High-Throughput All-Optical Determination of Nanorod Size and Orientation.

As a single-particle characterization technique, optical microscopy has transformed our understanding of structure-function relationships of plasmonic nanoparticles, but the need for ex-situ-correlated electron microscopy to obtain structural information handicaps an otherwise exceptional high-throughput technique. Here, we present an all-optical alternative to electron microscopy to accurately and quickly extract structural information about single gold nanorods (Au NRs) using calcite-assisted localization and kinetics (CLocK) microscopy. Color CLocK images of single Au NRs allow scattering from the longitudinal and transverse plasmon modes to be imaged simultaneously, encoding spectral data in CLocK images that can then be extracted to obtain Au NR size and orientation. Moreover, through the use of convolutional neural networks, Au NR length, width, and aspect ratio can be predicted directly from color CLocK images within ∼10% of the true value measured by electron microscopy.

Noninvasive assessment of human microvascular function in health and disease using incident dark-field microscopy.

Reduced peripheral microvascular reactivity is associated with an increased risk for major adverse cardiac events (MACEs). Tools for noninvasive assessment of peripheral microvascular function are limited, and existing technology is poorly validated in both healthy populations and patients with cardiovascular disease (CVD). Here, we used a handheld incident dark-field imaging tool (CytoCam) to test the hypothesis that, compared with healthy individuals (no risk factors for CVD), subjects formally diagnosed with coronary artery disease (CAD) or those with ≥2 risk factors for CAD (at risk) would exhibit impaired peripheral microvascular reactivity. A total of 17 participants (11 healthy, 6 at risk) were included in this pilot study. CytoCam was used to measure sublingual microvascular total vessel density (TVD), perfused vessel density (PVD), and microvascular flow index (MFI) in response to the topical application of acetylcholine (ACh) and sublingual administration of nitroglycerin (NTG). Baseline MFI and PVD were significantly reduced in the at-risk cohort compared with healthy individuals. Surprisingly, following the application of acetylcholine and nitroglycerin, both groups showed a significant improvement in all three microvascular perfusion parameters. These results suggest that, despite baseline reductions in both microvascular density and perfusion, human in vivo peripheral microvascular reactivity to both endothelial-dependent and -independent vasoactive agents remains intact in individuals with CAD or multiple risk factors for disease.NEW & NOTEWORTHY To our knowledge, this is the first study to comprehensively characterize in vivo sublingual microvascular structure and function (endothelium-dependent and -independent) in healthy patients and those with CVD. Importantly, we used an easy-to-use handheld device that can be easily translated to clinical settings. Our results indicate that baseline microvascular impairments in structure and function can be detected using the CytoCam technology, although reactivity to acetylcholine may be maintained even during disease in the peripheral microcirculation.

Unusual dark-field hyperspectral and confocal Raman microscopy features of a nanoporous gold electrode coated with porphyrazine complex.

Nanoporous gold electrodes are of great interest in electroanalytical chemistry, because of their unusual activity and large surface area. The electrochemical activity can be further improved by coating with molecular catalysts such as the tetraruthenated cobalt-tetrapyridylporphyrazines investigated in this work. The plasmonic enhancement of the scattered light at the nanoholes and borders modifies the electrode's optical characteristics, improving the transmission through the surface-enhanced Raman scattering (SERS) effect. When monitored by hyperspectral dark-field and confocal Raman microscopy, this effect allows probing of the porphyrazine species at the plasmonic nanholes, improving the understanding of the chemically modified gold electrodes.

Truncated M13 phage for smart detection of E. coli under dark field.

