Wide-field optical imaging of electrical charge and chemical reactions at the solid-liquid interface.

From molecules and particles to macroscopic surfaces immersed in fluids, chemical reactions often endow interfaces with electrical charge which in turn governs surface interactions and interfacial phenomena. The ability to measure the electrical properties of a material immersed in any solvent, as well as to monitor the spatial heterogeneity and temporal variation thereof, has been a long-standing challenge. Here, we describe an optical microscopy-based approach to probe the surface charge distribution of a range of materials, including inorganic oxide, polymer, and polyelectrolyte films, in contact with a fluid. The method relies on optical visualization of the electrical repulsion between diffusing charged probe molecules and the unknown surface to be characterized. Rapid image-based measurements enable us to further determine isoelectric points of the material as well as properties of its ionizable chemical groups. We further demonstrate the ability to optically monitor chemically triggered surface charge changes with millisecond time resolution. Finally, we present a scanning-surface probe technique capable of diffraction-limited imaging of spatial heterogeneities in chemical composition and charge over large areas. This technique will enable facile characterization of the solid-liquid interface with wide-ranging relevance across application areas from biology to engineering.

EAP45 association with budding HIV-1: Kinetics and domain requirements.

A number of viruses including HIV use the ESCRT system to bud from the infected cell. We have previously confirmed biochemically that ESCRT-II is involved in this process in HIV-1 and have defined the molecular domains that are important for this. Here, using SNAP-tag fluorescent labelling and both fixed and live cell imaging we show that the ESCRT-II component EAP45 colocalises with the HIV protein Gag at the plasma membrane in a temporal and quantitative manner, similar to that previously shown for ALIX and Gag. We show evidence that a proportion of EAP45 may be packaged within virions, and we confirm the importance of the N terminus of EAP45 and specifically the H0 domain in this process. By contrast, the Glue domain of EAP45 is more critical for recruitment during cytokinesis, emphasising that viruses have ways of recruiting cellular components that may be distinct from those used by some cellular processes. This raises the prospect of selective interference with the pathway to inhibit viral function while leaving cellular functions relatively unperturbed.

DNA Nanostructures for Targeted Antimicrobial Delivery.

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.

Frame localisation optical projection tomography.

We present a tomographic reconstruction algorithm (flOPT), which is applied to Optical Projection Tomography (OPT) images, that is robust to mechanical jitter and systematic angular and spatial drift. OPT relies on precise mechanical rotation and is less mechanically stable than large-scale computer tomography (CT) scanning systems, leading to reconstruction artefacts. The algorithm uses multiple (5+) tracked fiducial beads to recover the sample pose and the image rays are then back-projected at each orientation. The quality of the image reconstruction using the proposed algorithm shows an improvement when compared to the Radon transform. Moreover, when adding a systematic spatial and angular mechanical drift, the reconstruction shows a significant improvement over the Radon transform.

Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources.

Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized "EDEMP cycle" (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each.IMPORTANCEPseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer ("fluxomic") analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed ("fluxed") through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell's electron transfer pathways. Having access to this "blueprint" is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons.

Extrinsic cues trigger the local translation of specific mRNAs in growing axons via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a mechanism linking signals to local translation but it is not known how broadly this mechanism operates, nor whether it can selectively regulate mRNA translation. We report that receptor-ribosome coupling is employed by multiple guidance cue receptors and this interaction is mRNA-dependent. We find that different receptors associate with distinct sets of mRNAs and RNA-binding proteins. Cue stimulation of growing Xenopus retinal ganglion cell axons induces rapid dissociation of ribosomes from receptors and the selective translation of receptor-specific mRNAs. Further, we show that receptor-ribosome dissociation and cue-induced selective translation are inhibited by co-exposure to translation-repressive cues, suggesting a novel mode of signal integration. Our findings reveal receptor-specific interactomes and suggest a generalizable model for cue-selective control of the local proteome.

OptiJ: Open-source optical projection tomography of large organ samples.

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples.