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.

Mie Magnetic Structural Colors from Short Chains of TiO2 Nanospheres.

We demonstrate distinctive structural colors within a small footprint by using a short chain of nanospheres. Rather than using high-index materials like Si (n ∼ 4), which ensure strong modal confinement, TiO2 is employed. TiO2 has an intermediate index (n ∼ 2), promoting stronger modal coupling between the magnetic dipoles of each particle. This approach enables selective engineering of the magnetic response and yields larger spectral changes compared to that of Si. Despite the lower refractive index, the absence of absorption in TiO2 also produces higher scattering intensities than Si. We develop a quasistatic analytical model that describes the dipolar modal coupling in a trimer and use it to reveal distinct magnetic field strengths in the outer or central particle depending on the polarization of incident light. These results suggest pathways to manipulate the magnetic field in chains of particles and create vibrant structural colors with simple configurations.

An alternative method to the Takagi-Taupin equations for studying dark-field X-ray microscopy of deformed crystals.

This study introduces an alternative method to the Takagi-Taupin equations for investigating the dark-field X-ray microscopy (DFXM) of deformed crystals. In scenarios where dynamical diffraction cannot be disregarded, it is essential to assess the potential inaccuracies of data interpretation based on the kinematic diffraction theory. Unlike the Takagi-Taupin equations, this new method utilizes an exact dispersion relation, and a previously developed finite difference scheme with minor modifications is used for the numerical implementation. The numerical implementation has been validated by calculating the diffraction of a diamond crystal with three components, wherein dynamical diffraction is applicable to the first component and kinematic diffraction pertains to the remaining two. The numerical convergence is tested using diffraction intensities. In addition, the DFXM image of a diamond crystal containing a stacking fault is calculated using the new method and compared with the experimental result. The new method is also applied to calculate the DFXM image of a twisted diamond crystal, which clearly shows a result different from those obtained using the Takagi-Taupin equations.

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.

Microvascular effects of a mixed meal tolerance test: a model validation study.

PURPOSE: Endothelial dysfunction is a pathophysiological change preceding many cardiovascular events. Measuring improvements of endothelial function is challenging when function is already optimal, which may be remediated using a physiological challenge. This study aimed to determine whether imaging assessments can detect microvascular effects of a mixed meal tolerance test (MMTT). METHODS: Twenty healthy volunteers (age ≥45 and ≤70 years) underwent two MMTTs at the beginning (Day 1) and end (Day 84) of a twelve-week period. Imaging methods included laser speckle contrast imaging (LSCI) combined with post-occlusive reactive hyperaemia (PORH) and local thermal hyperaemia (LTH) challenges, passive leg movement ultrasonography (PLM), and sidestream dark field microscopy (SDFM). Measurements were conducted pre-MMTT and at 5 timepoints post-MMTT for PLM and SDFM and 3 timepoints post-MMTT for PORH and LTH. RESULTS: No consistent effects of the MMTT were detected on LSCI LTH, PLM and SDFM endpoints. LSCI PORH maximum perfusion was significantly suppressed 46, 136, and 300 min post-MMTT administration on Day 1, while residual perfusion decreased significantly 46 and 136 min post-MMTT on Day 1. However, when repeated on Day 84, PORH endpoints were not significantly affected by the MMTT. CONCLUSION: SDFM, PLM and LSCI LTH endpoints displayed high intra-subject variability and did not detect consistent effects of MMTT. LSCI PORH endpoints displayed the lowest intra-subject variability of all assessed endpoints and were affected by the MMTT on Day 1, but not on Day 84. Further standardization of methods or more robust challenges to affect vascular endpoints may be needed.

Changes in microcirculation of small intestine end-to-end anastomoses in an experimental model.

INTRODUCTION: Sufficient perfusion is essential for a safe intestinal anastomosis. Impaired microcirculation may lead to increased bacterial translocation and anastomosis insufficiency. Thus, it is important to estimate well the optimal distance of the anastomosis line from the last mesenterial vessel. However, it is still empiric. In this experiment the aim was to investigate the intestinal microcirculation at various distances from the anastomosis in a pig model. MATERIALS AND METHODS: On 8 anesthetized pigs paramedian laparotomy and end-to-end jejuno-jejunostomy were performed. Using Cytocam-IDF camera, microcirculatory recordings were taken before surgery at the planned suture line, and 1 to 3 mesenterial vessel mural trunk distance from it, and at the same sites 15 and 120 min after anastomosis completion. After the microcirculation monitoring, anastomosed and intact bowel segments were removed to test tensile strength. RESULTS: The proportion and the density of the perfused vessels decreased significantly after anastomosis completion. The perfusion rate increased gradually distal from the anastomosis, and after 120 min these values seemed to be normalized. Anastomosed bowels had significantly lower maximal tensile strength and higher slope of tensile strength curves than intact controls. CONCLUSION: Alterations in microcirculation and tensile strength were observed. After completing the anastomosis, the improvement in perfusion increased gradually away from the wound edge. The IDF device was useful to monitor intestinal microcirculation providing data to estimate better the optimal distance of the anastomosis from the last order mesenteric vessel.