BACKGROUND: The urgent need for affordable and rapid detection methodologies for foodborne pathogens, particularly Escherichia coli (E. coli), highlights the importance of developing efficient and widely accessible diagnostic systems. Dark field microscopy, although effective, requires specific isolation of the target bacteria which can be hindered by the high cost of producing specialized antibodies. Alternatively, M13 bacteriophage, which naturally targets E. coli, offers a cost-efficient option with well-established techniques for its display and modification. Nevertheless, its filamentous structure with a large length-diameter ratio contributes to nonspecific binding and low separation efficiency, posing significant challenges. Consequently, refining M13 phage methodologies and their integration with advanced microscopy techniques stands as a critical pathway to improve detection specificity and efficiency in food safety diagnostics. METHODS: We employed a dual-plasmid strategy to generate a truncated M13 phage (tM13). This engineered tM13 incorporates two key genetic modifications: a partial mutation at the N-terminus of pIII and biotinylation at the hydrophobic end of pVIII. These alterations enable efficient attachment of tM13 to diverse E. coli strains, facilitating rapid magnetic separation. For detection, we additionally implemented a convolutional neural network (CNN)-based algorithm for precise identification and quantification of bacterial cells using dark field microscopy. RESULTS: The results obtained from spike-in and clinical sample analyses demonstrated the accuracy, high sensitivity (with a detection limit of 10 CFU/µL), and time-saving nature (30 min) of our tM13-based immunomagnetic enrichment approach combined with AI-enabled analytics, thereby supporting its potential to facilitate the identification of diverse E. coli strains in complex samples. CONCLUSION: The study established a rapid and accurate detection strategy for E. coli utilizing truncated M13 phages as capture probes, along with a dark field microscopy detection platform that integrates an image processing model and convolutional neural network.

Balancing the Efficiency and Sensitivity of Defect Inspection of Non-Patterned Wafers with TDI-Based Dark-Field Scattering Microscopy.

In semiconductor manufacturing, defect inspection in non-patterned wafer production lines is essential to ensure high-quality integrated circuits. However, in actual production lines, achieving both high efficiency and high sensitivity at the same time is a significant challenge due to their mutual constraints. To achieve a reasonable trade-off between detection efficiency and sensitivity, this paper integrates the time delay integration (TDI) technology into dark-field microscopy. The TDI image sensor is utilized instead of a photomultiplier tube to realize multi-point simultaneous scanning. Experiments illustrate that the increase in the number of TDI stages and reduction in the column fixed pattern noise effectively improve the signal-to-noise ratio of particle defects without sacrificing the detecting efficiency.

A Universal Approach to Determine the Atomic Layer Numbers in Two-Dimensional Materials Using Dark-Field Optical Contrast.

Two-dimensional (2D) materials possess unique properties primarily due to the quantum confinement effect, which highly depends on their thicknesses. Identifying the number of atomic layers in these materials is a crucial, yet challenging step. However, the commonly used optical reflection method offers only very low contrast. Here, we develop an approach that shows unprecedented sensitivity by analyzing the brightness of dark-field optical images. The brightness of the 2D material edges has a linear dependence on the number of atomic layers. The findings are modeled by Rayleigh scattering, and the results agree well with the experiments. The relative contrast of single-layer graphene can reach 70% under white-light incident conditions. Furthermore, different 2D materials were successfully tested. By adjusting the exposure conditions, we can identify the number of atomic layers ranging from 1 to over 100. Finally, this approach can be applied to various substrates, even transparent ones, making it highly versatile.

Probing Twist-Induced Endocytotic Membrane Fission using Anisotropic Gold Homodimers.

Membrane fission involves a crucial step of lipid remodeling, in which the dynamin collar constricts and severs the tubulated lipid membrane at the neck of budding vesicles. Nevertheless, the difficulty in accurately determining the rotational dynamics of live endocytotic vesicles poses a limit on the elucidation of dynamin-induced membrane remodeling for endocytotic vesicle scission. Herein, we designed a DNA-modified gold homodimer (AuHD)-based anisotropic plasmonic probe with uniform surface chemistry, minimizing orientational fluctuation within vesicle encapsulation. Using AuHDs as cargos to image the dynamics of cargo-containing vesicles during endocytosis, we showed that, prior to detachment from plasma membrane, the cargo-containing vesicles underwent multiple intermittent twists of ~4° angular orientation relative to plasma membrane with a ~0.2 s dwell time. These findings suggest that the membrane torques resulting from dynamin actions in vivo constitute the pathway to membrane fission, potentially shedding light on how dynamin-mediated lipid remodeling orchestrates membrane fission.

