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Circulating tumor cells (CTCs) have emerged as promising circulating biomarkers for non-invasive cancer diagnosis and management. Isolation and detection of CTCs in clinical samples are challenging due to the extreme rarity and high heterogeneity of CTCs. Here, we describe a poly(ethylene oxide) (PEO) concentration gradient-based microfluidic method for rapid, label-free, highly efficient isolation of CTCs directly from whole blood samples. Stable concentration gradients of PEO were formed within the microchannel by co-injecting the side fluid (blood sample spiked with 0.025% PEO) and center fluid (0.075% PEO solution). The competition between the elastic lift force and the inertial lift force enabled size-based separation of large CTCs and small blood cells based on their distinct migration patterns. The microfluidic device could process 1 mL of blood sample in 30 min, with a separation efficiency of >90% and an enrichment ratio of >700 for tumor cells. The isolated CTCs from blood samples were enumerated by immunofluorescence staining, allowing for discrimination of breast cancer patients from healthy donors with an accuracy of 84.2%. The concentration gradient-based microfluidic separation provides a powerful tool for label-free isolation of CTCs for a wide range of clinical applications.
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Neoplasias de la Mama , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Humanos , Femenino , Microfluídica , Óxido de Etileno , Separación Celular/métodos , Células Neoplásicas Circulantes/patología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Línea Celular TumoralRESUMEN
Metamaterial-assisted illumination nanoscopy (MAIN) has been proven to be a promising approach for super-resolution microscopy with up to a 7-fold improvement in imaging resolution. Further resolution enhancement is possible in principle, however, has not yet been demonstrated due to the lack of high-quality ultrathin layered hyperbolic metamaterials (HMMs) used in the MAIN. Here, we fabricate a low-loss composite HMM consisting of high-quality bilayers of Al-doped Ag and MgO with a nominal thickness of 2.5 nm, and then use it to demonstrate an ultrathin layered hyperbolic metamaterial-assisted illumination nanoscopy (ULH-MAIN) with a 14-fold imaging resolution improvement. This improvement of resolution is achieved in fluorescent beads super-resolution experiments and verified with scanning electron microscopy. The ULH-MAIN presents a simple super-resolution imaging approach that offers distinct benefits such as low illumination power, low cost, and a broad spectrum of selectable probes, making it ideal for dynamic imaging of life science samples.
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Iluminación , MicroscopíaRESUMEN
In this Letter, we propose a new quantitative phase imaging methodology named Fourier optical spin splitting microscopy (FOSSM). FOSSM relies on a metasurface located at the Fourier plane of a polarized microscope to separate the object image into two replicas of opposite circularly polarized states. The bias retardation between the two replicas is tuned by translating the metasurface or rotating the analyzer. Combined with a polarized camera, FOSSM can easily achieve single-shot quantitative phase gradient imaging, which greatly reduces the complexity of current phase microscope setups, paving the way for the next generation high-speed real-time multifunctional microscopy.
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Microscopía , Microscopía/métodosRESUMEN
Optical edge detection is a useful method for characterizing boundaries, which is also in the forefront of image processing for object detection. As the field of metamaterials and metasurface is growing fast in an effort to miniaturize optical devices at unprecedented scales, experimental realization of optical edge detection with metamaterials remains a challenge and lags behind theoretical proposals. Here, we propose a mechanism of edge detection based on a Pancharatnam-Berry-phase metasurface. We experimentally demonstrated broadband edge detection using designed dielectric metasurfaces with high optical efficiency. The metasurfaces were fabricated by scanning a focused laser beam inside glass substrate and can be easily integrated with traditional optical components. The proposed edge-detection mechanism may find important applications in image processing, high-contrast microscopy, and real-time object detection on compact optical platforms such as mobile phones and smart cameras.
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Determining the axial position of an emitter with nanoscale precision is critical to a fundamental imaging methodology. While there are many advanced optical techniques being applied to high-resolution imaging, high-axial-resolution topography imaging of living cells is particularly challenging. Here, we present an application of metamaterial-assisted photobleaching microscopy (MAPM) with high-axial resolution to characterize morphological properties of living cells. Quantitative imaging of changes in the morphology of live cells is obtained by topographic and statistical analysis. The time-lapse topography image using the metamaterial-induced photostability provides information about growth factor induced changes in the cell morphology with high-axial resolution.
