Rapid fabrication of physically robust hydrogels.

Hydrogel materials show promise for diverse applications, particular as biocompatible materials due to their high water content. Despite advances in hydrogel technology in recent years, their application is often severely limited by inadequate mechanical properties and time-consuming fabrication processes. Here we report a rapid hydrogel preparation strategy that achieves the simultaneous photo-crosslinking and establishment of biomimetic soft-hard material interface microstructures. These biomimetic interfacial-bonding nanocomposite hydrogels are prepared within seconds and feature clearly separated phases but have a strongly bonded interface. Due to effective interphase load transfer, biomimetic interfacial-bonding nanocomposite gels achieve an ultrahigh toughness (138 MJ m-3) and exceptional tensile strength (15.31 MPa) while maintaining a structural stability that rivals or surpasses that of commonly used elastomer (non-hydrated) materials. Biomimetic interfacial-bonding nanocomposite gels can be fabricated into arbitrarily complex structures via three-dimensional printing with micrometre-level precision. Overall, this work presents a generalizable preparation strategy for hydrogel materials and acrylic elastomers that will foster potential advances in soft materials.

Bioluminescence assay for rapid detection of live Staphylococcus aureus based on the enrichment of egg yolk antibody modified magnetic metal organic framework immunobeads.

Specific and rapid detection of live Staphylococcus aureus (S.A) in environmental and food samples is critically important for protecting human health. In order to fulfill this purpose, two kinds of novel egg yolk antibody (IgY) immobilized immunomagnetic beads (IMBs; mSiO2-IgY and mMOF-IgY), with core-shell mSiO2 and mMOF as substrate, were prepared for selectively enriching S.A from samples. Furthermore, the IMBs with captured S.A were collected and re-dissolved in 0.5 mL PBS. After that, a cotton swab coated with sodium dodecylsulfate (SDS) was put in the solution to lyse S.A cells and emit ATP bioluminescence of the luciferin/luciferase system. Finally, a portable bioluminescence detector was used for quantification of ATP corresponding to S.A concentration. The results demonstrated that mMOF-IgY can enrich more S.A than mSiO2-IgY and emit a stronger signal. The reasons may be due to the higher immobilization amount of IgY on the IMBs. Under optimal conditions, the calibration line of S.A concentration was 10-105 CFU mL-1 by mMOF-IgY within 30 min. The low detection limit of S.A was 3 CFU mL-1. The results demonstrated that the assay takes much shorter time than plate counting. Its portability and excellent detection capability are suitable for rapid monitoring of specific pathogens in foods.

Structural basis of the C-terminal domain of SARS-CoV-2 N protein in complex with GMP reveals critical residues for RNA interaction.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein performs multiple functions during the viral life cycle, particularly in binding to the viral genomic RNA to form a helical ribonucleoprotein complex. Here, we present that the C-terminal domain of SARS-CoV-2 N protein (N-CTD) specifically interacts with polyguanlylic acid (poly(G)). The crystal structure of the N-CTD in complex with 5'-guanlylic acid (GMP, also known as guanosine monophosphate) was determined at a resolution of approximately 2.0 Å. A novel GMP-binding pocket in the N-CTD was illustrated. Residues Arg259 and Lys338 were identified to play key roles in binding to GMP through mutational analysis. These two residues are absolutely conserved in the other two highly pathogenic CoVs, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Overall, our findings expand the structural information on N protein interacting with guanylate and reveal a conserved GMP-binding pocket as a potential antiviral target.

Standardizing image assessment in optical diffraction tomography.

Optical diffraction tomography (ODT) has gradually become a popular label-free imaging technique that offers diffraction-limited resolution by mapping an object's three-dimensional (3D) refractive index (RI) distribution. However, there is a lack of comprehensive quantitative image assessment metrics in ODT for studying how various experimental conditions influence image quality, and subsequently optimizing the experimental conditions. In this Letter, we propose to standardize the image assessment in ODT by proposing a set of metrics, including signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and structural distinguishability (SD). To test the feasibility of the metrics, we performed experiments on angle-scanning ODT by varying the number of illumination angles, RI contrast of samples, sample feature sizes, and sample types (e.g., standard polystyrene beads and 3D printed structures) and evaluated the RI tomograms with SNR, CNR, and SD. We further quantitatively studied how image quality can be improved, and tested the image assessment metrics on subcellular structures of living cells. We envision the proposed image assessment metrics may greatly benefit end-users for assessing the RI tomograms, as well as experimentalists for optimizing ODT instruments.

