Human-vision-inspired cluster identification for single-molecule localization microscopy.

Single-molecule localization microscopy has enabled scientists to visualize cellular structures at the nanometer scale. However, researchers are facing great challenges in analyzing images presented by point clouds. Existing algorithms for cluster identification are coordinate-based analyses requiring users to input cutoff thresholds based on the distance or density of the point cloud. These thresholds are often one's best guess with repeated visual inspections, making the cluster assignment user-dependent. Here, we present a cluster identification algorithm mimicking the modulation transfer function of human vision. This approach does not require any input parameters and produces visually satisfactory cluster assignments. We tested this algorithm by identifying the clusters of the fusion proteins of the Nipah virus on its host cells. This algorithm was further extended to analyze three-dimensional point clouds using virus-like particles as an example.

Structured illumination microscopy with a phase-modulated spinning disk for optical sectioning.

Among various super-resolution microscopic techniques, structured illumination microscopy (SIM) stands out for live-cell imaging because of its higher imaging speed. However, conventional SIM lacks optical sectioning capability. Here we demonstrate a new, to the best of our knowledge, approach using a phase-modulated spinning disk (PMSD) that enhances the optical sectioning capability of SIM. The PMSD consists of a pinhole array for confocal imaging and a transparent polymer layer for light phase modulation. The light phase modulation was designed to cancel the zeroth-order diffracted beam and create a sharp lattice illumination pattern using the interference of four first-order diffracted beams. In the detection optical path, the PMSD serves as a spatial filter to physically reject about 80% of the out-of-focus signals, an approach that allows for real-time optical reconstruction of super-resolved images with enhanced contrast. Furthermore, the simplicity of the design makes it easy to upgrade a conventional fluorescence microscope to a PMSD SIM system.

The nanoscale organization of Nipah virus matrix protein revealed by super-resolution microscopy.

The matrix proteins (M) of many enveloped RNA viruses mediate virus assembly and budding. However, it remains poorly understood how M are involved in virus budding and how they interact with envelope proteins. Here, we show that the expression level of Nipah (NiV) M in particles produced by the host cells deviates from a gamma distribution and does not reflect that of the host cells, indicating assembly of the NiV-M in the process. Our data reveal that NiV-M affects the circularity of the particles while the NiV envelope proteins do not. The organization of NiV envelope proteins on the membrane of the particles is similar to those that do not express NiV-M, suggesting that NiV-M does not directly interact with the envelope proteins during assembly and budding.

Conditional Generative Adversarial Network for Spectral Recovery to Accelerate Single-Cell Raman Spectroscopic Analysis.

Raman spectroscopy is a powerful tool to investigate cellular heterogeneity. However, Raman spectra for single-cell analysis are hindered by a low signal-to-noise ratio (SNR). Here, we demonstrate a simple and reliable spectral recovery conditional generative adversarial network (SRGAN). SRGAN reduced the data acquisition time by 1 order of magnitude (i.e., 30 vs 3 s) by improving the SNR by a factor of ∼6. We classified five major foodborne bacteria based on single-cell Raman spectra to further evaluate the performance of SRGAN. Spectra processed using SRGAN achieved an identification accuracy of 94.9%, compared to 60.5% using unprocessed Raman spectra. SRGAN can accelerate spectral collection to improve the throughput of Raman spectroscopy and enable real-time monitoring of single living cells.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Predicted Structure of Fully Activated Tas1R3/1R3' Homodimer Bound to G Protein and Natural Sugars: Structural Insights into G Protein Activation by a Class C Sweet Taste Homodimer with Natural Sugars.

The Tas1R3 G protein-coupled receptor constitutes the main component of sweet taste sensory response in humans via forming a heterodimer with Tas1R2 or a homodimer with Tas1R3. The Tas1R3/1R3' homodimer serves as a low-affinity sweet taste receptor, stimulating gustducin G protein (GGust) signaling in the presence of a high concentration of natural sugars. This provides an additional means to detect the taste of natural sugars, thereby differentiating the flavors between natural sugars and artificial sweeteners. We report here the predicted 3D structure of active state Tas1R3/1R3' homodimer complexed with heterotrimeric GGust and sucrose. We discovered that the GGust makes ionic anchors to intracellular loops 1 and 2 of Tas1R3 while the Gα-α5 helix engages the cytoplasmic region extensively through salt bridge and hydrophobic interactions. We show that in the activation of this complex the Venus flytrap domains of the homodimer undergo a remarkable twist up to ∼100° rotation around the vertical axis to adopt a closed-closed conformation while the intracellular region relaxes to an open-open conformation. We find that binding of sucrose to the homodimer stabilizes a preactivated conformation with a largely open intracellular region that recruits and activates the GGust. Upon activation, the Gα subunit spontaneously opens up the nucleotide-binding site, making nucleotide exchange facile for signaling. This activation of GGust promotes the interdomain twist of the Venus flytrap domains. These structures and transformations could potentially be a basis for the design of new sweeteners with higher activity and less unpleasant flavors.