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Fotoblanqueo , Microscopía FluorescenteRESUMEN
The past two decades have witnessed a dramatic progress in the development of novel super-resolution fluorescence microscopy technologies. Here, we report a new fluorescence imaging method, called metamaterial-assisted photobleaching microscopy (MAPM), which possesses a nanometer-scale axial resolution and is suitable for broadband operation across the entire visible spectrum. The photobleaching kinetics of fluorophores can be greatly modified via a separation-dependent energy transfer process to a nearby metamaterial. The corresponding photobleaching rate is thus linked to the distance between the fluorophores and the metamaterial layer, leading to a reconstructed image with exceptionally high axial resolution. We apply the MAPM technology to image the HeLa cell membranes tagged with fluorescent proteins and demonstrate an axial resolution of â¼2.4 nm with multiple colors. MAPM utilizes a metamaterial-coated substrate to achieve super-resolution without altering anything else in a conventional microscope, representing a simple solution for fluorescence imaging at nanometer axial resolution.
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Exosomal microRNAs (miRNAs) are reliable and noninvasive biomarkers for the early diagnosis of cancer. Yet, accurate and feasible detection of exosomal miRNAs is often hampered by the low abundance of miRNAs in exosomes and the requirement for RNA extraction in large sample volumes. Here we show a thermophoretic sensor implemented with nanoflares for in situ detection of exosomal miRNAs, without resorting to either RNA extraction or target amplification. Thermophoretic accumulation of nanoflare-treated exosomes leads to an amplified fluorescence signal upon the binding of exosomal miRNAs to nanoflares, allowing for direct and quantitative measurement of exosomal miRNAs down to 0.36 fM in 0.5 µL serum samples. One of the best markers, exosomal miR-375, showed an accuracy of 85% for detection of estrogen receptor-positive breast cancer at early stages (stages I, II). This work provides a feasible tool to improve the diagnosis of cancer.
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Biomarcadores de Tumor/sangre , Exosomas/química , Nanopartículas del Metal/química , MicroARNs/sangre , Espectrometría de Fluorescencia/métodos , Adulto , Anciano , Biomarcadores de Tumor/genética , Neoplasias de la Mama/sangre , Carbocianinas/química , Línea Celular Tumoral , ADN/química , ADN/genética , Colorantes Fluorescentes/química , Oro/química , Oro/efectos de la radiación , Humanos , Rayos Infrarrojos , Nanopartículas del Metal/efectos de la radiación , MicroARNs/genética , Persona de Mediana Edad , Hibridación de Ácido Nucleico , TemperaturaRESUMEN
Extracellular vesicles (EVs) are heavily implicated in diverse pathological processes. Due to their small size, distinct biogenesis, and heterogeneous marker expression, isolation and detection of single EV subpopulations are difficult. Here, we develop a λ-DNA- and aptamer-mediated approach allowing for simultaneous size-selective separation and surface protein analysis of individual EVs. Using a machine learning algorithm to EV signature based on their size and marker expression, we demonstrate that the isolated microvesicles are more efficient than exosomes and apoptotic bodies in discriminating breast cell lines and Stage II breast cancer patients with varied immunohistochemical expression of HER2. Our method provides an important tool to assess the EV heterogeneity at the single EV level with potential value in clinical diagnostics.
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Aptámeros de Nucleótidos/química , ADN/química , Vesículas Extracelulares/química , Humanos , Receptor ErbB-2/químicaRESUMEN
By means of density functional theory calculations, we successfully predict two stable 2D triangular borophenes, namely B3H and B6O. Our results indicate that B3H is a Dirac material and its cone point is located at the K point of the Brillouin zone (BZ). B6O is identified as having a node-line ring and Dirac cones together. Its node-line ring formed by the intersection of the extended energy band from the two Dirac cones located on K point. This modified 2D borophene has great thermal and dynamic stability due to the electron transfer from the triangular boron lattice to the O atoms. The electronic structure of B6O nanofilm demonstrates novel properties such as two Dirac cones, more than 1.3 eV linear dispersion bands at some points of the BZ, as well as excellent transport properties for the extremely high mobility brought by the combination of the node-line semimetal and Dirac cones. Our study may motivate potential applications of 2D materials in nanoelectronics.
