Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without ...perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform ...multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.
vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke ...analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.
Prions are believed to propagate when an assembly of prion protein (PrP) enters a cell and replicates to produce two or more fibrils, leading to an exponential increase in PrP aggregate number with ...time. However, the molecular basis of this process has not yet been established in detail. Here, we use single-aggregate imaging to study fibril fragmentation and elongation of individual murine PrP aggregates from seeded aggregation in vitro. We found that PrP elongation occurs via a structural conversion from a PK-sensitive to PK-resistant conformer. Fibril fragmentation was found to be length-dependent and resulted in the formation of PK-sensitive fragments. Measurement of the rate constants for these processes also allowed us to predict a simple spreading model for aggregate propagation through the brain, assuming that doubling of the aggregate number is rate-limiting. In contrast, while α-synuclein aggregated by the same mechanism, it showed significantly slower elongation and fragmentation rate constants than PrP, leading to much slower replication rate. Overall, our study shows that fibril elongation with fragmentation are key molecular processes in PrP and α-synuclein aggregate replication, an important concept in prion biology, and also establishes a simple framework to start to determine the main factors that control the rate of prion and prion-like spreading in animals.
A novel methodology, based on the use of phosphorescence imaging, is applied to determine the local through-thickness velocity profile of lubricant in an elastohydrodynamic contact. The technique has ...spatial and temporal resolutions of 40 μm and 340 μs respectively and thus allows lubricant rheology to be investigated at conditions close to service conditions. The capability of the newly-developed method is verified by examining the flow of 5P4E polyphenyl ether, a lubricant base fluid used in very high temperature applications and is well-known for its high viscosity-pressure coefficient. Experimental results highlight the effect of the contact pressure on the velocity profile of this fluid in lubricated contacts. At low pressures, the velocity profile of 5P4E is close to linear, characteristic of Couette flow. As the local pressure increases, its velocity profile progressively deviates from a Couette profile and shear banding is evident at high pressure.
The effect of interfacial slip on the friction and film thickness in an elastohydrodynamic (EHD) contact was directly evaluated. Experiments showed that the film thickness and friction decrease upon ...the application of an oleophobic coating given a sufficient pressure, as opposed to bare glass. Direct measurements of the slip velocity enabled the determination of a power law relationship between pressure and slip length. This implied the existence of spatial heterogeneity of the flow in the tribological contact due to the pressure distribution, which was confirmed by experiments. The power law relationship could also be used to predict the film thickness and friction based on conservation of mass and by using a rheological model for the lubricant. The film thickness and friction predictions were compared to experimental results. The former matched experimental observations. The latter however, underestimated the reduction in friction due to slip by a factor of two, suggesting the necessity of further work to elucidate the interplay between lubricant flow, rheology and friction.
Abstract
Diffractive optical elements (DOEs) have a wide range of applications in optics and photonics, thanks to their capability to perform complex wavefront shaping in a compact form. However, ...widespread applicability of DOEs is still limited, because existing fabrication methods are cumbersome and expensive. Here, we present a simple and cost-effective fabrication approach for solid, high-performance DOEs. The method is based on conjugating two nearly refractive index-matched solidifiable transparent materials. The index matching allows for extreme scaling up of the elements in the axial dimension, which enables simple fabrication of a template using commercially available 3D printing at tens-of-micrometer resolution. We demonstrated the approach by fabricating and using DOEs serving as microlens arrays, vortex plates, including for highly sensitive applications such as vector beam generation and super-resolution microscopy using MINSTED, and phase-masks for three-dimensional single-molecule localization microscopy. Beyond the advantage of making DOEs widely accessible by drastically simplifying their production, the method also overcomes difficulties faced by existing methods in fabricating highly complex elements, such as high-order vortex plates, and spectrum-encoding phase masks for microscopy.
This work presents for the first time through-thickness velocity profiles obtained in an EHL contact by photobleached imaging. The velocity profile was inferred by following the evolution of the ...shape of a photobleached plug formed through the thickness of the fluorescently doped lubricant, oligomer polybutene (PB), in the contact when shear was applied. The proposed methodology was validated by successfully obtaining the expected linear profile with PB experiencing Couette flow. The methodology was then applied to PB in an EHL contact. The variation of the profiles within the contact area was also investigated. The velocity profile of PB in an EHL contact severely deviates from the common linear assumption and exhibits inhomogeneous shear: three regions of varying shear rate have been observed. The phenomenon is shown to be neither due to thermal nor diffusion effects. PB also shows significant slip at the glass-liquid interface. The amount of slip varies with position in the contact. Possible causes, such as pressure-induced viscosity enhancement, as well as the significance of the findings and the benefits of the technique are discussed. The linear velocity profile in an EHL contact is usually assumed for both the film thickness and friction predictions. The profile has, however, never been measured experimentally until now. This work enables the validation of conventional assumptions and the study of flow heterogeneity of lubricants in a contact. This facilitates an improved understanding of the rheology of confined lubricant and hence more accurate predictions of tribological properties.
Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques ...vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity.
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