Campylobacter jejuni Antimicrobial Resistance Profiles and Mechanisms Determined Using a Raman Spectroscopy-Based Metabolomic Approach.

Rapid identification of antimicrobial resistance (AMR) profiles and mechanisms is critical for clinical management and drug development. However, the current AMR detection approaches take up to 48 h to obtain a result. Here, we demonstrate a Raman spectroscopy-based metabolomic approach to rapidly determine the AMR profile of Campylobacter jejuni, a major cause of foodborne gastroenteritis worldwide. C. jejuni isolates with susceptible and resistant traits to ampicillin and tetracycline were subjected to different antibiotic concentrations for 5 h, followed by Raman spectral collection and chemometric analysis (i.e., second-derivative transformation analysis, hierarchical clustering analysis [HCA], and principal-component analysis [PCA]). The MICs obtained by Raman-2nd derivative transformation agreed with the reference agar dilution method for all isolates. The AMR profile of C. jejuni was accurately classified by Raman-HCA after treating bacteria with antibiotics at clinical susceptible and resistant breakpoints. According to PCA loading plots, susceptible and resistant strains showed different Raman metabolomic patterns for antibiotics. Ampicillin-resistant isolates had distinctive Raman signatures of peptidoglycan, which is related to cell wall synthesis. The ratio of saturated to unsaturated fatty acids in the lipid membrane layer of ampicillin-resistant isolates was higher than in susceptible ones, indicating more rigid envelope structure under ampicillin treatment. In comparison, tetracycline-resistant isolates exhibited prominent Raman spectral features associated with proteins and nucleic acids, demonstrating more active protein synthesis than susceptible strains with the presence of tetracycline. Taken together, Raman spectroscopy is a powerful metabolic fingerprinting technique for simultaneously revealing the AMR profiles and mechanisms of foodborne pathogens. IMPORTANCE Metabolism plays the central role in bacteria to mediate the early response against antibiotics and demonstrate antimicrobial resistance (AMR). Understanding the whole-cell metabolite profiles gives rise to a more complete AMR mechanism insight. In this study, we have applied Raman spectroscopy and chemometrics to achieve a rapid, accurate, and easy-to-operate investigation of bacterial AMR profiles and mechanisms. Raman spectroscopy reduced the analysis time by an order of magnitude to obtain the same results achieved through traditional culture-based antimicrobial susceptibility approaches. It offers great benefits as a high-throughput screening method in food chain surveillance and clinical diagnostics. Meanwhile, the AMR mechanisms toward two representative antibiotic classes, namely, ampicillin and tetracycline, were revealed by Raman spectroscopy at the metabolome level. This approach is based on bacterial phenotypic responses to antibiotics, providing information complementary to that obtained by conventional genetic methods such as genome sequencing. The knowledge obtained from Raman metabolomic data can be used in drug discovery and pathogen intervention.

Arcobacter Identification and Species Determination Using Raman Spectroscopy Combined with Neural Networks.

