Considering that most molecules absorb light across a broad range of the electromagnetic spectrum, the OA interrogations can be extended to a large number of exogenously administered small molecules, particulate agents, and genetically encoded labels. This unique property further makes contrast moieties used in other imaging modalities amenable for OA sensing.
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Meios de Contraste , Técnicas Fotoacústicas , Técnicas Fotoacústicas/métodos , Humanos , Meios de Contraste/química , Animais , Imagem Multimodal/métodos , Imageamento por Ressonância Magnética/métodosRESUMO
Optoacoustic (OA) imaging has achieved tremendous progress with state-of-the-art systems providing excellent functional and molecular contrast, centimeter scale penetration into living tissues, and ultrafast imaging performance, making it highly suitable for handheld imaging in the clinics. OA can greatly benefit from efficient integration with ultrasound (US) imaging, which remains the routine method in bedside clinical diagnostics. However, such integration has not been straightforward since the two modalities typically involve different image acquisition strategies. Here, we present a new, to our knowledge, hybrid optoacoustic ultrasound (OPUS) imaging approach employing a spherical array with dedicated segments for each modality to enable volumetric OA imaging merged with conventional B-mode US. The system performance is subsequently showcased in healthy human subjects. The new OPUS approach hence represents an important step toward establishing OA in point-of-care diagnostic settings.
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Técnicas Fotoacústicas , Humanos , Técnicas Fotoacústicas/métodos , Ultrassonografia/métodos , Diagnóstico por Imagem , Voluntários SaudáveisRESUMO
Noninvasive monitoring of cardiac development can potentially prevent cardiac anomalies in adulthood. Mouse models provide unique opportunities to study cardiac development and disease in mammals. However, high-resolution noninvasive functional analyses of murine embryonic cardiac models are challenging because of the small size and fast volumetric motion of the embryonic heart, which is deeply embedded inside the uterus. In this study, a real time volumetric optoacoustic spectroscopy (VOS) platform for whole-heart visualization with high spatial (100 µm) and temporal (10 ms) resolutions is developed. Embryonic heart development on gestational days (GDs) 14.5-17.5 and quantify cardiac dynamics using time-lapse-4D image data of the heart is followed. Additionally, spectroscopic recordings enable the quantification of the blood oxygenation status in heart chambers in a label-free and noninvasive manner. This technology introduces new possibilities for high-resolution quantification of embryonic heart function at different gestational stages in mammalian models, offering an invaluable noninvasive method for developmental biology.
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Coração , Técnicas Fotoacústicas , Animais , Camundongos , Técnicas Fotoacústicas/métodos , Coração/embriologia , Coração/diagnóstico por imagem , Análise Espectral/métodos , Feminino , GravidezRESUMO
Tendon injuries resulting from accidents and aging are increasing globally. However, key tendon functional parameters such as microvascularity and oxygen perfusion remain inaccessible via the currently available clinical diagnostic tools, resulting in disagreements on optimal treatment options. Here, a new noninvasive method for anatomical and functional characterization of human tendons based on multispectral optoacoustic tomography (MSOT) is reported. Healthy subjects are investigated using a hand-held scanner delivering real-time volumetric images. Tendons in the wrist, ankle, and lower leg are imaged in the near-infrared optical spectrum to utilize endogenous contrast from Type I collagen. Morphology of the flexor carpi ulnaris, carpi radialis, palmaris longus, and Achilles tendons are reconstructed in full. The functional roles of the flexor digitorium longus, hallicus longus, and the tibialis posterior tendons have been visualized by dynamic tracking during toe extension-flexion motion. Furthermore, major vessels and microvasculature near the Achilles tendon are localized, and the global increase in oxygen saturation in response to targeted exercise is confirmed by perfusion studies. MSOT is shown to be a versatile tool capable of anatomical and functional tendon assessments. Future studies including abnormal subjects can validate the method as a viable noninvasive clinical tool for tendinopathy management and healing monitoring.