Shaping Copper Oxide Layers on Gold Nanoparticle Ensembles by Controlled Electrodeposition with Single Particle Scatterometry.

Electrodeposition of copper on gold nanoelectrode ensembles result in the formation of uniform copper oxide layers on individual nanoparticles. A linear sweep of voltammetric change induces three distinct morphologies dependent upon particle density. Ex situ imaging and in situ scatterometry at a single-particle level identifies multi-step electrochemical growth sequences that deviated from classical nucleation and growth pathways. In addition, the study demonstrated the possibility of synthesizing sophisticated structures based on the symmetry of nanoelectrodes. This result guides the nanoscale morphology control of electrode ensembles with potential application in electrocatalysis and sensing.

Circular Polarization Conversion in Single Plasmonic Spherical Particles.

Temporal and spectral behaviors of plasmons determine their ability to enhance the characteristics of metamaterials tailored to a wide range of applications, including electric-field enhancement, hot-electron injection, sensing, as well as polarization and angular momentum manipulation. We report a dark-field (DF) polarimetry experiment on single particles with incident circularly polarized light in which gold nanoparticles scatter with opposite handedness at visible wavelengths. Remarkably, for silvered nanoporous silica microparticles, the handedness conversion occurs at longer visible wavelengths, only after adsorption of molecules on the silver. Finite element analysis (FEA) allows matching the circular polarization (CP) conversion to dominant quadrupolar contributions, determined by the specimen size and complex susceptibility. We hypothesize that the damping accompanying the adsorption of molecules on the nanostructured silver facilitates the CP conversion. These results offer new perspectives in molecule sensing and materials tunability for light polarization conversion and control of light spin angular momentum at submicroscopic scale.

Surface Curvature Dominated Guest-Induced Nonequilibrium Deformations of Single Covalent Organic Framework-300 Particles.

Understanding the guest-induced dynamic deformation process of covalent organic frameworks (COFs) is vitally important to further increase their stimulus-response performances. Here we report on the dark-field microscopic (DFM) imaging approach to in situ monitor the guest-induced deformation evolution of individual COF-300 crystals in real time. We observe not only transient and nonequilibrium intermediate deformation states but also local surface curvature-driven diverse adsorption behaviours of single COF-300 particles for dichloromethane (DCM), undergoing one, two, and multiple expansion-contraction deformations as well as contraction-to-expansion transition. The surface curvature-dominated deformations are ascribed to the significant differences in the adsorption capacity for DCM at the curved tip and flat side regions, in which DCM can be adsorbed preferentially by curved tip regions of COF-300.

Label-free identification of microplastics in human cells: dark-field microscopy and deep learning study.

The development of an automatic method of identifying microplastic particles within live cells and organisms is crucial for high-throughput analysis of their biodistribution in toxicity studies. State-of-the-art technique in the data analysis tasks is the application of deep learning algorithms. Here, we propose the approach of polystyrene microparticle classification differing only in pigmentation using enhanced dark-field microscopy and a residual neural network (ResNet). The dataset consisting of 11,528 particle images has been collected to train and evaluate the neural network model. Human skin fibroblasts treated with microplastics were used as a model to study the ability of ResNet for classifying particles in a realistic biological experiment. As a result, the accuracy of the obtained classification algorithm achieved up to 93% in cell samples, indicating that the technique proposed will be a potent alternative to time-consuming spectral-based methods in microplastic toxicity research.

Intensity-Based Single Particle Plasmon Sensing.

Plasmon sensors respond to local changes of their surrounding environment with a shift in their resonance wavelength. This response is usually detected by measuring light scattering spectra to determine the resonance wavelength. However, single wavelength detection has become increasingly important because it simplifies the setup, increases speed, and improves statistics. Therefore, we investigated theoretically how the sensitivity toward such single wavelength scattering intensity changes depend on the material and shape of the plasmonic sensor. Surprisingly, simple equations describe this intensity sensitivity very accurately and allow us to distinguish the various contributions: Rayleigh scattering, dielectric contrast, plasmon shift, and frequency-dependent plasmon bulk damping. We find very good agreement of theoretical predictions and experimental data obtained by single particle spectroscopy.