Rapid and accurate identification of Arcobacter is of great importance because it is considered an emerging food- and waterborne pathogen and potential zoonotic agent. Raman spectroscopy can differentiate bacteria based on Raman scattering spectral patterns of whole cells in a fast, reagentless, and easy-to-use manner. We aimed to detect and discriminate Arcobacter bacteria at the species level using confocal micro-Raman spectroscopy (785 nm) coupled with neural networks. A total of 82 reference and field isolates of 18 Arcobacter species from clinical, environmental, and agri-food sources were included. We determined that the bacterial cultivation time and growth temperature did not significantly influence the Raman spectral reproducibility and discrimination capability. The genus Arcobacter could be successfully differentiated from the closely related genera Campylobacter and Helicobacter using principal-component analysis. For the identification of Arcobacter to the species level, an accuracy of 97.2% was achieved for all 18 Arcobacter species using Raman spectroscopy combined with a convolutional neural network (CNN). The predictive capability of Raman-CNN was further validated using an independent data set of 12 Arcobacter strains. Furthermore, a Raman spectroscopy-based fully connected artificial neural network (ANN) was constructed to determine the actual ratio of a specific Arcobacter species in a bacterial mixture ranging from 5% to 100% by biomass (regression coefficient >0.99). The application of both CNN and fully connected ANN improved the accuracy of Raman spectroscopy for bacterial species determination compared to the conventional chemometrics. This newly developed approach enables rapid identification and species determination of Arcobacter within an hour following cultivation.IMPORTANCE Rapid identification of bacterial pathogens is critical for developing an early warning system and performing epidemiological investigation. Arcobacter is an emerging foodborne pathogen and has become more important in recent decades. The incidence of Arcobacter species in the agro-ecosystem is probably underestimated mainly due to the limitation in the available detection and characterization techniques. Raman spectroscopy combined with machine learning can accurately identify Arcobacter at the species level in a rapid and reliable manner, providing a promising tool for epidemiological surveillance of this microbe in the agri-food chain. The knowledge elicited from this study has the potential to be used for routine bacterial screening and diagnostics by the government, food industry, and clinics.

Microcavity-coupled fiber Bragg grating with tunable reflection spectra and speed of light.

After a fiber Bragg grating (FBG) is fabricated, the reflection spectrum of the FBG is generally not tunable without mechanical deformation or temperature adjustment. Here we present a microcavity-coupled FBG with both a tunable reflection lineshape and dispersion using electromagnetically induced transparency. The Fano interference of light in the FBG and the microcavity allows for dramatic modification of the reflection spectrum. The phase of the reflected spectrum is continuously tunable between 0 and 2π to produce various Fano lineshapes. The dispersion of the output light is adjustable from normal dispersion to abnormal dispersion, consequently providing an adjustable speed of light. Additionally, it allows the FBG to switch from a notch filter to a bandpass filter at the resonant wavelength, which is not possible in a conventional uniform FBG.

Orbital angular momentum transition of light using a cylindrical vector beam.

We demonstrate that using a single cylindrical vector (CV) beam in two-mode fibers, the orbital angular momentum of light can be switched among -1, 0, and 1. The input CV beam can be a conventional radial and azimuthal polarization distribution or a generalized CV beam, and we first use and verify that a rocking-long period fiber grating generates the tunable generalized CV beam. Because of using a single CV beam as the light source, this approach not only provides an increased stability compared to the conventional superposed eigenmodes method, but also builds a bridge between the polarization singularity beams and the phase singularity beams.

Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups.

Inorganic polyphosphate (polyP) released by human platelets has recently been shown to activate blood clotting and identified as a potential target for the development of novel antithrombotics. Recent studies have shown that polymers with cationic binding groups (CBGs) inhibit polyP and attenuate thrombosis. However, a good molecular-level understanding of the binding mechanism is lacking for further drug development. While molecular dynamics (MD) simulation can provide molecule-level information, the time scale required to simulate these large biomacromolecules makes classical MD simulation impractical. To overcome this challenge, we employed metadynamics simulations with both all-atom and coarse-grained force fields. The force field parameters for polyethylene glycol (PEG) conjugated CBGs and polyP were developed to carry out coarse-grained MD simulations, which enabled simulations of these large biomacromolecules in a reasonable time scale. We found that the length of the PEG tail does not impact the interaction between the (PEG) n-CBG and polyP. As expected, increasing the number of the charged tertiary amine groups in the head group strengthens its binding to polyP. Our simulation shows that (PEG) n-CBG initially form aggregates, mostly with the PEG in the core and the hydrophilic CBG groups pointing toward water; then the aggregates approach the polyP and sandwich the polyP to form a complex. We found that the binding of (PEG) n-CBG remains intact against various lengths of polyP. Binding thermodynamics for two of the (PEG) n-CBG/polyP systems simulated were measured by isothermal titration calorimetry to confirm the key finding of the simulations that the length PEG tail does not influence ligand binding to polyP.

High-resolution broadband sum frequency generation vibrational spectroscopy using intrapulse interference.

We theoretically propose a new approach to obtain high-spectral-resolution sum frequency generation (SFG) vibrational spectra using intrapulse interference. By introducing a π-step phase modulation to the broadband 800 nm pulse, the broadband 800 nm laser pulse splits into two distinguishable pulses in the time domain with a fixed time delay. The resolution of the intrapulse interference SFG can be better than 1 cm-1 and is limited only by the spectral resolution of the spectrometer. This approach can accurately retrieve the amplitude and the relative phase of vibrational peaks. Additionally, the sensitivity of SFG is enhanced by adopting femtosecond IR and femtosecond visible pulses.