Promoted Photothermal Catalytic CO Hydrogenation by Using TiC-Supported Co-Fe5 C2 Catalysts.

Photothermal catalytic CO hydrogenation offers the potential to synthesize light hydrocarbons by using solar energy. However, the selectivity and activity of the reaction are still far below those achieved in conventional thermal catalytic processes. Herein, we report that the Co-modified Fe5 C2 on TiC catalyst promotes photothermal catalytic CO hydrogenation with a 59 % C2+ selectivity in the produced hydrocarbons and a 30 % single-pass CO conversion at a high gas hourly space-time velocity of 12 000 mL g-1 h-1 . Using in-situ-irradiated XPS, we show that light-induced hot electron injection from TiC to Fe5 C2 modulates the chemical state of Fe, thereby increasing the CO-to-C2+ conversion. This work suggests that it is possible for plasmon-mediated surface chemistry to enhance the activity and selectivity of photothermal catalytic reactions.

Visible-light activatable coumarin-based phototriggers for fluorescence imaging with ultra-high photolysis efficiency.

Photoactivated fluorophores (PAFs) are powerful imaging tools for observing subcellular structures and tracking dynamic biological processes. However, photoremovable protecting groups (PPGs) widely used to construct PAFs suffer from the drawbacks of short-wavelength excitation and/or low photolysis efficiency. Herein, a class of coumarin-based PPGs with electron-rich thiophene derived substitutions at the C3-position of a coumarin scaffold were prepared. The modification not only leads to the redshift of the absorption band to the blue light region (400-500 nm), but also the increases of uncaging quantum yield (Φu) as well as molar extinction coefficient (εmax), thus enhancing the photolysis efficiency (Φu × Îµmax) up to 34.2 × 103 M-1 cm-1. The exceptionally high photolysis efficiency enables efficient photolysis in blue light as weak as 2 mW cm-2 or in blue light from a Luminol chemiluminescence system. Based on the excellent photolysis properties, the PAF constructed by the new PPG exhibits fast photoactivation and a low background signal, and the resulting fluorescence images display a signal-to-noise ratio greater than 780. It is anticipated that the superior photolysis performance makes the PPGs a novel platform for the construction of photo responsive systems in a variety of applications.

Complex wave and phase retrieval from a single off-axis interferogram.

Single-frame off-axis holographic reconstruction is promising for quantitative phase imaging. However, reconstruction accuracy and contrast are degraded by noise, frequency spectrum overlap of the interferogram, severe phase distortion, etc. In this work, we propose an iterative single-frame complex wave retrieval based on an explicit model of object and reference waves. We also develop a phase restoration algorithm that does not resort to phase unwrapping. Both simulation and real experiments demonstrate higher accuracy and robustness compared to state-of-the-art methods, for both complex wave estimation and phase reconstruction. Importantly, the allowed bandwidth for the object wave is significantly improved in realistic experimental conditions (similar amplitudes for object and reference waves), which makes it attractive for large field-of-view and high-resolution imaging applications.

Total variation and spatial iteration-based 3D structured illumination microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) plays an essential role in biological volumetric imaging with the capabilities of improving lateral and axial resolution. However, the traditional linear 3D algorithm is sensitive to noise and generates artifacts, while the low temporal resolution hinders live-cell imaging. In this paper, we propose a novel 3D-SIM algorithm based on total variation (TV) and fast iterative shrinkage threshold algorithm (FISTA), termed TV-FISTA-SIM. Compared to conventional algorithms, TV-FISTA-SIM achieves higher reconstruction fidelity with the least artifacts, even when the signal-to-noise ratio (SNR) is as low as 5 dB, and a faster reconstruction rate. Through simulation, we have verified that TV-FISTA-SIM can effectively reduce the amount of required data with less deterioration. Moreover, we demonstrate TV-FISTA-SIM for high-quality multi-color 3D super-resolution imaging, which can be potentially applied to live-cell imaging applications.

