Are the soft, liquid-like structures detected around bacteria by ambient dynamic atomic force microscopy capsules?

High-resolution imaging of bacterial capsules by microscopy is of paramount importance in microbiology due to their role in pathogenesis. This is, however, quite a challenging task due to their delicate nature. In this context, recent reports have claimed successful exploitation of the capacity of atomic force microscopy (AFM) for imaging of extremely deformable (even liquid) surfaces under ambient conditions to detect bacterial capsules in the form of tiny amounts of liquid-like substances around bacteria. In order to further explore this supposed capacity of AFM, in this work, three staphylococcal strains have been scrutinized for the presence of capsules using such an AFM-based approach with a phosphate buffer and water as the suspending liquids. Similar results were obtained with the three strains. AFM showed the presence of liquid-like substances identical to those attributed to bacterial capsules in the previous literature. Extensive imaging and chemical analysis point out the central role of the suspending liquid (buffer) in the formation of these substances. The phenomenon has been reproduced even by using nonliving particles, a finding that refutes the biological origin of the liquid-like substances visualized around the cells. Deliquescence of major components of biological buffers, such as K(2)HPO(4), CaCl(2), or HEPES, is proposed as the fundamental mechanism of the formation of these ultrasmall liquid-like structures. Such an origin could explain the high similarity of our results obtained with three very different strains and also the high similarity of these results to others reported in the literature based on other bacteria and suspending liquids. Finally, possible biological/biomedical implications of the presence of these ultrasmall amounts of liquids wrapping microorganisms are discussed.

On the role of RhoA/ROCK signaling in contact guidance of bone-forming cells on anisotropic Ti6Al4V surfaces.

Patterned surfaces direct cell spatial dynamics, yielding cells oriented along the surface geometry, in a process known as contact guidance. The Rho family of GTPases controls the assembly of focal adhesions and cytoskeleton dynamics, but its role in modulating bone-cell alignment on patterned surfaces remains unknown. This article describes the interactions of two human cell types involved in osseointegration, specifically mesenchymal stem cells and osteoblasts, with submicron- or nano-scale Ti6Al4V grooved surfaces generated by mechanical abrasion. The surface chemistry of the alloy was not affected by grinding, ensuring that the differences found in cellular responses were exclusively due to changes in topography. Patterned surfaces supported cell growth and stimulated mesenchymal stem cell viability. Anisotropic surfaces promoted cell orientation and elongation along the grates. Both cell types oriented on nanometric surfaces with grooves of 150 nm depth and 2 µm width. The number of aligned cells increased by approximately 30% on submicrometric grooves with sizes of about 1 µm depth and 10 µm width. Cells were treated with drugs that attenuate the activities of the GTPase RhoA and one of its downstream effectors, Rho-associated kinase (ROCK), and contact guidance of treated cells on the grooved surfaces was investigated. The data indicate that the RhoA/ROCK pathway is a key modulator of both mesenchymal stem cell and osteoblast orientation on nanometric surface features. RhoA and its effector participate in the alignment of mesenchymal stem cells on submicrometric grooves, but not of osteoblasts. These findings show that RhoA/ROCK signaling is involved in contact guidance of bone-related cells on metallic substrates, although to a varying extent depending on the specific cell type and the dimensions of the pattern.

Ionic liquid microdroplets as versatile lithographic molds for sculpting curved topographies on soft materials surfaces.

Soft lithography comprises a set of approaches for shaping the surface of soft materials such as PDMS on the microscopic scales. These procedures usually begin with the development of templates/masters normally generated by electron or photolithography techniques. However, the richness in available shapes is limited, usually producing shapes containing sharp parts. Innovation is called for to develop reliable approaches capable of imparting well-defined 3D curved shapes to these solids, a topology that is somehow unnatural for solid surfaces. Here we report on the use of tiny drops of room-temperature ionic liquid, organic liquids that have attracted increasing amounts of attention in recent years because of their unique chemical properties) as a versatile platform for imprinting PDMS with tunable 3D curved geometry, which is out of reach of conventional lithographic techniques and ranges from almost flat depressions to almost closed cavities on the millimeter to micrometer scale. The concept exploits a peculiar combination of physical properties displayed by ionic liquids as their null volatility and their polarity, together with some unique properties of liquid surfaces as their virtually null surface roughness. Proof-of-concept experiments show their application as chemical microreactors and ultrasmooth optical lenses. This all-liquid method is simple, low-cost, versatile, maskless, tension-free, and easily scalable, so we envision a community-wide application in numerous modern physical, chemical, biological, and engineering settings.

AFM probing in aqueous environment of Staphylococcus epidermidis cells naturally immobilised on glass: physico-chemistry behind the successful immobilisation.

AFM probing of microbial cells in liquid environments usually requires them to be physically or chemically attached to a solid surface. The fixation mechanisms may influence the nanomechanical characterization done by force curve mapping using an AFM. To study the response of a microbial cell surface to this kind of local measurement this study attempts to overcome the problem associated to the uncertainties introduced by the different fixation treatments by analysing the surface of Staphylococcus epidermidis cells naturally (non-artificially mediated) immobilised on a glass support surface. The particularities of this natural bacterial fixation process for AFM surface analysis are discussed in terms of theoretical predictions of the XDLVO model applied to the systems bacteria/support substratum and bacteria/AFM tip immersed in water. In this sense, in the first part of this study the conditions for adequate natural fixation of three S. epidermidis strains have been analyzed by taking into account the geometries of the bacterium, substrate and tip. In the second part, bacteria are probed without the risk of any possible artefacts due to the mechanical or chemical fixation procedures. Forces measured over the successfully adhered cells have (directly) shown that the untreated bacterial surface suffers from a combination of both reversible and non-reversible deformations during acquisition of force curves all taken under the same operational conditions. This is revealed directly through high-resolution tapping-mode imaging of the bacterial surface immediately following force curve mapping. The results agree with the two different types of force curves that were repeatedly obtained. Interestingly, one type of these force curves suggests that the AFM tip is breaking (rather than pushing) the cell surface during acquisition of the force curve. In this case, adhesive peaks were always observed, suggesting a mechanical origin of the measured pull-off forces. The other type of force curves shows no adhesive peaks and exhibits juxtaposing of approaching and retraction curves, reflecting elastic deformations.