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Técnicas Fotoacústicas , Tendões , Humanos , Técnicas Fotoacústicas/métodos , Tendões/diagnóstico por imagem , Adulto , Masculino , Tomografia/métodos , Feminino , Traumatismos dos Tendões/diagnóstico por imagemRESUMO
Monitoring brain responses to ultrasonic interventions is becoming an important pillar of a growing number of applications employing acoustic waves to actuate and cure the brain. Optical interrogation of living tissues provides a unique means for retrieving functional and molecular information related to brain activity and disease-specific biomarkers. The hybrid optoacoustic imaging methods have further enabled deep-tissue imaging with optical contrast at high spatial and temporal resolution. The marriage between light and sound thus brings together the highly complementary advantages of both modalities toward high precision interrogation, stimulation, and therapy of the brain with strong impact in the fields of ultrasound neuromodulation, gene and drug delivery, or noninvasive treatments of neurological and neurodegenerative disorders. In this review, we elaborate on current advances in optical and optoacoustic monitoring of ultrasound interventions. We describe the main principles and mechanisms underlying each method before diving into the corresponding biomedical applications. We identify areas of improvement as well as promising approaches with clinical translation potential.
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Encéfalo , Diagnóstico por Imagem , Humanos , Ultrassonografia , Encéfalo/diagnóstico por imagemRESUMO
Major biological discoveries are made by interrogating living organisms with light. However, the limited penetration of un-scattered photons within biological tissues limits the depth range covered by optical methods. Deep-tissue imaging is achieved by combining light and ultrasound. Optoacoustic imaging exploits the optical generation of ultrasound to render high-resolution images at depths unattainable with optical microscopy. Recently, laser ultrasound has been suggested as a means of generating broadband acoustic waves for high-resolution pulse-echo ultrasound imaging. Herein, an approach is proposed to simultaneously interrogate biological tissues with light and ultrasound based on layer-by-layer coating of silica optical fibers with a controlled degree of transparency. The time separation between optoacoustic and ultrasound signals collected with a custom-made spherical array transducer is exploited for simultaneous 3D optoacoustic and laser ultrasound (OPLUS) imaging with a single laser pulse. OPLUS is shown to enable large-scale anatomical characterization of tissues along with functional multi-spectral imaging of chromophores and assessment of cardiac dynamics at ultrafast rates only limited by the pulse repetition frequency of the laser. The suggested approach provides a flexible and scalable means for developing a new generation of systems synergistically combining the powerful capabilities of optoacoustics and ultrasound imaging in biology and medicine.
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Lasers , Microscopia , UltrassonografiaRESUMO
Optoacoustic (OA) imaging is based on optical excitation of biological tissues with nanosecond-duration laser pulses and detection of ultrasound (US) waves generated by thermoelastic expansion following light absorption. The image quality and fidelity of OA images critically depend on the extent of tomographic coverage provided by the US detector arrays. However, full tomographic coverage is not always possible due to experimental constraints. One major challenge concerns an efficient integration between OA and pulse-echo US measurements using the same transducer array. A common approach toward the hybridization consists in using standard linear transducer arrays, which readily results in arc-type artifacts and distorted shapes in OA images due to the limited angular coverage. Deep learning methods have been proposed to mitigate limited-view artifacts in OA reconstructions by mapping artifactual to artifact-free (ground truth) images. However, acquisition of ground truth data with full angular coverage is not always possible, particularly when using handheld probes in a clinical setting. Deep learning methods operating in the image domain are then commonly based on networks trained on simulated data. This approach is yet incapable of transferring the learned features between two domains, which results in poor performance on experimental data. Here, we propose a signal domain adaptation network (SDAN) consisting of i) a domain adaptation network to reduce the domain gap between simulated and experimental signals and ii) a sides prediction network to complement the missing signals in limited-view OA datasets acquired from a human forearm by means of a handheld linear transducer array. The proposed method showed improved performance in reducing limited-view artifacts without the need for ground truth signals from full tomographic acquisitions.