Single-Shot Three-Dimensional Orientation Imaging of Nanorods Using Spin to Orbital Angular Momentum Conversion.

The key information about any nanoscale system relates to the orientations and conformations of its parts. Unfortunately, these details are often hidden below the diffraction limit, and elaborate techniques must be used to optically probe them. Here we present imaging of the 3D rotation motion of metal nanorods, restoring the distinct nanorod orientations in the full extent of azimuthal and polar angles. The nanorods imprint their 3D orientation onto the geometric phase and space-variant polarization of the light they scatter. We manipulate the light angular momentum and generate optical vortices that create self-interference images providing the nanorods' angles via digital processing. After calibration by scanning electron microscopy, we demonstrated time-resolved 3D orientation imaging of sub-100 nm nanorods under Brownian motion (frame rate up to 500 fps). We also succeeded in imaging nanorods as nanoprobes in live-cell imaging and reconstructed their 3D rotational movement during interaction with the cell membrane (100 fps).

Dark-Field Microscopic Study of Cellular Uptake of Carbon Nanodots: Nuclear Penetrability.

Carbon nanodots are fascinating candidates for the field of biomedicine, in applications such as bioimaging and drug delivery. However, the nuclear penetrability and process are rarely studied and lack understanding, which limits their applications for drug carriers, single-molecule detection and live cell imaging. In this study, we attempt to examine the uptake of CNDs in cells with a focus on the potential nuclear penetrability using enhanced dark-field microscopy (EDFM) associated with hyperspectral imaging (HSI) to quantitatively determine the light scattering signals of CNDs in the cells. The effects of both CND incubation time and concentration are investigated, and plausible nuclear penetration involving the nuclear pore complex (NPC) is discussed. The experimental results and an analytical model demonstrate that the CNDs' uptake proceeds by a concentration-dependent three-stage behavior and saturates at a CND incubation concentration larger than 750 µg/mL, with a half-saturated concentration of 479 µg/mL. These findings would potentially help the development of CNDs' utilization in drug carriers, live cell imaging and other biomedical applications.

Multivariate Imaging for Fast Evaluation of In Situ Dark Field Microscopy Hyperspectral Data.

Dark field scattering microscopy can create large hyperspectral data sets that contain a wealth of information on the properties and the molecular environment of noble metal nanoparticles. For a quick screening of samples of microscopic dimensions that contain many different types of plasmonic nanostructures, we propose a multivariate analysis of data sets of thousands to several hundreds of thousands of scattering spectra. By using non-negative matrix factorization for decomposing the spectra, components are identified that represent individual plasmon resonances and relative contributions of these resonances to particular microscopic focal volumes in the mapping data sets. Using data from silver and gold nanoparticles in the presence of different molecules, including gold nanoparticle-protein agglomerates or silver nanoparticles forming aggregates in the presence of acrylamide, plasmonic properties are observed that differ from those of the original nanoparticles. For the case of acrylamide, we show that the plasmon resonances of the silver nanoparticles are ideally suited to support surface enhanced Raman scattering (SERS) and the two-photon excited process of surface enhanced hyper Raman scattering (SEHRS). Both vibrational tools give complementary information on the in situ formed polyacrylamide and the molecular composition at the nanoparticle surface.

In Situ Imaging of Ion Motion in a Single Nanoparticle: Structural Transformations in Selenium Nanoparticles.

Intraparticle ion motions are critical to the structure and properties of nanomaterials, but rarely disclosed. Herein, in situ visualization of ion motions in a single nanoparticle is presented by dark-field microscopy imaging, which shows HgCl2 -induced structural transformation of amorphous selenium nanoparticles (SeNPs) with the main composition of Se8 . Owing to the high binding affinity with selenium and coulomb interactions, Hg2+ ions can permeate into the interior of SeNPs, making the amorphous Se8 turn to polycrystalline Hg3 Se2 Cl2 . As a proof of concept, SeNPs then serve as a highly effective scavenger for selective removal of Hg2+ ions from solution. This new finding offers visual proof for the photophysical process involving intraparticle ion motion, demonstrating that tracking the ion motions is a novel strategy to comprehend the formation mechanism with the purpose of developing new nanostructures like nanoalloys and nano metal compounds.