The Q225P Mutation in SigB Promotes Membrane Vesicle Formation in Staphylococcus aureus.

Both Gram-positive and Gram-negative bacteria release nano-sized lipid bilayered particles, known as membrane vesicles (MVs), into external environments. Although MVs play a variety of roles in bacterial physiology and pathogenesis, the mechanisms underlying MV formation in Gram-positive microorganisms such as Staphylococcus aureus remain obscure. Bacterial MV production can be induced in response to stress conditions, and the alternative sigma factor B (SigB) functions as a central regulator of the stress response in Gram-positive bacteria. In a previous study, we demonstrated that the SigB(Q225P) substitution mutation in S. aureus promotes biofilm formation. Here, we report that the SigB(Q225P) mutation also increases MV production in this important pathogen. LacZ reporter assays and electrophoretic mobility shift assays showed that the Q225P substitution reduces SigB binding to the promoter region of the thermonuclease gene (nuc), resulting in a significant reduction in Nuc expression. Deletion of nuc markedly enhances S. aureus MV generation, possibly due to the accumulation of nucleic acids. These results are not only important for understanding MV biogenesis in S. aureus, but also useful for the development of a S. aureus MV-based platform for MV application.

Phase-Separated Multienzyme Compartmentalization for Terpene Biosynthesis in a Prokaryote.

Liquid-liquid phase separation (LLPS) forms biomolecular condensates or coacervates in cells. Metabolic enzymes can form phase-separated subcellular compartments that enrich enzymes, cofactors, and substrates. Herein, we report the construction of synthetic multienzyme condensates that catalyze the biosynthesis of a terpene, α-farnesene, in the prokaryote E. coli. RGGRGG derived from LAF-1 was used as the scaffold protein to form the condensates by LLPS. Multienzyme condensates were then formed by assembling two enzymes Idi and IspA through an RIAD/RIDD interaction. Multienzyme condensates constructed inside E. coli cells compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α-farnesene production. This work demonstrates LLPS-driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of the biosynthesis of α-farnesene.

High-speed Fourier ptychographic microscopy for quantitative phase imaging.

Fourier ptychographic microscopy (FPM), as an emerging computational imaging method, has been applied to quantitative phase imaging with resolution bypassing the physical limit of the detection objective. Due to the weak illumination intensity and long image acquisition time, the achieved imaging speed in current FPM methods is still low, making them unsuitable for real-time imaging applications. We propose and demonstrate a high-speed FPM method based on using laser illumination and digital micro-mirror devices for illumination angle scanning. In this new, to the best of our knowledge, FPM method, we realized quantitative phase imaging and intensity imaging at over 42 frames per second (fps) with around 1 µm lateral resolution. The quantitative phase images have revealed membrane height fluctuations of red blood cells with nanometer-scale sensitivity, while the intensity images have resolved subcellular features in stained cancer tissue slices.

Sensing Sub-10 nm Wide Perturbations in Background Nanopatterns Using Optical Pseudoelectrodynamics Microscopy (OPEM).

Using light as a probe to investigate perturbations with deep subwavelength dimensions in large-scale wafers is challenging because of the diffraction limit and the weak Rayleigh scattering. In this Letter, we report on a nondestructive noninterference far-field imaging method, which is built upon electrodynamic principles (mechanical work and force) of the light-matter interaction, rather than the intrinsic properties of light. We demonstrate sensing of nanoscale perturbations with sub-10 nm features in semiconductor nanopatterns. This framework is implemented using a visible-light bright-field microscope with a broadband source and a through-focus scanning apparatus. This work creates a new paradigm for exploring light-matter interactions at the nanoscale using microscopy that can potentially be extended to many other problems, for example, bioimaging, material analysis, and nanometrology.

Regularized pseudo-phase imaging for inspecting and sensing nanoscale features.