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Tomografia Computadorizada por Raios X , Tomografia , Humanos , Tomografia/métodos , Ultrassonografia/métodos , Artefatos , Transdutores , Processamento de Imagem Assistida por Computador/métodos , Imagens de FantasmasRESUMO
Non-alcoholic fatty liver disease (NAFLD) is an umbrella term referring to a group of conditions associated to fat deposition and damage of liver tissue. Early detection of fat accumulation is essential to avoid progression of NAFLD to serious pathological stages such as liver cirrhosis and hepatocellular carcinoma. Methods: We exploited the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS), which combines the advantages of optical and acoustic contrasts, for an early-stage multi-parametric assessment of NAFLD in mice. Results: The multispectral optoacoustic imaging allowed for spectroscopic differentiation of lipid content, as well as the bio-distributions of oxygenated and deoxygenated hemoglobin in liver tissues in vivo. The pulse-echo (reflection) ultrasound (US) imaging further provided a valuable anatomical reference whilst transmission US facilitated the mapping of speed of sound changes in lipid-rich regions, which was consistent with the presence of macrovesicular hepatic steatosis in the NAFLD livers examined with ex vivo histological staining. Conclusion: The proposed multimodal approach facilitates quantification of liver abnormalities at early stages using a variety of optical and acoustic contrasts, laying the ground for translating the TROPUS approach toward diagnosis and monitoring NAFLD in patients.
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Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/patologia , Fígado/diagnóstico por imagem , Fígado/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , LipídeosRESUMO
Emerging evidence indicates crosstalk between the brain and hematopoietic system following cerebral ischemia. Here, we investigated metabolism and oxygenation in the spleen and spinal cord in a transient middle cerebral artery occlusion (tMCAO) model. Sham-operated and tMCAO mice underwent [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to assess glucose metabolism. Naïve, sham-operated and tMCAO mice underwent multispectral optoacoustic tomography (MSOT) assisted by quantitative model-based reconstruction and unmixing algorithms for accurate mapping of oxygenation patterns in peripheral tissues at 24 h after reperfusion. We found increased [18F]FDG uptake and reduced MSOT oxygen saturation, indicating hypoxia in the thoracic spinal cord of tMCAO mice compared with sham-operated mice but not in the spleen. Reduced spleen size was observed in tMCAO mice compared with sham-operated mice ex vivo. tMCAO led to an increase in the numbers of mature T cells in femoral bone marrow tissues, concomitant with a stark reduction in these cell subsets in the spleen and peripheral blood. The combination of quantitative PET and MSOT thus enabled observation of hypoxia and increased metabolic activity in the spinal cord of tMCAO mice at 24 h after occlusion compared to sham-operated mice.
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Background: Abnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. Methods: Fluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. Results: THK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinson's disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. Conclusion: We demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.
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Optoacoustic tomography (OAT) provides a non-invasive means to characterize cerebral hemodynamics across an entire murine brain while attaining multi-parametric readouts not available with other modalities. This unique capability can massively impact our understanding of brain function. However, OAT largely lacks the soft tissue contrast required for unambiguous identification of brain regions. Hence, its accurate registration to a reference brain atlas is paramount for attaining meaningful functional readings. Herein, we capitalized on the simultaneously acquired bi-modal data from the recently-developed hybrid magnetic resonance optoacoustic tomography (MROT) scanner in order to devise an image coregistration paradigm that facilitates brain parcellation and anatomical referencing. We evaluated the performance of the proposed methodology by coregistering OAT data acquired with a standalone system using different registration methods. The enhanced performance is further demonstrated for functional OAT data analysis and characterization of stimulus-evoked brain responses. The suggested approach enables better consolidation of the research findings thus facilitating wider acceptance of OAT as a powerful neuroimaging tool to study brain functions and diseases.