Single-Nanoparticle Coulometry Method with High Sensitivity and High Throughput to Study the Electrochemical Activity and Oscillation of Single Nanocatalysts.

To explore nanocatalysts with high electro-catalytic performance and less loading of precious metals, efforts have been made to develop electrochemical methods with high spatial resolution at the single nanoparticle level. Herein, a highly sensitive single-nanoparticle coulometry method is successfully developed to study the electrochemical activity and oscillation of single PtTe nanocatalysts. Based on microbattery reactions involving the formic acid electro-oxidation and the deposition of Ag on the single PtTe nanocatalyst surface, this method enables the transition from the undetectable sub-fA electric signal of the formic acid electro-oxidation into strong localized surface plasmon resonance scattering signal of Ag detected by dark-field microscopy. The lowest limiting current for a single nanocatalyst is found to be as low as 25.8 aA. Different trends of activity versus the formic acid concentration and types of activity of the single nanocatalyst have been discovered. Unveiled frequency-amplitude graph shows that the two electrochemical oscillation modes of low frequency with high amplitude and vice versa coexist in a single PtTe nanocatalyst, indicating the abundantly smooth surfaces and defects of nanocatalysts. This conducted study will open up the new avenue for further behavioral and mechanistic investigation of more types of nanocatalysts in the electrochemistry community.

Human leptospirosis in the Marche region: Over 10 years of surveillance.

We conducted a 10 years' retrospective study in 347 symptomatic individuals to assess the regional distribution of leptospirosis. A total of 173 individuals were diagnosed positive (49.8%): 11.5% were found positive to Leptospira by microscopic agglutination test positive, whereas 38.3% were found positive by microscopy analysis. The maximum peak of leptospirosis was reached in 2017 (n = 32). The most common serovars were Icterohaemorrhagiae and Poi.

Size-dependent light scattering of CoOOH nanoflakes for convenient and sensitive detection of alkaline phosphatase in human serum.

As a natural enzyme, alkaline phosphatase (ALP) plays an essential role in clinicopathological examinations and biomedical research, and is capable of hydrolyzing the phosphate group of l-ascorbic acid-2-phosphate (AAP) to yield l-ascorbic acid (L-AA). L-AA reduced cobalt oxyhydroxide (CoOOH) nanoflakes to Co2+ , leading to a smaller size and weaker light scattering, which could be monitored by electron microscopic images and optical spectra. The indirect detection of ALP was achieved by the reduced light scattering signal of CoOOH nanoflakes. Under optimal conditions, the decrease in scattering intensity was proportional to the ALP concentration over the range 0.1-160 U/L and the detection limit was 0.034 U/L (3σ/k). Compared with other assays, this proposed light scattering method was more convenient and economic for ALP sensing. The method was successfully applied to ALP analysis in human serum samples, and was similar to the results obtained by commercial kits.

Ultrahigh-Speed Imaging of Rotational Diffusion on a Lipid Bilayer.

We studied the rotational and translational diffusion of a single gold nanorod linked to a supported lipid bilayer with ultrahigh temporal resolution of two microseconds. By using a home-built polarization-sensitive dark-field microscope, we recorded particle trajectories with lateral precision of 3 nm and rotational precision of 4°. The large number of trajectory points in our measurements allows us to characterize the statistics of rotational diffusion with unprecedented detail. Our data show apparent signatures of anomalous diffusion such as sublinear scaling of the mean-squared angular displacement and negative values of angular correlation function at small lag times. However, a careful analysis reveals that these effects stem from the residual noise contributions and confirms normal diffusion. Our experimental approach and observations can be extended to investigate diffusive processes of anisotropic nanoparticles in other fundamental systems such as cellular membranes or other two-dimensional fluids.