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Characterization of changes to the cell surface during the life cycle of Streptomyces coelicolor: atomic force microscopy of living cells.

Cell surface changes that accompany the complex life cycle of Streptomyces coelicolor were monitored by atomic force microscopy (AFM) of living cells. Images were obtained using tapping mode to reveal that young, branching vegetative hyphae have a relatively smooth surface and are attached to an inert silica surface by means of a secreted extracellular matrix. Older hyphae, representing a transition between substrate and aerial growth, are sparsely decorated with fibers. Previously, a well-organized stable mosaic of fibers, called the rodlet layer, coating the surface of spores has been observed using electron microscopy. AFM revealed that aerial hyphae, prior to sporulation, possess a relatively unstable dense heterogeneous fibrous layer. Material from this layer is shed as the hyphae mature, revealing a more tightly organized fibrous mosaic layer typical of spores. The aerial hyphae are also characterized by the absence of the secreted extracellular matrix. The formation of sporulation septa is accompanied by modification to the surface layer, which undergoes localized temporary disruption at the sites of cell division. The characteristics of the hyphal surfaces of mutants show how various chaplin and rodlin proteins contribute to the formation of fibrous layers of differing stabilities. Finally, older spores with a compact rodlet layer develop surface concavities that are attributed to a reduction of intracellular turgor pressure as metabolic activity slows.

Bacterial Proteins↗

Analysis of immunolabeled cells by atomic force microscopy, optical microscopy, and flow cytometry.

In this study we investigated the applicability of the (silver-enhanced) immunogold labeling method for atomic force microscopy. Human lymphocytes were labeled with anti-CD3 conjugated to fluorescein isothiocyanate and a secondary antibody (goat anti-mouse) linked with 1- or 30-nm colloidal gold particles. Silver enhancement was applied on these labeled cells to increase the size of the labels. In a setup combining an inverted optical microscope and a stand-alone atomic force microscope, a direct correlation was made between the force and the fluorescent images. Additionally, we performed flow cytometric analysis. From the results we conclude that immunogold labeling using small labels (1 nm) in combination with silver enhancement (30 min) proves to be a reliable method for high-resolution cell surface antigen detection in atomic force microscopy.

Animals↗

Microscopic observation of unworn siloxane-hydrogel soft contact lenses by atomic force microscopy.

In the present study, samples of lotrafilcon A, balafilcon A, and galyfilcon A contact lenses were observed by atomic force microscopy (AFM) in tapping mode at areas ranging from 0.25 to 400 microm2. Mean roughness (Ra), root-mean-square roughness (Rms) and maximum roughness (Rmax) in nanometers were obtained for the three lens materials at different magnifications. The three contact lenses showed significantly different surface topography. However, roughness values were dependent of the surface area to be analyzed. For a 1 microm2 area, statistics revealed a significantly more irregular surface of balafilcon A (Ra = 6.44 nm; Rms = 8.30 nm; Rmax = 96.82 nm) compared with lotrafilcon A (Ra = 2.40 nm; Rms = 3.19 nm; Rmax = 40.89 nm) and galyfilcon A (Ra = 1.40 nm; Rms = 1.79 nm; Rmax = 15.33 nm). Ra and Rms were the most consistent parameters, with Rmax presenting more variability for larger surface areas. The higher roughness of balafilcon A is attributed to the plasma oxidation treatment used to improve wettability. Conversely, galyfilcon A displays a smoother surface. Present observations could have implications in clinical aspects of siloxane-hydrogel contact lens wear such as lens spoliation, resistance to bacterial adhesion, or mechanical interaction with the ocular surface.

Contact Lenses, Hydrophilic↗

Surface morphology and kinetic properties in rapid growth of EAFP protein crystals investigated by atomic force microscopy.