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Optoacoustic tomography is commonly performed with bulky and expensive short-pulsed solid-state lasers providing high per-pulse energies in the millijoule range. Light emitting diodes (LEDs) represent a cost-effective and portable alternative for optoacoustic signal excitation that can additionally provide excellent pulse-to-pulse stability. Herein, we introduce a full-view LED-based optoacoustic tomography (FLOAT) system for deep tissue in vivo imaging. It is based on a custom-made electronic unit driving a stacked array of LEDs, which attains 100 ns pulse width and highly stable (0.62 % standard deviation) total per-pulse energy of 0.48 mJ. The illumination source is integrated into a circular array of cylindrically-focused ultrasound detection elements to result in a full-view tomographic configuration, which plays a critical role in circumventing limited-view effects, enhancing the effective field-of-view and image quality for cross-sectional (2D) imaging. We characterized the FLOAT performance in terms of pulse width, power stability, excitation light distribution, signal-to-noise and penetration depth. FLOAT of the human finger revealed a comparable imaging performance to that achieved with the standard pulsed Nd:YAG laser. It is anticipated that this compact, affordable and versatile illumination technology will facilitate optoacoustic imaging developments in resource-limited settings for biological and clinical applications.
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Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed.
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AVC Isquêmico , Técnicas Fotoacústicas , Camundongos , Animais , Técnicas Fotoacústicas/métodos , Angiografia , Microvasos , Acústica , MamíferosRESUMO
Fast tracking of biological dynamics across multiple murine organs using the currently commercially available whole-body preclinical imaging systems is hindered by their limited contrast, sensitivity and spatial or temporal resolution. Spiral volumetric optoacoustic tomography (SVOT) provides optical contrast, with an unprecedented level of spatial and temporal resolution, by rapidly scanning a mouse using spherical arrays, thus overcoming the current limitations in whole-body imaging. The method enables the visualization of deep-seated structures in living mammalian tissues in the near-infrared spectral window, while further providing unrivalled image quality and rich spectroscopic optical contrast. Here, we describe the detailed procedures for SVOT imaging of mice and provide specific details on how to implement a SVOT system, including component selection, system arrangement and alignment, as well as the image processing methods. The step-by-step guide for the rapid panoramic (360°) head-to-tail whole-body imaging of a mouse includes the rapid visualization of contrast agent perfusion and biodistribution. The isotropic spatial resolution possible with SVOT can reach 90 µm in 3D, while alternative steps enable whole-body scans in less than 2 s, unattainable with other preclinical imaging modalities. The method further allows the real-time (100 frames per second) imaging of biodynamics at the whole-organ level. The multiscale imaging capacity provided by SVOT can be used for visualizing rapid biodynamics, monitoring responses to treatments and stimuli, tracking perfusion, and quantifying total body accumulation and clearance dynamics of molecular agents and drugs. Depending on the imaging procedure, the protocol requires 1-2 h to complete by users trained in animal handling and biomedical imaging.
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Processamento de Imagem Assistida por Computador , Técnicas Fotoacústicas , Tomografia Computadorizada de Feixe Cônico Espiral , Animais , Camundongos , Perfusão , Técnicas Fotoacústicas/métodos , Distribuição Tecidual , Tomografia Computadorizada de Feixe Cônico Espiral/métodos , Modelos Animais de Doenças , Meios de ContrasteRESUMO
High-intensity focused ultrasound (HIFU) enables localized ablation of biological tissues by capitalizing on the synergistic effects of heating and cavitation. Monitoring of those effects is essential for improving the efficacy and safety of HIFU interventions. Herein, we suggest a hybrid optoacoustic-ultrasound (OPUS) approach for real-time assessment of heating and cavitation processes while providing an essential anatomical reference for accurate localization of the HIFU-induced lesion. Both effects could clearly be observed by exploiting the temperature dependence of optoacoustic (OA) signals and the strong contrast of gas bubbles in pulse-echo ultrasound (US) images. The differences in temperature increase and its rate, as recorded with a thermal camera for different HIFU pressures, evinced the onset of cavitation at the expected pressure threshold. The estimated temperatures based on OA signal variations were also within 10-20 % agreement with the camera readings for temperatures below the coagulation threshold (â¼50 °C). Experiments performed in excised tissues as well as in a post-mortem mouse demonstrate that both heating and cavitation effects can be effectively visualized and tracked using the OPUS approach. The good sensitivity of the suggested method for HIFU monitoring purposes was manifested by a significant increase in contrast-to-noise ratio within the ablated region by > 10 dB and > 5 dB for the OA and US images, respectively. The hybrid OPUS-based monitoring approach offers the ease of handheld operation thus can readily be implemented in a bedside setting to benefit several types of HIFU treatments used in the clinics.