Local strain inhomogeneities during electrical triggering of a metal-insulator transition revealed by X-ray microscopy.

Electrical triggering of a metal-insulator transition (MIT) often results in the formation of characteristic spatial patterns such as a metallic filament percolating through an insulating matrix or an insulating barrier splitting a conducting matrix. When MIT triggering is driven by electrothermal effects, the temperature of the filament or barrier can be substantially higher than the rest of the material. Using X-ray microdiffraction and dark-field X-ray microscopy, we show that electrothermal MIT triggering leads to the development of an inhomogeneous strain profile across the switching device, even when the material does not undergo a pronounced, discontinuous structural transition coinciding with the MIT. Diffraction measurements further reveal evidence of unique features associated with MIT triggering including lattice distortions, tilting, and twinning, which indicate structural nonuniformity of both low- and high-resistance regions inside the switching device. Such lattice deformations do not occur under equilibrium, zero-voltage conditions, highlighting the qualitative difference between states achieved through increasing temperature and applying voltage in nonlinear electrothermal materials. Electrically induced strain, lattice distortions, and twinning could have important contributions in the MIT triggering process and drive the material into nonequilibrium states, providing an unconventional pathway to explore the phase space in strongly correlated electronic systems.

Dark-field microscopy studies of single silicon nanoparticles fabricated by e-beam evaporation technique: effect of thermal annealing, polarization of light and deposition parameters.

Herein, we report the dark-field microscopy studies on single silicon nanoparticles (SiNPs) fabricated using different deposition parameters in the electron beam evaporation technique. The morphology of the fabricated SiNPs is studied using theAtomic Force Microscope. Later, for the first time, the effect of thermal annealing and deposition parameters (i.e. beam current and deposition time) on the far-field scattering images and spectra of single SiNPs is studied using a transmission-mode dark-field optical microscope to estimate the wavelength locations and full-width at half maxima of the optical resonances of single SiNPs. Finally, the role of polarization of incident light on the optical resonances of single SiNPs is also studied by recording their scattering images and spectra.

Correcting directional dark field x-ray imaging artefacts using position dependent image deblurring and attenuation removal.

In recent years, a novel x-ray imaging modality has emerged that reveals unresolved sample microstructure via a "dark-field image", which provides complementary information to conventional "bright-field" images, such as attenuation and phase-contrast modalities. This x-ray dark-field signal is produced by unresolved microstructures scattering the x-ray beam resulting in localised image blur. Dark-field retrieval techniques extract this blur to reconstruct a dark-field image. Unfortunately, the presence of non-dark-field blur such as source-size blur or the detector point-spread-function can affect the dark-field retrieval as they also blur the experimental image. In addition, dark-field images can be degraded by the artefacts induced by large intensity gradients from attenuation and propagation-based phase contrast, particularly around sample edges. By measuring any non-dark-field blurring across the image plane and removing it from experimental images, as well as removing attenuation and propagation-based phase contrast, we show that a directional dark-field image can be retrieved with fewer artefacts and more consistent quantitative measures. We present the details of these corrections and provide "before and after" directional dark-field images of samples imaged at a synchrotron source. This paper utilises single-grid directional dark-field imaging, but these corrections have the potential to be broadly applied to other x-ray imaging techniques.

Characterization of extended defects in 2D materials using aperture-based dark-field STEM in SEM.

Quantitative diffraction contrast analysis with defined diffraction vectors is a well-established method in TEM for studying defects in crystalline materials. A comparable transmission technique is however not available in the more widely used SEM platforms. In this work, we transfer the aperture-based dark-field imaging method from the TEM to the SEM, thus enabling quantitative diffraction contrast studies at lower voltages in SEM. This is achieved in STEM mode by inserting a custom-made aperture between the sample and the STEM detector and centering the hole on a desired reflection. To select individual reflections for dark-field imaging, we use our Low Energy Nanodiffraction (LEND) setup [Schweizer et al., Ultramicroscopy 213, 112956 (2020)], which captures transmission diffraction patterns from a fluorescent screen positioned below the sample. The aperture-based dark-field STEM method is particularly useful for studying extended defects in 2D materials, where (i) stronger diffraction at the lower voltages used in SEM is advantageous, but (ii) two-beam conditions cannot be established, making quantitative diffraction contrast analysis with standard bright-field and annular dark-field detectors impossible. We demonstrate the method by studying basal plane dislocations in bilayer graphene, which have attracted considerable research interest due to their exceptional structural and electronic properties. Direct comparison of results obtained on identical dislocations by the established TEM method and by the new aperture-based dark-field STEM method in SEM shows that a reliable Burgers vector analysis is possible by applying the well-known g·b=0 invisibility criterion. We further use the LEND setup to acquire 4D-STEM data and show that the virtual dark-field images match well with those in aperture-based dark-field STEM images for reliable Burgers vector analysis.