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Efficient drug delivery remains a significant challenge in modern medicine and pharmaceutical research. Micrometer-scale robots have recently emerged as a promising solution to enhance the precision of drug administration through remotely controlled navigation within microvascular networks. Real-time tracking is crucial for accurate guidance and confirmation of target arrival. However, deep-tissue monitoring of microscopic structures in vivo is limited by the sensitivity and spatiotemporal resolution of current bioimaging techniques. In this study, biocompatible microrobots are synthesized by incorporating indocyanine green and iron oxide nanoparticles onto copper phosphate microflowers using a layer-by-layer approach, enhancing optoacoustic contrast and enabling magnetic navigation. Magnetic control of these particles under optoacoustic guidance is demonstrated in vivo. Furthermore, super-resolution optoacoustic imaging, achieved through individual particle tracking, is shown to enable the characterization of microvascular structures and quantification of blood flow. The combination of the microflowers' high carrying capacity, in vivo actuation, and high-resolution tracking capabilities opens new opportunities for precise microvascular targeting and localized administration of theranostic agents via intravascular routes., (© 2025 Wiley‐VCH GmbH.)

Introduction: Transcranial pulse stimulation (TPS) is increasingly being investigated as a promising potential treatment for Alzheimer's disease (AD). Although the safety and preliminary clinical efficacy of TPS short pulses have been supported by neuropsychological scores in treated AD patients, its fundamental mechanisms are uncharted., Methods: Herein, we used a multi-modal preclinical imaging platform combining real-time volumetric optoacoustic tomography, contrast-enhanced magnetic resonance imaging, and ex vivo immunofluorescence to comprehensively analyze structural and hemodynamic effects induced by TPS. Cohorts of healthy and AD transgenic mice were imaged during and after TPS exposure at various per-pulse energy levels., Results: TPS enhanced the microvascular network, whereas the blood-brain barrier remained intact following the procedure. Notably, higher pulse energies were necessary to induce hemodynamic changes in AD mice, arguably due to their impacted vessels., Discussion: These findings shed light on cerebrovascular dynamics induced by TPS treatment, and hence are expected to assist improving safety and therapeutic outcomes., Highlights: ·Transcranial pulse stimulation (TPS) facilitates transcranial wave propagation using short pulses to avoid tissue heating. ·Preclinical multi-modal imaging combines real-time volumetric optoacoustic (OA) tomography, contrast-enhanced magnetic resonance imaging (CE-MRI), and ex vivo immunofluorescence to comprehensively analyze structural and hemodynamic effects induced by TPS. ·Blood volume enhancement in microvascular networks was reproducibly observed with real-time OA imaging during TPS stimulation. ·CE-MRI and gross pathology further confirmed that the brain architecture was maintained intact without blood-brain barrier (BBB) opening after TPS exposure, thus validating the safety of the procedure. ·Higher pulse energies were necessary to induce hemodynamic changes in AD compared to wild-type animals, arguably due to their pathological vessels., (© 2025 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Optoacoustic imaging offers label-free multi-parametric characterization of cerebrovascular morphology and hemodynamics at depths and spatiotemporal resolution unattainable with optical microscopy. Effective imaging depth can greatly be enhanced by employing photons in the second near-infrared (NIR-II) window. However, diminished absorption by hemoglobin along with a lack of suitable contrast agents hinder an efficient application of the technique in this spectral range. Herein, copper sulfide (CuS) micro- and nano-formulations for multi-scale optoacoustic imaging in the NIR-II window are introduced. Dynamic contrast enhancement induced by intravenously administered CuS nanoparticles facilitated visualization of blood perfusion in murine cerebrovascular networks. The individual calcium carbonate microparticles carrying CuS are further shown to generate sufficient responses to enable super-resolution microvascular imaging and blood flow velocity mapping with localization optoacoustic tomography., (© 2024 The Author(s). Small Methods published by Wiley‐VCH GmbH.)

Localization optoacoustic tomography (LOT) has recently emerged as a transformative super-resolution technique breaking through the acoustic diffraction limit in deep-tissue optoacoustic (OA) imaging via individual localization and tracking of particles in the bloodstream. However, strong light absorption in red blood cells has previously restricted per-particle OA detection to relatively large microparticles, ≈5 µm in diameter. Herein, it is demonstrated that submicron-sized porous gold nanoparticles, ≈600 nm in diameter, can be individually detected for noninvasive super-resolution imaging with LOT. Ultra-high-speed bright-field microscopy revealed that these nanoparticles generate microscopic plasmonic vapor bubbles, significantly enhancing opto-acoustic energy conversion through a nano-to-micro size transformation. Comprehensive in vitro and in vivo tests further demonstrated the biocompatibility and biosafety of the particles. By reducing the detectable particle size by an order of magnitude, nanoLOT enables microangiographic imaging with a significantly reduced risk of embolisms from particle aggregation and opens new avenues to visualize how nanoparticles reach vascular and potentially extravascular targets. The performance of nanoLOT for non-invasive imaging of microvascular networks in the murine brain anticipates new insights into neurovascular coupling mechanisms and longitudinal microcirculatory changes associated with neurodegenerative diseases., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

