Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study.

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50-300 nm in diameter, which is below the resolution limit of a conventional light microscopy. To date, there is no standardized method of fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, a semi-automatic (threshold-based) method, and an automatic method based on user-friendly open source machine learning software. Images were obtained using three super resolution techniques - atomic force microscopy (AFM), scanning electron microscopy (SEM), and structured illumination microscopy (SIM). Parameters describing fenestrations such as diameter, area, roundness, frequency, and porosity were measured. Finally, we studied the user bias by comparison of the data obtained by five different users applying provided analysis methods.

Application of a layered model for determination of the elasticity of biological systems.

Elasticity of biological systems is considered to be an important property that might be related to functional or pathological changes. Therefore, careful study and detailed understanding of cell and tissue elasticity is crucial for correct description of their functioning. Atomic Force Microscopy (AFM) is a powerful technique, which allows for determination of the physical properties, such as elasticity, of soft-matter systems in nano-scale. An important step in AFM elasticity studies is a proper interpretation of experimental data. Two most frequently used theoretical schemes applied to determine elasticity are due to Hertz and Sneddon, which are effectively one-parameter models. In this work, we go beyond this approach. Firstly, as elasticity is a local property, we extract from the slope of experimental force-indentation curve an elasticity parameter, which varies with indentation depth. Then secondly, we find best approximation of this parameter by applying the two-layer model with four effective parameters, as proposed by Kovalev. This method is employed to the experimental data taken on murine liver sinusoidal endothelial cells in non-alcoholic fatty liver disease model. The obtained results show additional effects, not seen within the traditional, simplified scheme. Namely, the elasticity of the first layer does not change its value in the model of non-alcoholic fatty liver disease, but the increase of stiffness is noticed in second layer. The second goal of this article is to reveal and discuss the differences between traditional approaches and the one being presented. The deviations from the original assumptions are analysed and the corresponding restrictions on utility of theoretical models are presented.

Fluorine-programmed nanozipping to tailored nanographenes on rutile TiO2 surfaces.

The rational synthesis of nanographenes and carbon nanoribbons directly on nonmetallic surfaces has been an elusive goal for a long time. We report that activation of the carbon (C)-fluorine (F) bond is a reliable and versatile tool enabling intramolecular aryl-aryl coupling directly on metal oxide surfaces. A challenging multistep transformation enabled by C-F bond activation led to a dominolike coupling that yielded tailored nanographenes directly on the rutile titania surface. Because of efficient regioselective zipping, we obtained the target nanographenes from flexible precursors. Fluorine positions in the precursor structure unambiguously dictated the running of the "zipping program," resulting in the rolling up of oligophenylene chains. The high efficiency of the hydrogen fluoride zipping makes our approach attractive for the rational synthesis of nanographenes and nanoribbons directly on insulating and semiconducting surfaces.

Atomic Force Microscopy Reveals the Dynamic Morphology of Fenestrations in Live Liver Sinusoidal Endothelial Cells.

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Morphology and force probing of primary murine liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) represent unique type of endothelial cells featured by their characteristic morphology, ie, lack of a basement membrane and presence of fenestrations-transmembrane pores acting as a dynamic filter between the vascular space and the liver parenchyma. Delicate structure of LSECs membrane combined with a submicron size of fenestrations hinders their visualization in live cells. In this work, we apply atomic force microscopy contact mode to characterize fenestrations in LSECs. We reveal the structure of fenestrations in live LSECs. Moreover, we show that the high-resolution imaging of fenestrations is possible for the glutaraldehyde-fixed LSECs. Finally, thorough information about the morphology of LSECs including great contrast in visualization of sieve plates and fenestrations is provided using Force Modulation mode. We show also the ability to precisely localize the cell nuclei in fixed LSECs. It can be helpful for more precise description of nanomechanical properties of cell nuclei using atomic force microscopy. Presented methodology combining high-quality imaging of fixed cells with an additional nanomechanical information of both live and fixed LSECs provides a unique approach to study LSECs morphology and nanomechanics that could foster understanding of the role of LSECs in maintaining liver homeostasis.

Nanomechanical sensing of the endothelial cell response to anti-inflammatory action of 1-methylnicotinamide chloride.

BACKGROUND: There is increasing evidence that cell elastic properties should change considerably in response to chemical agents affecting the physiological state of the endothelium. In this work, a novel assay for testing prospective endothelium-targeted agents in vitro is presented. MATERIALS AND METHODS: The proposed methodology is based on nanoindentation spectroscopy using an atomic force microscope tip, which allows for quantitative evaluation of cell stiffness. As an example, we chose a pyridine derivative, 1-methylnicotinamide chloride (MNA), known to have antithrombotic and anti-inflammatory properties, as reported in recent in vivo experiments. RESULTS: First, we determined a concentration range of MNA in which physiological parameters of the endothelial cells in vitro are not affected. Then, cell dysfunction was induced by incubation with tumor necrosis factor-alpha (TNF-α) and the cellular response to MNA treatment after TNF-α incubation was studied. In parallel to the nanoindentation spectroscopy, the endothelium phenotype was characterized using a fluorescence spectroscopy with F-actin labeling, and biochemical methods, such as secretion measurements of both nitric oxide (NO), and prostacyclin (PGI2) regulatory agents. CONCLUSION: We found that MNA could reverse the dysfunction of the endothelium caused by inflammation, if applied in the proper time and to the concentration scheme established in our investigations. A surprisingly close correlation was found between effective Young's modulus of the cells and actin polymerization/depolymerization processes in the endothelium cortical cytoskeleton, as well as NO and PGI2 levels. These results allow us to construct the physiological model of sequential intracellular pathways activated in the endothelium by MNA.

