Aleks Ponjavic

• PhD
• Fellow at University of Leeds

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer–promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, de...

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir‐PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an a...

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid preci...

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir‐PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an a...

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (res...

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 th...

Significance Despite the success of T cell–redirecting immunotherapies for blood cancers, toxic, off-target side effects prevent their safe application to other diseases. Part of the challenge lies in our incomplete understanding of T cell receptor (TCR) triggering. Although we know signaling requires TCR phosphorylation, how extracellular TCR/liga...

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 o...

Detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular respo...

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. We introduce the structure-specific chemical antibody; a Y shaped, bioinspired small molecule with a dimeric region to mimic avidity, a...

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 (...

We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views,...

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 μm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization...

Enhancer-promoter dynamics are critical for the spatiotemporal control of gene expression, but it remains unclear how these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. Here, we show that the intact NuRD complex increases CTCF/Cohesin binding and the probability of the interacti...

Cell-cell contacts often underpin signaling between cells. For immunology, the binding of a T cell receptor to an antigen-presenting pMHC initiates downstream signaling and an immune response. Although this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many, often contradictory observations...

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching. While the initial emphasis in the 3D...

Cell-cell contact often underpins signalling between cells. Contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm. Protein binding and accumulation leads to the contact becoming crowded, reducing the rate of protein diffusion, even for unbound proteins. Here we show that, by tracking quantum dots of...

Significance One approach to testing biological theories is to determine if they are predictive. We have developed a simple, theoretical treatment of T cell receptor (TCR) triggering that relies on just two physical principles: ( i ) the time TCRs spend in cell–cell contacts depleted of large tyrosine phosphatases and ( ii ) constraints on the size...

Proteasomes are molecular machineries responsible for regulated protein degradation and general homeostasis. The distribution of this degradation capacity is reflected by the cellular localisation of proteasomal particles. Here we combine super-resolution imaging, single-particle tracking (SPT) and single-cell patch clamp techniques to investigate...

We present a sensitive inverted light sheet microscope, capable of single-molecule fluorescence imaging of cells in 96-well plates. Light sheet microscope designs are often complex and costly, requiring custom-made sample chambers that are incompatible with standard cell culture samples. To overcome this limitation, we have developed single-objecti...

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 fragmen...

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...

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling protei...

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Of fundamental importance, they are transient structures that generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very litt...

To the editor — Full understanding of lymphocyte activation will require thorough characterization of the ‘resting’ state and how it changes. Surfaces coated with the cationic homopolymer poly-L-lysine (PLL) are widely used for total internal reflection fluorescence (TIRF) imaging of the organization of surface proteins on resting lymphocytes^1,2,3...

Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing...

Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localisation at the cell membrane. Here, by employing...

The ultrashear viscometer (USV) has been adapted and employed to investigate the permanent shear thinning of polystyrene solutions in a series of phthalate ester base fluids. The permanent shear stability index based on viscosities measured at 10⁶ s⁻¹, PSSI(10⁶), has been found to be a convenient way to express the magnitude of permanent shear thin...

Presentation at the ISDB conference, Singapore, 2017

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...

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...

Molybdenum dialkyldithiocarbamates (MoDTCs) are very effective friction modifier additives for use in engine oils and other lubricants. However, as engine oils age during extended drain intervals, MoDTCs can lose some or all of their ability to reduce friction and this is generally believed to result from their oxidative degradation. In this study,...

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 m...

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...

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...

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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...

The rheology of submicron thick polymer melt is examined under high normal pressure conditions by a recently developed photobleached-fluorescence imaging velocimetry technique. In particular, the validity and limitation of Reynold equation solution, which suggests a linear through-thickness velocity profile, is investigated. Polybutene (PB) is shea...

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 de...

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 ap...

This paper documents a computational study of flow profiles in confined fluids. The study is motivated by experimental evidence for deviation from Couette flow found by one of the authors (JSW). The computational study examines several possible stress-strain relations. Since a linear profile is the only possible solution for a constant stress layer...

The knowledge of the through-thickness flow profile of lubricants confined between two rubbing surfaces is necessary for the friction prediction of lubricated engineering systems. While it is crucial to materials selection and engineering design, little work on the direct measurement of lubricant flow has been performed in elastohydrodynamic lubric...

The through-thickness velocity profile of a sub-micron thick polymer melt, under - elastohydrodynamic (EHL) conditions, has been recently obtained for the first time by the authors using photobleached imaging technique. In this work, the developed technique is applied to investigate the effect of shear rate on the evolution of velocity profile in p...

Much effort has been spent recently on experimentally proving the existence of interfacial slip of a Newtonian fluid. A constant limitation is the proximity to the surface at which the velocity of a fluid can be measured. A new technique is developed to maximise this proximity. The objective is to acquire velocity measurements of a fluid as close a...

Under certain conditions, surface-active molecules are known to self-organise into SAMs according to two main driving forces: molecular surface adsorption via diffusive/convective transport, and surface reorganisation and growth. For the latter in-situ methods are required to deconvolute the complex underlying kinetics and dynamics. To this end, a...