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Dark-field microscopy (DFM) is a powerful label-free and high-contrast imaging technique due to its ability to reveal features of transparent specimens with inhomogeneities. However, owing to the Abbe's diffraction limit, fine structures at sub-wavelength scale are difficult to resolve. In this work, we report a single image super resolution DFM scheme using a convolutional neural network (CNN). A U-net based CNN is trained with a dataset which is numerically simulated based on the forward physical model of the DFM. The forward physical model described by the parameters of the imaging setup connects the object ground truths and dark field images. With the trained network, we demonstrate super resolution dark field imaging of various test samples with twice resolution improvement. Our technique illustrates a promising deep learning approach to double the resolution of DFM without any hardware modification.
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Recent advancements in optical metamaterials have opened new possibilities in the exciting field of super-resolution microscopies. The far-field metamaterial-assisted illumination nanoscopies (MAINs) have, very recently, enhanced the lateral resolution to one-fifteenth of the optical wavelength. However, the axial localization accuracy of fluorophores in the MAINs remains rarely explored. Here, a MAIN with a nanometer-scale axial localization accuracy is demonstrated by monitoring the distance-dependent photobleaching dynamics of the fluorophores on top of an organic hyperbolic metamaterial (OHM) substrate under a wide-field single-objective microscope. With such a regular experimental configuration, 3D imaging of various biological samples with the resolution of ≈40 nm in the lateral dimensions and ≈5 nm in the axial dimension is realized. The demonstrated imaging modality enables the resolution of the 3D morphology of nanoscopic cellular structures with a significantly simplified experimental setup.
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Whether in the field of medical care, or in people's daily life and health protection, the importance of masks has been paid more and more attention. Acne, the most common complication after wearing masks, which is also called maskne, has been successfully introduced into the common language as a common topic of dermatologist consultations. This study aims to study the changes of microflora in maskne patients and healthy controls before and after wearing masks. In the summer of 2023, we collected a total of 50 samples from 15 maskne patients and 10 healthy controls before and after wearing surgical masks for a long time. 16 S ribosomal DNA sequencing and identification technology with V3-V4 variable region were adopted to explore the microbiome changes caused by mask wearing, analyze the changes in microbial diversity, and make interaction network. LDA effect size analysis was used to identify which bacteria showed significant changes in their relative abundance from phylum to genus. After wearing a mask, the microbiome of the maskne patients changed significantly more than that of the healthy controls, with both α diversity and ß diversity lower than those of maskne patients before wearing masks and those of healthy controls after wearing masks. Co-occurrence network analysis showed that compared with other groups, the network of maskne patients after wearing masks for a long time had the lowest connectivity and complexity, but the highest clustering property, while the opposite was true for healthy controls. Many microbes that are potentially beneficial to the skin decreased significantly after wearing a mask. There was almost no difference in healthy controls before and after wearing a mask.
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Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.
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Fluorescence super-resolution microscopy has, over the last two decades, been extensively developed to access deep-subwavelength nanoscales optically. Label-free super-resolution technologies however have only achieved a slight improvement compared to the diffraction limit. In this context, we demonstrate a label-free imaging method, i.e., hyperbolic material enhanced scattering (HMES) nanoscopy, which breaks the diffraction limit by tailoring the light-matter interaction between the specimens and a hyperbolic material substrate. By exciting the highly confined evanescent hyperbolic polariton modes with dark-field detection, HMES nanoscopy successfully shows a high-contrast scattering image with a spatial resolution around 80 nm. Considering the wavelength at 532 nm and detection optics with a 0.6 numerical aperture (NA) objective lens, this value represents a 5.5-fold resolution improvement beyond the diffraction limit. HMES provides capabilities for super-resolution imaging where fluorescence is not available or challenging to apply.
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Microscopía FluorescenteRESUMEN
Optical analog signal processing technology has been widely studied and applied in a variety of science and engineering fields, with the advantages of overcoming the low-speed and high-power consumption associated with its digital counterparts. Much attention has been given to emerging metasurface technology in the field of optical imaging and processing systems. Here, we demonstrate, for the first time, broadband two-dimensional spatial differentiation and high-contrast edge imaging based on a dielectric metasurface across the whole visible spectrum. This edge detection method works for both intensity and phase objects simply by inserting the metasurface into a commercial optical microscope. This highly efficient metasurface performing a basic optical differentiation operation opens up new opportunities in applications of fast, compactible and power-efficient ultrathin devices for data processing and biological imaging.