Complex Formations between Surfactants and Polyelectrolytes of the Same Charge on a Water Surface.

The mechanism of complex formation between surfactants and polyelectrolytes with the same charge on the water surface was investigated using molecular dynamics simulations and phase-sensitive sum-frequency generation vibrational spectroscopy. Although complex formation between highly charged surfactants and polyelectrolytes of the same charge is generally expected to be prohibited by the electrostatic repulsive force, our study shows that it is possible to form thermodynamically stable complexes when excess ions are present in the solution. We found that anionic partially hydrolyzed polyacrylamide (HPAM) could interact with anionic sodium dodecyl sulfate (SDS) on a water surface in the presence of salts. With excess Na+ ions in the solution, the charge screening effect allows HPAM to weakly interact with SDS via hydrogen bonds. In the presence of divalent Ca2+ ions, the surfactant and the polymer are strongly coupled by forming Ca2+ ion bridges and hydrogen bonds. Our calculation shows that the presence of Ca2+ ions creates a steep binding energy of ∼30 kJ/mol near the water surface. These results were qualitatively verified using phase-sensitive sum-frequency generation vibrational spectroscopy.

Real-time 3D stabilization of a super-resolution microscope using an electrically tunable lens.

Single-molecule localization microscopy (SMLM) has become an essential tool for examining a wide variety of biological structures and processes. However, the relatively long acquisition time makes SMLM prone to drift-induced artifacts. Here we report an optical design with an electrically tunable lens (ETL) that actively stabilizes a SMLM in three dimensions and nearly eliminates the mechanical drift (RMS ~0.7 nm lateral and ~2.7 nm axial). The bifocal design that employed fiducial markers on the coverslip was able to stabilize the sample regardless of the imaging depth. The effectiveness of the ETL was demonstrated by imaging endosomal transferrin receptors near the apical surface of B-lymphocytes at a depth of 8 µm. The drift-free images obtained with the stabilization system showed that the transferrin receptors were present in distinct but heterogeneous clusters with a bimodal size distribution. In contrast, the images obtained without the stabilization system showed a broader unimodal size distribution. Thus, this stabilization system enables a more accurate analysis of cluster topology. Additionally, this ETL-based stabilization system is cost-effective and can be integrated into existing microscopy systems.

In-line polarization rotator based on the quantum-optical analogy.

An in-line polarization rotator (PR) is proposed based on the quantum-optical analogy (QOA). The proposed PR possesses an auxiliary E7 liquid crystal (LC) waveguide in the vicinity of the single-mode fiber (SMF) core. Because of the matched core size, the PR demonstrates good compatibility with the established backbone networks which are composed of conventional SMFs. With optimized parameters for the auxiliary waveguide, the PR offers a near 100% polarization conversion efficiency at the 1550 nm band with a bandwidth of ∼30 nm, a length of ∼4625.9 µm with a large tolerance of ∼550 µm, and a tolerance of the input light polarization angle and rotation angle of the E7 LC of ∼π/30 and ∼π/36 rad, respectively. The performance was verified by the full-vector finite-element method. The proposed PR can be easily fabricated based on the existing photonics crystal fiber manufacturing process, making it a potentially inexpensive device for applications in modern communication systems. Moreover, the QOA, compared with the previous supermode-theory design method, allows a designer to consider several waveguides separately. Therefore, various unique characteristics can be met simultaneously which is consistent with the trend of modern fiber design.

Interactions of Sulfobetaine Zwitterionic Surfactants with Water on Water Surface.

We carried out a combined study using surface tension, phase-sensitive frequency generation (SFG) vibrational spectroscopy, and MD simulations to investigate the industrially relevant zwitterionic surfactant N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (DDAPS) on water surface. The SFG Im(χ(2)) spectra showed that the interaction between DDAPS and water was different from those between biologically relevant zwitterionic phospholipids and water. While zwitterionic phospholipids were found to be anionic-like and flipped water molecules with their OHs pointing toward the air, DDAPS oriented water molecules with their OHs mostly pointing toward the liquid water. We built a new force field for the MD simulation which produced the correct surface tension of water with various DDPAS coverage. The MD simulation showed that the head groups of DDPAS were nearly parallel to the water surface. When the surface coverage of DDPAS was increased, the averaged tilting angle of DDPAS's tails decreased, but it had little effect on the orientation of the headgroup. The sulfobetaine zwitterionic surfactant was found to be more cationic-like because the positively charged group was more capable of orienting interfacial water.