High resolution LT-STM imaging of PTCDA molecules assembled on an InSb(001) c(8 × 2) surface.

The self-assembling of 3,4,9,10-perylene-tetracarboxylic-dianhydride (PTCDA) molecules deposited on an InSb(001) c(8 × 2) surface at sub-monolayer quantities has been investigated at low temperature (77 K) using scanning tunnelling microscopy. Sub-molecular resolution was obtained on PTCDA molecules. The results reveal that individual PTCDA molecules are arranged on the substrate in chains parallel to the [110] crystallographic direction, correlated with characteristic features of the low temperature InSb(001) c(8 × 2) surface electronic structure. A structural model for PTCDA molecules adsorbed on InSb is proposed.

PTCDA molecules on a KBr/InSb system: a low temperature STM study.

We have used scanning tunnelling microscopy (STM) at 77 K to investigate 3,4,9,10-perylene-tetracarboxylic dianhydride (PTCDA) molecules adsorbed on an ultrathin (1-2 monolayer (ML)) film of KBr grown on a c(8 × 2)InSb(001) substrate. The molecules are stabilized both at the KBr steps and on the terraces. On the 1 ML film the PTCDA molecules appear predominantly as single entities, whereas on the 2 ML film formation of molecular clusters is preferred. Differences in the adsorption configurations indicate that the interaction between the molecules and the surface differs significantly for the cases of 1 and 2 ML films. We present images of the molecules obtained with sub-molecular resolution for both filled and empty state sampling modes. We argue that the highest occupied molecular orbital (the lowest unoccupied molecular orbital) is responsible for intramolecular contrast in filled (empty) state images of the molecules, even though they are deformed due to strong interaction with the substrate.

PTCDA molecules on an InSb(001) surface studied with atomic force microscopy.

PTCDA (3,4,9,10-perylene-tetracarboxylic-dianhydride) molecular structures assembled on an InSb(001) c(8 × 2) reconstructed surface have been studied using frequency modulated atomic force microscopy. The high-resolution imaging of the structures is possible through repulsive interactions, using the constant height scanning mode. During initial stages of growth the [110] diffusion channel dominates as indicated by formation of long PTCDA molecular chains parallel to the [110] crystallographic direction on the InSb surface. For a single monolayer coverage a wetting layer of PTCDA is formed. Finally it is shown that the PTCDA/InSb is a promising system for building molecular nanostructures by manipulation of single molecules with the AFM tip.

X-ray tomographic imaging of crystal structure at the atomic level.

A direct nondiffractive tomographic algorithm is proposed for the determination of the crystal structure from real-space projections obtained by illuminating the sample with white x rays. This approach was applied to the pattern of the directional fine structure in absorption of white x rays recorded for a GaP crystal and allowed for a determination of the electron density distribution within the unit cell.

Site-selective holographic imaging of iron arrangements in magnetite.

Complex gamma-ray holograms were recorded by tuning to the nuclear absorption lines of 57Fe in magnetite corresponding to different hyperfine fields. The numerical reconstruction of the holograms to real space provided three-dimensional images of local iron arrangements in octahedral and tetrahedral sublattices of magnetite. This direct site-selective imaging of atomic structure was performed using a tabletop experimental setup.

Atomic structure of InSb(001) and GaAs(001) surfaces imaged with noncontact atomic force microscopy.

Noncontact atomic force microscopy (NC-AFM) has been used to study the c(8x2) InSb(001) and the c(8x2) GaAs(001) surfaces prepared by sputter cleaning and annealing. Atomically resolved tip-surface interaction maps display different characteristic patterns depending on the tip front atom type. It is shown that representative AFM maps can be interpreted consistently with the most recent structural model of A(III)B(V)(001) surface, as corresponding to the A(III) sublattice, to the B(V) sublattice, or to the combination of both sublattices.

Surface topography dependent desorption of alkali halides

Electron-stimulated desorption of the (100)KBr surface has been investigated in vacuum with noncontact atomic force microscopy and mass spectroscopy. It has been found that both desorption components (K and Br) show oscillatory dependence on the electron dose with the oscillation amplitude decaying gradually. These results correspond with periodically varying, as a result of a layer-by-layer desorption, surface topography. It is proposed that the surface terrace edges act as traps for excited F centers diffusing in the crystal. The oscillating density of terrace edges varies surface recombination/reflection rates for the F centers and modulates the balance between surface and bulk deexcitation of the crystal.

Bombardment Induced Electron-Capture Processes at Sodium Halide Surfaces.

Discrete features observed in the energy distribution of electrons emitted from ion-bombarded sodium halide surfaces can be attributed to a new type of collisional deexcitation mechanism. Such a mechanism involves sodium atoms in bombardment-excited autoionizing states that are the result of cascade collisions within the crystal lattice. This deexcitation process, in contrast to that for a metal, is not simply a consequence of the inner-shell lifetime of the initial collisionally excited sodium Na+* ion. Rather, the deexcitation consists of a sequence of lattice collisions during which the excited Na+* ion captures an electron to form the inner-shell-excited Na0* states responsible for the observed transitions. The formation of such autoionizing Na0* states is described within the framework of a new model in which excitation processes and localized collisional electron-transfer mechanisms are taken into account. These localized electron-transfer processes make possible new channels for electronic deexcitation, chemical dissociation, and defect production; they are critical for understanding inelastic ion-surface collisions in solids.