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The dynamics of photons in fluorescent molecules plays a key role in fluorescence imaging, optical sensing, organic photovoltaics, and displays. Photobleaching is an irreversible photodegradation process of fluorophores, representing a fundamental limitation in relevant optical applications. Chemical reagents are used to suppress the photobleaching rate but with exceptionally high specificity for each type of fluorophore. Here, using organic hyperbolic materials (OHMs), an optical platform to achieve unprecedented fluorophore photostability without any chemical specificity is demonstrated. A more than 500-fold lengthening of the photobleaching lifetime and a 230-fold increase in the total emitted photon counts are observed simultaneously. These exceptional improvements solely come from the low-loss hyperbolic dispersion of OHM films and the large resultant Purcell effect in the visible spectral range. The demonstrated OHM platform may open up a new paradigm in nanophotonics and organic plasmonics for super-resolution imaging and the engineering of light-matter interactions at the nanoscale.
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Resolution capability of the linear structured illumination microscopy (SIM) plays a key role in its applications in physics, medicine, biology, and life science. Many advanced methodologies have been developed to extend the resolution of structured illumination by using subdiffraction-limited optical excitation patterns. However, obtaining SIM images with a resolution beyond 40 nm at visible frequency remains as an insurmountable obstacle due to the intrinsic limitation of spatial frequency bandwidth of the involved materials and the complexity of the illumination system. Here, a low-loss natural organic hyperbolic material (OHM) that can support record high spatial-frequency modes beyond 50k0 , i.e., effective refractive index larger than 50, at visible frequencies is reported. OHM-based speckle structured illumination microscopy demonstrates imaging resolution at 30 nm scales with enhanced fluorophore photostability, biocompatibility, easy to use and low cost. This study will open up a new route in super-resolution microscopy by utilizing OHM films for various applications including bioimaging and sensing.
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Iluminación/instrumentación , Iluminación/métodos , Nanotecnología/instrumentación , Nanotecnología/métodos , Diseño de Equipo , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodosRESUMEN
Structured illumination microscopy (SIM) is one of the most powerful and versatile optical super-resolution techniques. Compared with other super-resolution methods, SIM has shown its unique advantages in wide-field imaging with high temporal resolution and low photon damage. However, traditional SIM only has about 2 times spatial resolution improvement compared to the diffraction limit. In this work, we propose and experimentally demonstrate an easily-implemented, low-cost method to extend the resolution of SIM, named speckle metamaterial-assisted illumination nanoscopy (speckle-MAIN). A metamaterial structure is introduced to generate speckle-like sub-diffraction-limit illumination patterns in the near field with improved spatial frequency. Such patterns, similar to traditional SIM, are then used to excite objects on top of the surface. We demonstrate that speckle-MAIN can bring the resolution down to 40 nm and beyond. Speckle-MAIN represents a new route for super-resolution, which may lead to important applications in bio-imaging and surface characterization.
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Microscopía/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodosRESUMEN
Hyperbolic media strengthen numerous attractive applications in optics such as super-resolution imaging, enhanced spontaneous emission, and nanoscale waveguiding. Natural hyperbolic materials exist at visible frequencies; however, implementations of these materials suffer substantial compromises resulting from the high loss in the currently available candidates. Here, the first experimental and theoretical investigation of regioregular poly(3-alkylthiophenes) (rr-P3ATs), a naturally low-loss organic hyperbolic material (OHM) in the visible frequency range, is shown. These hyperbolic properties arise from a highly ordered structure of layered electron-rich conjugated thiophene ring backbones separated by insulating alkyl side chains. The optical and electronic properties of the rr-P3AT can be tuned by controlling the degree of crystallinity and alkyl side chain length. First-principles calculations support the experimental observations, which result from the rr-P3AT's structural and optical anisotropy. Conveniently, rr-P3AT-based OHMs are facile to fabricate, flexible, and biocompatible, which may lead to tremendous new opportunities in a wide range of applications.
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The strongly enhanced and confined subwavelength optical fields near plasmonic nanoantennas have been extensively studied not only for the fundamental understanding of light-matter interactions at the nanoscale but also for their emerging practical application in enhanced second harmonic generation, improved inelastic electron tunneling, harvesting solar energy, and photocatalysis. However, owing to the deep subwavelength nature of plasmonic field confinement, conventional optical imaging techniques are incapable of characterizing the optical performance of these plasmonic nanoantennas. Here, we demonstrate super-resolution imaging of â¼20 nm optical field confinement by monitoring randomly moving dye molecules near plasmonic nanoantennas. This Brownian optical microscopy is especially suitable for plasmonic field characterization because of its capabilities for polarization sensitive wide-field super-resolution imaging.