Re-evaluating the surface tension analysis of polyelectrolyte-surfactant mixtures using phase-sensitive sum frequency generation spectroscopy.

Surface tension (ST) has been the most important measure of a molecule's surface activity. However, in many cases the complex behaviors of ST are challenging to interpret. For example, aqueous solutions of sodium docecyl sulfate (SDS) and poly(diallyldimethylammonium chloride) (PDADMAC) show dramatic changes in ST when the concentration of SDS varies. Although surfactants are generally described as "substances that reduce surface tension", new evidence shows that ST may have little changes when a significant amount of SDS is present at the water surface. The decrease of surface entropy resulting from a better ordering of interfacial molecules, such as water, counteracts the decrease of surface enthalpy and is able to keep the ST nearly unchanged. The dramatic ST decrease and recovery of the SDS-PDADMAC mixtures was discovered to be a result of a surface charge reversal. Similar surface charge reversal was also observed in cationic surfactant and anionic polyelectrolyte mixtures.

Cardiomyocyte ryanodine receptor clusters expand and coalesce after application of isoproterenol.

Earlier work has shown that ventricular ryanodine receptors (RyR2) within a cluster rearrange on phosphorylation as well as with a number of other stimuli. Using dSTORM, we investigated the effects of 300 nmol/liter isoproterenol on RyR2 clusters. In rat ventricular cardiomyocytes, there was a symmetrical enlargement of RyR2 cluster areas, a decrease in the edge-to-edge nearest neighbor distance, and distribution changes that suggested movement to increase the cluster areas by coalescence. The surface area covered by the phosphorylated clusters was significantly greater than in the control cells, as was the cluster density. This latter change was accompanied by a decreased cluster fragmentation, implying that new tetramers were brought into the sarcoplasmic reticulum. We propose a possible mechanism to explain these changes. We also visualized individual RyR2 tetramers and confirmed our earlier electron-tomographic finding that the tetramers are in a disorganized but non-random array occupying about half of the cluster area. Multiclusters, cluster groups defined by the maximum distance between their members, were analyzed for various distances. At 100 nm, the areas occupied by the multiclusters just exceeded those of the single clusters, and more than half of the multiclusters had only a single subcluster that could initiate a spark. Phosphorylation increased the size of the multiclusters, markedly so for distances >100 nm. There was no relationship between the number of subclusters in a group and the area covered by it. We conclude that isoproterenol induces rapid, significant, changes in the molecular architecture of excitation-contraction coupling.

3D structured illumination microscope using a spinning disk [Invited].

Three-dimensional (3D) structured illumination microscopy (SIM) improves spatial resolution by a factor of two in both lateral and axial directions. However, the adoption of 3D SIM is limited by low imaging speed, susceptibility to out-of-focus light, and likelihood of reconstruction errors. Here we present a novel approach for 3D SIM using a spinning disk. The disk generates a 3D lattice illumination pattern on the sample and optically reconstructs super-resolved images in real time. This technique achieves a 2-times resolution improvement with a speed up to 100 frames per second while physically rejecting 90% of the background signal.

Role of interfacial water on protein adsorption at cross-linked polyethylene oxide interfaces.

Sum frequency generation (SFG) vibrational spectroscopy was used to study the structure of water at cross-linked PEO film interfaces in the presence of human serum albumin (HSA) protein. Although PEO is charge neutral, the PEO film/water interface exhibited an SFG signal of water similar to that of a highly charged water/silica interface, signifying the presence of ordered water. Ordered water molecules were observed not only at the water/PEO interface, but also within the PEO film. It indicates that the PEO and water form an ordered hydrogen-bonded network extending from the bulk PEO film into liquid water, which can provide an energy barrier for protein adsorption. Upon exposure to the protein solution, the SFG spectra of water at the water/PEO interface remained nearly unperturbed. For comparison, the SFG spectra of water/silica and water/polystyrene interfaces were also studied with and without HSA in the solution. The SFG spectra of the interfacial water were correlated with the amount of protein adsorbed on the surfaces using fluorescence microscopy, which showed that the amount of protein adsorbed on the PEO film was about 10 times less than that on a polystyrene film and 3 times less than that on silica.