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Optoacoustic (OA, photoacoustic) imaging synergistically combines rich optical contrast with the resolution of ultrasound within light-scattering biological tissues. Contrast agents have become essential to boost deep-tissue OA sensitivity and fully exploit the capabilities of state-of-the-art OA imaging systems, thus facilitating the clinical translation of this modality. Inorganic particles with sizes of several microns can also be individually localized and tracked, thus enabling new applications in drug delivery, microrobotics, or super-resolution imaging. However, significant concerns have been raised regarding the low bio-degradability and potential toxic effects of inorganic particles. Bio-based, biodegradable nano- and microcapsules consisting of an aqueous core with clinically-approved indocyanine green (ICG) and a cross-linked casein shell obtained in an inverse emulsion approach are introduced. The feasibility to provide contrast-enhanced in vivo OA imaging with nanocapsules as well as localizing and tracking individual larger microcapsules of 4-5 µm is demonstrated. All components of the developed capsules are safe for human use and the inverse emulsion approach is known to be compatible with a variety of shell materials and payloads. Hence, the enhanced OA imaging performance can be exploited in multiple biomedical studies and can open a route to clinical approval of agents detectable at a single-particle level.
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Verde de Indocianina , Nanocápsulas , Humanos , Cápsulas , Emulsões , Verde de Indocianina/farmacologiaRESUMO
Optoacoustic tomography has been established as a powerful modality for preclinical imaging. However, efficient whole-body imaging coverage has not been achieved owing to the arduous requirement for continuous acoustic coupling around the animal. In this work, we introduce panoramic (3600) head-to-tail 3D imaging of mice with spiral volumetric optoacoustic tomography (SVOT). The system combines multi-beam illumination and a dedicated head holder enabling uninterrupted acoustic coupling for whole-body scans. Image fidelity is optimized with self-gated respiratory motion rejection and dual speed-of-sound reconstruction algorithms to attain spatial resolution down to 90 µm. The developed system is thus highly suitable for visualizing rapid biodynamics across scales, such as hemodynamic changes in individual organs, responses to treatments and stimuli, perfusion, total body accumulation, or clearance of molecular agents and drugs with unmatched contrast, spatial and temporal resolution.
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Progress in brain research critically depends on the development of next-generation multi-modal imaging tools capable of capturing transient functional events and multiplexed contrasts noninvasively and concurrently, thus enabling a holistic view of dynamic events in vivo. Here we report on a hybrid magnetic resonance and optoacoustic tomography (MROT) system for murine brain imaging, which incorporates an MR-compatible spherical matrix array transducer and fiber-based light illumination into a 9.4â T small animal scanner. An optimized radiofrequency coil has further been devised for whole-brain interrogation. System's utility is showcased by acquiring complementary angiographic and soft tissue anatomical contrast along with simultaneous dual-modality visualization of contrast agent dynamics in vivo.
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Imaging modalities capable of visualizing the human brain have led to major advances in neurology and brain research. Multi-spectral optoacoustic tomography (MSOT) has gained importance for studying cerebral function in rodent models due to its unique capability to map changes in multiple hemodynamic parameters and to directly visualize neural activity within the brain. The technique further provides molecular imaging capabilities that can facilitate early disease diagnosis and treatment monitoring. However, transcranial imaging of the human brain is hampered by acoustic attenuation and other distortions introduced by the skull. Here, we demonstrate non-invasive transcranial MSOT angiography of pial veins through the temporal bone of an adult healthy volunteer. Time-of-flight (TOF) magnetic resonance angiography (MRA) and T1-weighted structural magnetic resonance imaging (MRI) were further acquired to facilitate anatomical registration and interpretation. The superior middle cerebral vein in the temporal cortex was identified in the MSOT images, matching its location observed in the TOF-MRA images. These initial results pave the way toward the application of MSOT in clinical brain imaging.