Aleks Ponjavic

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping.

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.

Through-Thickness Velocity Profile Measurements in an Elastohydrodynamic Contact

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.

In situ viscosity measurement of confined liquids

The viscosity of liquids governs crucial physical and engineering phenomena, ranging from diffusion and transport processes of nutrients and chemicals, to the generation of friction and the physics of damping. Engineering fluids frequently experience local conditions that change their bulk rheological properties. While viscosity data can easily be acquired using conventional rheometers, the results are not always applicable to fluids under engineering conditions. This is particularly the case for fluids being sheared at high pressure under severe confinement, which experience very high shear stresses and often show extensive shear thinning. There is a lack of suitable methods for measuring fluid viscosity under such conditions. This work describes a novel in situ viscosity measurement technique to fill this gap. It involves the quantification of the fluorescence lifetime of a fluorescent dye that is sensitive to viscosity. The capability of the developed technique is verified by taking measurements in submicron thick films of two model fluids confined in a ball on flat contact. Viscosity measurements were successfully performed at pressures up to 1.2 GPa and shear rates up to 105 s−1. Spatial heterogeneity in viscosity caused by variations in pressure within the thin fluid film could be observed using the technique. It was also possible to detect differences in the rheological responses of a Newtonian and a non-Newtonian fluid. These first in situ high pressure, high shear viscosity measurements demonstrate the versatility of the proposed technique in providing information on the viscosity in conditions where contemporary techniques are insufficient. More importantly it highlights the complexity of the rheology of engineering fluids and provides a means of verifying existing theories by performing in situ measurements. Information on local viscosity is crucial for understanding the physics of confined fluids and to facilitate improvements in engineering technology.

The effect of boundary slip on elastohydrodynamic lubrication

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.

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.

Flow measurements of a polyphenyl ether oil in an elastohydrodynamic contact.

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.

Nanoscopic Cellular Imaging: Confinement Broadens Understanding

In recent years, single-molecule fluorescence imaging has been reconciling a fundamental mismatch between optical microscopy and subcellular biophysics. However, the next step in nanoscale imaging in living cells can be accessed only by optical excitation confinement geometries. Here, we review three methods of confinement that can enable nanoscale imaging in living cells: excitation confinement by laser illumination with beam shaping; physical confinement by micron-scale geometries in bacterial cells; and nanoscale confinement by nanophotonics.

Capturing resting T cells: the perils of PLL

Supported by a Royal Society University Research Fellowship (UF120277 to S.F.L.) and Research Professorship (RP150066 to D.K.); the EPSRC (EP/L027631/1 to A.P.,); the Wellcome Trust (098274/Z/12/Z to S.J.D., and WT101609MA to R.A.F.); PA Cephalosporin Fund (C.E.); the Wolfson Imaging Centre Oxford (funded by the Wolfson Foundation and Wellcome Trust; 104924/14/Z/14); the Micron Advanced BioImaging Unit (Wellcome Trust Strategic Award 091911); the Medical Research Council (MC_UU_12010/Unit Programmes G0902418 and MC_UU_12025); an MRC/BBSRC/EPSRC award (MR/K01577X/1); and a Marie Sklodowska-Curie Intra-European grant (707348 to I.U.).

4D imaging reveals stage dependent random and directed cell motion during somite morphogenesis

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Although transient structures they are of fundamental importance as they generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about cellular dynamics underlying the morphological transitions during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi-photon imaging of transgenic chick embryos, where all cells express a membrane-bound GFP. We specifically focussed on the dynamic cellular changes in two principle regions within the somite, the medial and lateral domains, to investigate extensive morphological transformations. Furthermore, by using quantitative analysis and cell tracking, we capture for the first time a directed movement of dermomyotomal progenitor cells towards the rostro-medial domain of the dermomyotome, where skeletal muscle formation initiates.

Direct Observation of Murine Prion Protein Replication in Vitro

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.