The whole process of EAFP protein monoclinic crystal growth with an extremely fast rate has been observed by atomic force microscopy. The results showed that the patterns of the growth images in rapidly growing crystals are complicated. The two-dimensional multi-layered stacks of growth steps are characteristic of higher supersaturation and the growth of steps proceeds in a manner of strong anisotropic spiral dislocations dominantly under lower supersaturation conditions. The complex dislocation sources, including multiple dislocation and multi-interacting single dislocation sources, the constant step-split and the propagation of trooped steps were observed on the {100} surfaces of growing EAFP crystals. The step height of each layer generated either by two-dimensional nucleation at higher supersaturation or by screw dislocation at lower supersaturation is about 2-3 nm, which corresponds to the length of the crystallographic unit cell. Although the rate of advancement for each growth step is similar to that of other protein crystal growth, the unique way of the propagation distinct with the trooped steps, by which a bundle of steps are strapped together, would be responsible for the rapid growth of EAFP crystals. All features show a possible mechanism by which the fast growth of EAFP crystals could be attained. The structural basis of the growth mechanism is also discussed.

Eucommiaceae↗

Atomic force microscopy of plant cell walls, plant cell wall polysaccharides and gels.

Methods developed for the routine imaging of polysaccharides by atomic force microscopy (AFM) have been used to image plant polysaccharides from higher plants (pectin) and algae (carrageenan). These methods have been extended to image K-carrageenan association in hydrated films. Finally, AFM has been used to image polysaccharide architecture in moist plant cell walls. Simple experimental and image processing methods have been used to enhance molecular structure in 'rough' cell wall surfaces.

Cell Wall↗

Atomic force microscopy evaluation of aqueous interfaces of immobilized hyaluronan.

Hyaluronan (HA) was immobilized on aminated glass surfaces in three different ways: by simple ionic interaction and by covalent linking at low density and at full density. In agreement with previous reports, in vitro experiments show that the outcome of fibroblast adhesion tests is markedly affected by the details of the coupling procedure, suggesting that different interfacial forces are operating at the aqueous/HA interface in the three cases investigated. The interfacial properties of the HA-coated surfaces were probed by force-distance curves obtained with the atomic force microscope (AFM). This approach readily shows significant differences among the tested samples, which are directly related to the coupling strategy and to results of cell adhesion tests. In particular, the range of interaction between the tip and the surface is much lower when HA is covalently linked than when it is ionically coupled, suggesting a more compact surface structure in the former case. Increasing HA surface density minimizes the interaction force between the surface and the AFM tip, likely reflecting more complete shielding by the HA chains of the underlying substrate. In summary, these measurements clearly show the different nature of the aqueous interfaces tested, and underline the role of this analytical approach in the development and control of finely tuned biomaterial surfaces.

Animals↗

Atomic force microscopy investigation of a chlorella virus, PBCV-1.

A virus PBCV-1, which infects certain fresh water algae and has been shown by transmission and cryo-electron microscopy to exist as a triskaidecahedron, was imaged using atomic force microscopy (AFM). From AFM the particles have diameters of about 190nm and the overall structure is in all important respects consistent with existing models. The surface lattice of the virion is composed of trimeric capsid proteins distributed according to p3 symmetry to create a honeycomb arrangement of raised edges forming quasi-hexagonal cells. At the pentagonal vertices are five copies of a different protein forming an exact pentagon, and this has yet another unique protein in its center. The apical protein exhibits some unusual mechanical properties in that it can be made to retract into the virion interior when subjected to AFM tip pressure. When PBCV-1 virions degrade, they give rise to small, uniform, spherical, and virus like particles (VLP) consistent with T=1 or 3 icosahedral products. Also observed upon disintegration are strands of linear dsDNA. Fibers of unknown function are also occasionally seen associated with some virions.

Chlorella↗

Tapping-mode atomic force microscopy in fluid of hydrated extracellular matrix.