Recovering tiny nanoscale features using a general optical imaging system is challenging because of poor signal to noise ratio. Rayleigh scattering implies that the detectable signal of an object of size d illuminated by light of wavelength λ is proportional to d6/λ4, which may be several orders of magnitude weaker than that of additive and multiplicative perturbations in the background. In this article, we solve this fundamental issue by introducing the regularized pseudo-phase, an observation quantity for polychromatic visible light microscopy that seems to be more sensitive than conventional intensity images for characterizing nanoscale features. We achieve a significant improvement in signal to noise ratio without making any changes to the imaging hardware. In addition, this framework not only retains the advantages of conventional denoising techniques, but also endows this new measurand (i.e., the pseudo-phase) with an explicit physical meaning analogous to optical phase. Experiments on a NIST reference material 8820 sample demonstrate that we can measure nanoscale defects, minute amounts of tilt in patterned samples, and severely noise-polluted nanostructure profiles with the pseudo-phase framework even when using a low-cost bright-field microscope.

Optical inspection of nanoscale structures using a novel machine learning based synthetic image generation algorithm.

In this paper, we present a novel interpretable machine learning technique that uses unique physical insights about noisy optical images and a few training samples to classify nanoscale defects in noisy optical images of a semiconductor wafer. Using this technique, we not only detected both parallel bridge defects and previously undetectable perpendicular bridge defects in a 9-nm node wafer using visible light microscopy [Proc. SPIE9424, 942416 (2015)], but we also accurately classified their shapes and estimated their sizes. Detection and classification of nanoscale defects in optical images is a challenging task. The quality of images is affected by diffraction and noise. Machine learning techniques can reduce noise and recognize patterns using a large training set. However, for detecting a rare "killer" defect, acquisition of a sufficient training set of high quality experimental images can be prohibitively expensive. In addition, there are technical challenges involved in using electromagnetic simulations and optimization of the machine learning algorithm. This paper proposes solutions to address each of the aforementioned challenges.

Safe Staphylococcal Platform for the Development of Multivalent Nanoscale Vesicles against Viral Infections.

Many viruses often have closely related yet antigenically distinct serotypes. An ideal vaccine against viral infections should induce a multivalent and protective immune response against all serotypes. Inspired by bacterial membrane vesicles (MVs) that carry different protein components, we constructed an agr locus deletion mutant of the Staphylococcus aureus strain (RN4220-Δagr) to reduce potential toxicity. Nanoscale vesicles derived from this strain (ΔagrMVs) carry at least four major components that can deliver heterologous antigens. These components were each fused with a triple FLAG tag, and the tagged proteins could be incorporated into the ΔagrMVs. The presentation levels were (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, and (2.89 ± 0.74)% of the total ΔagrMV proteins for Mntc-FLAG, PdhB-FLAG, PdhA-FLAG, and Eno-FLAG, respectively. With two DENV envelope E domain III proteins (EDIIIconA and EDIIIconB) as models, the DENV EDIIIconA and EDIIIconB delivered by two staphylococcal components were stably embedded in the ΔagrMVs. Administration of such engineered ΔagrMVs in mice induced antibodies against all four DENV serotypes. Sera from immunized mice protected Vero cells and suckling mice from a lethal challenge of DENV-2. This study will open up new insights into the preparation of multivalent nanosized viral vaccines against viral infections.

Dynamic spatial filtering using a digital micromirror device for high-speed optical diffraction tomography.

Optical diffraction tomography (ODT) is an emerging microscopy technique for three-dimensional (3D) refractive index (RI) mapping of transparent specimens. Recently, the digital micromirror device (DMD) based scheme for angle-controlled plane wave illumination has been proposed to improve the imaging speed and stability of ODT. However, undesired diffraction noise always exists in the reported DMD-based illumination scheme, which leads to a limited contrast ratio of the measurement fringe and hence inaccurate RI mapping. Here we present a novel spatial filtering method, based on a second DMD, to dynamically remove the diffraction noise. The reported results illustrate significantly enhanced image quality of the obtained interferograms and the subsequently derived phase maps. And moreover, with this method, we demonstrate mapping of 3D RI distribution of polystyrene beads as well as biological cells with high accuracy. Importantly, with the proper hardware configuration, our method does not compromise the 3D imaging speed advantage promised by the DMD-based illumination scheme. Specifically, we have been able to successfully obtain interferograms at over 1 kHz speed, which is critical for potential high-throughput label-free 3D image cytometry applications.