Effect of UV irradiation on the surface Gibbs energy of Ti6Al4V and thermally oxidized Ti6Al4V.

Thermal oxidation of Ti6Al4V increases the thickness, modifies the structure, and changes the amount of alloying elements of the surface titanium dioxide layer with respect to the spontaneous passive layer of Ti6Al4V. The effects on the surface properties of Ti6Al4V and thermally oxidized Ti6Al4V after different periods of UV irradiation have been studied by measurement of water, formamide, and diiodomethane contact angles. The rate of modification of the water contact angle with the irradiation time is dependent on the surface treatment, but the water adhesion work, after an initial energetic step, follows a similar trend for both. Application of the Young equation together with the van Oss approach allowed evaluation of the surface Gibbs energy of the alloys. Similar to the water adhesion work, the surface Gibbs energy dependence on the irradiation time follows a similar trend for both samples and it is due to the change of the electron-donor parameter of the acid-base component. Also, a linear relationship common for both samples has been obtained between the cosines of the water contact angle and the formamide or diiodomethane contact angle. These facts indicate that the surface modification continuously produced by the UV irradiation is similar all along the process and similar for both samples after an energetic threshold for the thermally oxidized sample. It has been also tested that the hydrophilic-hydrophobic conversion is reversible for Ti6Al4V and Ti6Al4V thermally treated.

Sensitivity of surface roughness parameters to changes in the density of scanning points in multi-scale AFM studies. Application to a biomaterial surface.

In the field of biomaterials surfaces, the ability of the atomic force microscope (AFM) to access the surface structure at unprecedented spatial (vertical and lateral) resolution, is helping in a better understanding on how topography affects the overall interaction of biological cells with the material surface. Since cells in a wide range of sizes are in contact with the biomaterial surface, a quantification of the surface structure in such a wide range of dimensional scales is needed. With the advent of the AFM, this can be routinely done in the lab. In this work, we show that even when it is clear that such a scale-dependent study is needed, AFM maps of the biomaterial surface taken at different scanning lengths are not completely consistent when they are taken at the same scanning resolution, as it is usually done: AFM images of different scanning areas have different point-to-point physical distances. We show that this effect influences the quantification of the average (R(a)) and rms (R(q)) roughness parameters determined at different length scales. This is the first time this inconsistency is reported and should be taken into account when roughness is measured in this way. Since differences will be in general in the range of nanometres, this is especially interesting for those processes involving the interaction of the biomaterial surface with small biocolloids as bacteria, while this effect should not represent any problems for larger animal cells.

Looking at the micro-topography of polished and blasted Ti-based biomaterials using atomic force microscopy and contact angle goniometry.

Surface topography of polished and blasted samples of a Ti6Al4V biomaterial has been studied using an atomic force microscope. Surface RMS roughness and surface area have been measured at different scales, from 1 to 50 microm, while at distances below 10 microm the surface RMS roughness in both kinds of samples is not very different, this difference becomes significant at larger scanning sizes. This means that the surface roughness scale that could have a main role in cell adhesion varies depending on the size, shape and flexibility of participating cells. This consideration suggests that in cell-material interaction studies, surface roughness should not be considered as an absolute and independent property of the material, but should be measured at scales in the order of the cell sizes, at least if a microscopic interpretation of the influence of roughness on the adhesion is intended. The microscopic information is contrasted with that coming from a macroscopic approach obtained by contact angle measurements for polar and non-polar liquids whose surface tension is comprised in a broad range. Despite the very large differences of contact angles among liquids for each surface condition, a similar increase for the blasted surface with respect to the polished has been found. Interpretation of these results are in accordance with the microscopic analysis done through the use of a functional roughness parameter, namely the valley fluid retention index, evaluated from the AFM images, which has been shown not to correlate with the RMS roughness, one of the most commonly used roughness parameter.

Optical interference artifacts in contact atomic force microscopy images.

Atomic force microscopy images are usually affected by different kinds of artifacts due to either the microscope design and operation mode or external environmental factors. Optical interferences between the laser light reflected off the top of the cantilever and the light scattered by the surface in the same direction is one of the most frequent sources of height artifact in contact (and occasionally non-contact) images. They are present when imaging highly reflective surfaces, or even when imaging non-reflective materials deposited onto reflective ones. In this study interference patterns have been obtained with a highly polished stainless steel planchet. The influence of these artifacts in surface roughness measurements is discussed, and a semi-quantitative method based on the fast Fourier transform technique is proposed to remove the artifacts from the images. This method improves the results obtained by applying the usual flattening routines.

Study of inhomogeneities in sources prepared for alpha-particle spectrometry using scanning probe microscopy.

For high-resolution alpha-particle spectrometry, sources of high quality must be prepared by methods giving the thinnest and most homogeneous deposit possible on a suitable support. Surface characteristics of several types of alpha-particle sources were studied using a scanning probe microscope. Major inhomogeneities were observed, which means that the materials and techniques used for thc preparation of sources must be improved.