Two-Dimensional Flow Nanometry of Biological Nanoparticles for Accurate Determination of Their Size and Emission Intensity

Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and molecular composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging, which is a severe limitation. Surface-sensitive microscopy allows one to precisely determine fluorescence or scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. We here show that attaching BNPs (specifically, vesicles and functionalized gold NPs) to a supported lipid bilayer, subjecting them to a hydrodynamic flow, and tracking their motion via surface-sensitive imaging enable to determine their diffusion coefficients and flow-induced drift velocities and to accurately quantify both BNP size and emission intensity. For vesicles, the high accuracy is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity.Comment: 28 pages, 5 figure

A fast open-source Fiji-macro to quantify virus infection and transfection on single-cell level by fluorescence microscopy

The ability to automatically analyze large quantities of image data is a valuable tool for many biochemical assays, as it rapidly provides reliable data. Here, we describe a fast and robust Fiji macro for the analysis of cellular fluorescence microscopy images with single-cell resolution. The macro presented here was validated by successful reconstruction of fluorescent and non-fluorescent cell mixing ratios (for fluorescence fractions ranging between 0 and 100%) and applied to quantify the efficiency of transfection and virus infection inhibition. It performed well compared with manually obtained image quantification data. Its use is not limited to the cases shown here but is applicable for most monolayered cellular assays with nuclei staining. We provide a detailed description of how the macro works and how it is applied to image data. It can be downloaded free of charge and may be used by and modified according to the needs of the user. • Rapid, simple, and reproducible segmentation of eukaryotic cells in confluent cellular assays • Open-source software for use without technical or computational expertise • Single-cell analysis allows identification and quantification of virus infected cell populations and infection inhibitio

Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10nm and 5ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin

Particle Diffusivity and Free-Energy Profiles in Hydrogels from Time-Resolved Penetration Data

A combined experimental and theoretical method to simultaneously determine diffusivity and free-energy profiles of particles that penetrate into inhomogeneous hydrogel systems is presented. As the only input, arbitrarily normalized concentration profiles from fluorescence intensity data of labeled tracer particles for different penetration times are needed. The method is applied to dextran molecules of varying size that penetrate into hydrogels of polyethylene-glycol chains with different lengths that are covalently cross-linked by hyperbranched polyglycerol hubs. Extracted dextran bulk diffusivities agree well with fluorescence correlation spectroscopy data obtained separately. Empirical scaling laws for dextran diffusivities and free energies inside the hydrogel are identified as a function of the dextran mass. An elastic free-volume model that includes dextran as well as polyethylene-glycol linker flexibility quantitively describes the repulsive dextran-hydrogel interaction free energy, which is of steric origin, and furthermore suggests that the hydrogel mesh-size distribution is rather broad and particle penetration is dominated by large hydrogel pores. Particle penetration into hydrogels for steric particle-hydrogel interactions is thus suggested to be governed by an elastic size-filtering mechanism that involves the tail of the hydrogel pore-size distribution

Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders

Mucin-Inspired, High Molecular Weight Virus Binding Inhibitors Show Biphasic Binding Behavior to Influenza A Viruses

Multivalent binding inhibitors are a promising new class of antivirals that prevent virus infections by inhibiting virus binding to cell membranes. The design of these inhibitors is challenging as many properties, for example, inhibitor size and functionalization with virus attachment factors, strongly influence the inhibition efficiency. Here, virus binding inhibitors are synthesized, the size and functionalization of which are inspired by mucins, which are naturally occurring glycosylated proteins with high molecular weight (MDa range) and interact efficiently with various viruses. Hyperbranched polyglycerols (hPGs) with molecular weights ranging between 10 and 2600 kDa are synthesized, thereby hitting the size of mucins and allowing for determining the impact of inhibitor size on the inhibition efficiency. The hPGs are functionalized with sialic acids and sulfates, as suggested from the structure of mucins, and their inhibition efficiency is determined by probing the inhibition of influenza A virus (IAV) binding to membranes using various methods. The largest, mucin-sized inhibitor shows potent inhibition at pm concentrations, while the inhibition efficiency decreases with decreasing the molecular weight. Interestingly, the concentration-dependent IAV inhibition shows a biphasic behavior, which is attributed to differences in the binding affinity of the inhibitors to the two IAV envelope proteins, neuraminidase, and hemagglutinin

Повторный гидравлический разрыв пласта в горизонтальных скважинах с нецементируемым хвостовиком

Myocardial perfusion imaging with 99mTc-tetrofosmin is based on the assumption of a linear correlation between myocardial blood flow (MBF) and tracer uptake. However, it is known that 99mTc-tetrofosmin uptake is directly related to energy-depen-dent transport processes, such as Na/H ion channel activity, as well as cellular and mitochondrial membrane potentials. Therefore, cellular alterations that affect these energy-depen-dent transport processes ought to influence 99mTc-tetrofosmin uptake independently of blood flow. Because metabolism (18F-FDG)–perfusion (99mTc-tetrofosmin) mismatch myocardium (MPMM) reflects impaired but viable myocardium showing cel-lular alterations, MPMM was chosen to quantify the blood flow– independent effect of cellular alterations on 99mTc-tetrofosmin uptake. Therefore, we compared microsphere-equivalent MBF (MBF_micr; 15O-water PET) and 99mTc-tetrofosmin uptake i

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells

<p>Abstract</p> <p>Background</p> <p>3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenvironment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them.</p> <p>Methods</p> <p>In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay.</p> <p>Results</p> <p>Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures.</p> <p>Conclusions</p> <p>Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to <it>in vivo </it>engraftment of stem and progenitor cells in the context of regenerative medicine.</p