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., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Small extracellular vesicles (sEVs) have attracted tremendous interest in recent years due to their exceptional properties for therapeutic and diagnostic applications. Although much research was focused on the quantity and content of sEVs, less efforts have been put into discovering the interaction between sEVs and cells. Here we engineered multicompartment particles, termed vesicosomes, by deposition of sEVs derived from MCF7, CHO cells and human plasma onto the surface of polyelectrolyte (PE)-coated silica (SiO 2 ) microparticles. Uptake of the PE-coated SiO 2 microparticles by parent cells was significantly enhanced by coating them with sEVs, compared to PE-coated SiO 2 microparticles independent of the terminated polyelectrolyte layer. This study highlights the emerging role of sEVs membrane receptors in the sEV-cells interaction and demonstrates the potential application of sEV-like multicompartment particles as therapeutic carriers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no relevant financial or non-financial competing interests to report., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Detailed characterization of microvascular alterations requires high-resolution 3D imaging methods capable of providing both morphological and functional information. Existing optical microscopy tools are routinely used for microangiography, yet offer suboptimal trade-offs between the achievable field of view and spatial resolution with the intense light scattering in biological tissues further limiting the achievable penetration depth. Herein, a new approach for volumetric deep-tissue microangiography based on stereovision combined with super-resolution localization imaging is introduced that overcomes the spatial resolution limits imposed by light diffusion and optical diffraction in wide-field imaging configurations. The method capitalizes on localization and tracking of flowing fluorescent particles in the second near-infrared window (NIR-II, ≈1000-1700 nm), with the third (depth) dimension added by triangulation and stereo-matching of images acquired with two short-wave infrared cameras operating in a dual-view mode. The 3D imaging capability enabled with the proposed method facilitates a detailed visualization of microvascular networks and an accurate blood flow quantification. Experiments performed in tissue-mimicking phantoms demonstrate that high resolution is preserved up to a depth of 4 mm in a turbid medium. Transcranial microangiography of the entire murine cortex and penetrating vessels is further demonstrated at capillary level resolution., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Rapid volumetric in vivo visualization of circulating microparticles can facilitate new biomedical applications, such as blood flow characterization or targeted drug delivery. However, existing imaging modalities generally lack the sensitivity to detect the weak signals generated by individual micrometer-sized particles distributed across millimeter- to centimeter-scale depths in living mammalian tissues. Also, the temporal resolution is typically insufficient to track the particles in an entire three-dimensional region. Herein, we introduce a new type of monodisperse (4 μm) silica-core microparticle coated with a shell formed by a multilayered structure of carbon nanotubes (CNT) and gold nanoparticles (AuNP) to provide strong optoacoustic (OA) absorption-based contrast. We capitalize on the unique advantages of a state-of-the-art high-frame-rate OA tomography system to visualize and track the motion of these core-shell particles individually and volumetrically as they flow throughout the mouse brain vasculature. The feasibility of localizing individual solid particles smaller than red blood cells opens new opportunities for mapping the blood flow velocity, enhancing the resolution and visibility of OA images, and developing new biosensing assays.

Extrinsically administered light-absorbing agents may greatly enhance the sensitivity and imaging performance of optoacoustic tomography (OAT). Beyond the use of targeted contrast agents in functional and molecular imaging applications, tracking of highly absorbing microparticles has recently been shown to facilitate super-resolution volumetric angiography and mapping of blood flow. However, in vivo characterization of new types of microparticulate absorbing agents is often hindered due to their potential toxicity, incompatible dimensions, or sub-optimal extinction spectrum shadowed by strong background absorption of hemoglobin. Herein, we used an intracardiac perfusion mouse model to individually track the perfusion of absorbing particles through the cerebral vasculature by acquiring a sequence of high-frame-rate 3D OAT images. The particles were injected in the left ventricle of the heart after substitution of blood by an artificial cerebrospinal fluid post mortem, which has further contributed to minimizing the background OAT signals induced by hemoglobin absorption. The presented approach can greatly aid the development of new microparticulate contrast agents with optimized performance for various OAT imaging applications.

There has been growing interest in recent years in developing multifunctional materials for studying the structure interface in biological systems. In this regard, the multimodal systems, which possess activity in the near-infrared (NIR) region, become even more critical for the possibility of improving examined biotissue depth and, eventually, data analysis. Herein, we engineered bi-modal contrast agents by integrating carbon nanotubes (CNT) and gold nanoparticles (AuNP) around silica microspheres using the Layer-by-Layer self-assembly method. The experimental studies revealed that microspheres with CNT sandwiched between AuNP exhibit strong absorption in the visible and NIR regions and high optoacoustic contrast (OA, also called photoacoustics) and Raman scattering when illuminated with 532 nm and 785 nm lasers, respectively. The developed microspheres demonstrated amplification of the signal in the OA flow cytometry at the laser wavelength of 1064 nm. This finding was further validated with ex vivo brain tissue using a portable Raman spectrometer and imaging with the Raster-scanning OA mesoscopy technique. The obtained data suggest that the developed contrast agents can be promising in applications of localization OA tomography (LOT), OA flow cytometry, and multiplex SERS detection., (Copyright © 2020. Published by Elsevier B.V.)