Nonlinear Focal Modulation Microscopy.

We demonstrate nonlinear focal modulation microscopy (NFOMM) to achieve superresolution imaging. Traditional approaches to superresolution that utilize point scanning often rely on spatially reducing the size of the emission pattern by directly narrowing (e.g., through minimizing the detection pinhole in Airyscan, Zeiss) or indirectly peeling its outer profiles [e.g., through depleting the outer emission region in stimulated emission depletion (STED) microscopy]. We show that an alternative conceptualization that focuses on maximizing the optical system's frequency shifting ability offers advantages in further improving resolution while reducing system complexity. In NFOMM, a spatial light modulator and a suitably intense laser illumination are used to implement nonlinear focal-field modulation to achieve a transverse spatial resolution of ∼60 nm (∼λ/10). We show that NFOMM is comparable with STED microscopy and suitable for fundamental biology studies, as evidenced in imaging nuclear pore complexes, tubulin and vimentin in Vero cells. Since NFOMM is readily implemented as an add-on module to a laser-scanning microscope, we anticipate wide utility of this new imaging technique.

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy.

Unlike most optical coherence microscopy (OCM) systems, dynamic speckle-field interferometric microscopy (DSIM) achieves depth sectioning through the spatial-coherence gating effect. Under high numerical aperture (NA) speckle-field illumination, our previous experiments have demonstrated less than 1 µm depth resolution in reflection-mode DSIM, while doubling the diffraction limited resolution as under structured illumination. However, there has not been a physical model to rigorously describe the speckle imaging process, in particular explaining the sectioning effect under high illumination and imaging NA settings in DSIM. In this paper, we develop such a model based on the diffraction tomography theory and the speckle statistics. Using this model, we calculate the system response function, which is used to further obtain the depth resolution limit in reflection-mode DSIM. Theoretically calculated depth resolution limit is in an excellent agreement with experiment results. We envision that our physical model will not only help in understanding the imaging process in DSIM, but also enable better designing such systems for depth-resolved measurements in biological cells and tissues.

Digital micromirror device-based common-path quantitative phase imaging.

We propose a novel common-path quantitative phase imaging (QPI) method based on a digital micromirror device (DMD). The DMD is placed in a plane conjugate to the objective back-aperture plane for the purpose of generating two plane waves that illuminate the sample. A pinhole is used in the detection arm to filter one of the beams after sample to create a reference beam. Additionally, a transmission-type liquid crystal device, placed at the objective back-aperture plane, eliminates the specular reflection noise arising from all the "off" state DMD micromirrors, which is common in all DMD-based illuminations. We have demonstrated high sensitivity QPI, which has a measured spatial and temporal noise of 4.92 nm and 2.16 nm, respectively. Experiments with calibrated polystyrene beads illustrate the desired phase measurement accuracy. In addition, we have measured the dynamic height maps of red blood cell membrane fluctuations, showing the efficacy of the proposed system for live cell imaging. Most importantly, the DMD grants the system convenience in varying the interference fringe period on the camera to easily satisfy the pixel sampling conditions. This feature also alleviates the pinhole alignment complexity. We envision that the proposed DMD-based common-path QPI system will allow for system miniaturization and automation for a broader adaption.

Tomographic phase microscopy: principles and applications in bioimaging [Invited].

Tomographic phase microscopy (TPM) is an emerging optical microscopic technique for bioimaging. TPM uses digital holographic measurements of complex scattered fields to reconstruct three-dimensional refractive index (RI) maps of cells with diffraction-limited resolution by solving inverse scattering problems. In this paper, we review the developments of TPM from the fundamental physics to its applications in bioimaging. We first provide a comprehensive description of the tomographic reconstruction physical models used in TPM. The RI map reconstruction algorithms and various regularization methods are discussed. Selected TPM applications for cellular imaging, particularly in hematology, are reviewed. Finally, we examine the limitations of current TPM systems, propose future solutions, and envision promising directions in biomedical research.