Fragments of native, hydrated rat tail tendon were imaged by tapping-mode atomic force microscopy while immersed in fluid. The specimens were soft and sensitive to the operating parameters, and with minimal imaging pressure the collagen fibrils appeared covered by irregular blobs or by filamentous material. A slight increase in pressure caused the underlying fibril surface to appear, with an evident D-period, gap- and overlap-zones and three intraperiod ridges. Fibrils often ran parallel and in phase, implying some coupling mechanism. Longitudinal subfibrils, 8-9 nm thick, occasionally appeared. The simultaneous acquisition of the "tapping amplitude" along with the usual "height" channel clearly confirmed the presence of longitudinal subfibrils, indicative of the inner architecture of the fibril.

Animals↗

What can we learn from atomic force microscopy adhesion measurements with single drug particles?

Frequently solid dosage form formulation manufacture and delivery depend critically on the control and exploitation of interparticulate interactions. Traditional approaches to understand such interactions rely on indirect assessments of adhesion or consider the behaviour of large numbers of particles. In recent years, the possibility of characterizing and perhaps quantifying forces of adhesion between individual micron and sub-micron sized particles has become viable using the atomic force microscope. This has significant potential in formulation development, particularly in the optimization of inhalation and other solid-dosage form based therapies. However, before a widespread acceptance of this approach by pharmaceutical scientists and industry can proceed a number of issues remain to be considered. These include how can single particle events be mapped on to bulk behaviour, the need to understand the sometimes wide variations in adhesion data observed and can formulations be compared quantitatively and perhaps be screened by this approach?

Adhesiveness↗

Nanotribological characterization of human hair and skin using atomic force microscopy.

Healthy hair and skin is highly desired. Characterization of their morphological, frictional, and adhesive properties (tribological properties) is essential to enhance understanding of hair and skin and to advance the science. Literature on the tribological characterization of hair and skin is scarce to date. The paper presents nanotribological data and analysis on hair (Caucasian, Asian, and African hair at virgin, chemo-mechanically damaged, and treated conditions) and synthetic hair and skin, as well as roughness data of human skin replica. Roughness statistics are presented to characterize the vertical and spatial surface parameters. Average coefficient of friction values were determined for each ethnicity and hair type, and are discussed. The directionality dependence of friction is also discussed. Magnitude and spatial distribution of adhesive force are used to estimate thickness and distribution of the conditioner film.

Asian People↗

Collagen adsorption and structure on polymer surfaces observed by atomic force microscopy.

The structure and adsorption patterns of type I and type III collagen were imaged on various polymer substrates with atomic force microscopy. Type I collagen had higher adsorption on polystyrene than on a series of polymethacrylates and formed a network of tightly, interwoven strands. Upon adsorption to different polymethacrylates, with varying side chain lengths, the collagen molecules formed long, branching fibrils. Types I and III collagen had different adsorption patterns, in some cases, on the identical substrate material. For example, instead of forming a tightly packed network, type III forms long, branching fibers on the polystyrene surface. On other materials, such as poly(n-butyl methacrylate), the two types of collagen showed similar adsorption pattern and structure. Adsorbed collagen was also imaged on various blends of polystyrene and polymethacrylates to determine how the polymer surface chemical structure and surface topography mediates protein adsorption.

Adsorption↗

Unraveling the nanostructure of supramolecular assemblies of hydrogen-bonded rosettes on graphite: an atomic force microscopy study.

The self-organization of multicomponent tetrarosette assemblies into ordered nanostructures on graphite surfaces has been studied by atomic force microscopy (AFM). Real-space information on the level of individual molecules allowed us to analyze the underlying structure in unprecedented detail. In highly ordered nanorod domains, tetrarosettes 1(3) x (DEB)(12) arrange in the form of parallel rows with a spacing of 4.6 +/- 0.1 nm. High resolution AFM revealed the internal packing of the tetrarosette assemblies in these rows, which can be described by an oblique lattice with a = 2.5 +/- 0.3 nm, b = 5.0 +/- 0.1 nm, and gamma = 122 +/- 3 degrees. The results, together with recent improvements in synthetic approaches, contribute to the development of a general strategy to develop H-bonding-based nanostructures with molecular precision.

Graphite↗