Fluorescence imaging of cells using long-range electromagnetic surface waves for excitation.

We present a depth-localized illumination technique for wide-field fluorescence microscopy, based on long-range optical surface waves. This technique allows one to excite the fluorescence only in a thin near-substrate layer of the specimen. Our experimental setup is compatible with both upright and inverted microscopes. It provides fluorescent microscopic images, which are superior to the epifluorescence ones in signal-to-noise ratio, contrast, and detail. We demonstrate the applicability of our technique for imaging both bacterial and eukaryotic cells (E. coli and HeLa, respectively).

Application of the Johnson-Kendall-Roberts model in AFM-based mechanical measurements on cells and gel.

The force-distance curves (FCs) obtained by the atomic force microscope (AFM) with colloid probes contain information about both the viscoelastic properties and adhesion of a sample. Here, we processed both the approach and retraction parts of FCs obtained on polyacrylamide gels (in water or PBS) and Vero cells (in a culture medium). The Johnson-Kendall-Roberts model was applied to the retraction curves to account for the adhesion. The effects of loading rate, holding time and indentation depth on adhesion force and Young's modulus, calculated from approach and retraction curves, were studied. It was shown that both bulk and local interfacial viscoelasticity can affect the observed approach-retraction hysteresis and measured parameters. The addition of 1% bovine serum albumin (BSA) decreased adhesion of the probe to the PAA gel surface, so interfacial viscoelasticity effects were diminished. On the contrary, the adhesiveness of Vero cells increased after BSA addition, indicating the complex nature of the cell-probe interaction.

Mechanical properties of fibroblasts depend on level of cancer transformation.

Recently, it was revealed that tumor cells are significantly softer than normal cells. Although this phenomenon is well known, it is connected with many questions which are still unanswered. Among these questions are the molecular mechanisms which cause the change in stiffness and the correlation between cell mechanical properties and their metastatic potential. We studied mechanical properties of cells with different levels of cancer transformation. Transformed cells in three systems with different transformation types (monooncogenic N-RAS, viral and cells of tumor origin) were characterized according to their morphology, actin cytoskeleton and focal adhesion organization. Transformation led to reduction of cell spreading and thus decreasing the cell area, disorganization of actin cytoskeleton, lack of actin stress fibers and decline in the number and size of focal adhesions. These alterations manifested in a varying degree depending on type of transformation. Force spectroscopy by atomic force microscopy with spherical probes was carried out to measure the Young's modulus of cells. In all cases the Young's moduli were fitted well by log-normal distribution. All the transformed cell lines were found to be 40-80% softer than the corresponding normal ones. For the cell system with a low level of transformation the difference in stiffness was less pronounced than for the two other systems. This suggests that cell mechanical properties change upon transformation, and acquisition of invasive capabilities is accompanied by significant softening.

Atomic force microscopic study of the structure of high-density polyethylene deformed in liquid medium by crazing mechanism.

A procedure has been developed for the direct atomic force microscopic (AFM) examination of the native structure of high-density polyethylene (HDPE) deformed in an adsorption-active liquid medium (AALM) by the crazing mechanism. The AFM investigation has been carried out in the presence of a liquid medium under conditions preventing deformed films from shrinkage. Deformation of HDPE in AALM has been shown to proceed through the delocalized crazing mechanism and result in the development of a fibrillar-porous structure. The structural parameters of the crazed polymer have been determined. The obtained AFM images demonstrate a nanosized nonuniformity of the deformation and enable one to observe the structural rearrangements that take place in the deformed polymer after removal of the liquid medium and stress relaxation. A structural similarity has been revealed between HDPE deformed in the AALM and hard elastic polymers.

The effects of confluency on cell mechanical properties.

Mechanical properties of cells depend on various external and internal factors, like substrate stiffness and surface modifications, cell ageing and disease state. Some other currently unknown factors may exist. In this study we used force spectroscopy by AFM, confocal microscopy and flow cytometry to investigate the difference between single non-confluent and confluent (in monolayer) Vero cells. In all cases the stiffness values were fitted by log-normal rather than normal distribution. Log-normal distribution was also found for an amount of cortical actin in cells by flow cytometry. Cells in the monolayer were characterized by a significantly lower (1.4-1.7 times) Young's modulus and amount of cortical actin than in either of the single non-confluent cells or cells migrating in the experimental wound. Young's modulus as a function of indentation speed followed a weak power law for all the studied cell states, while the value of the exponent was higher for cells growing in monolayer. These results show that intercellular contacts and cell motile state significantly influence the cell mechanical properties.

[The effect of poly(3-hydroxybutyrate) modification by poly(ethylene glycol) on the viability of cells grown on the polymer films].

A biodegradable polymer of bacterial origin, poly(3-hydroxybutyrate) (PHB), is intensively studied as biomaterial for tissue engineering. However, factors determining its biocompatibility still require better understanding. To analyze the PHB films biocompatibility, the polymer material was modified by hydrophilic polymer, poly(ethylene glycol) 300 (PEG). The blends PHB/PEG with different PEG content (10, 20, 30 and 50%) were produced by subsequent incubation in water resulted in removal of 95% PEG. The surface roughness and hydrophilicity were studied by atomic force microscopy (AFM) and contact angle "water-polymer" measurement, respectively. The film biocompatibility on cell culture of COS-1 fibroblasts was studied in vitro. It was shown that both roughness and hydrophobicity are directly proportional to initial PEG content in the PHB/PEG blends. The growth rate of COS-1 fibroblasts on polymer films is determined by combination of two basic physicochemical properties of the polymer surface: the roughness and hydrophilicity. The optimal roughness requred for COS-1 cells growth is the average roughness more than 25 nm, whereas the limit values of the contact angle "water-polymer" that was responsible for relatively high cell viability were not found. These data indicate that the film surface roughness had the greatest effect on the cell growth, whereas the increase in the polymer surface hydrophilicity caused the additional positive effect on viability of attached cells. Thus, the modification of PHB polymer material by PEG resulted in the improved viability of cells cultivated on the polymer films in vitro. The obtained data can be used for development of such medical devices as surgeon patches and periodontal membranes.

Atomic force microscopy of living and fixed Xenopus laevis embryos.

Xenopus laevis embryos are a rather simple and at the same time a very interesting animal model, which is widely used for research in developmental biology. Intensive coordinated cell movements take place during the multi-cellular organism development. Little is known of the cellular, molecular and biomechanical mechanisms of these movements. The conceptual framework for analysis of cell interactions within integrated populations is poorly developed. We have used atomic force microscopy (AFM) to observe the surface of fixed X. laevis embryos at different stages of their development. We have developed a new sample preparation protocol for these observations. The obtained images were compared with scanning electronic microscopy (SEM) data. Cell rearrangement during morphogenesis in vivo was also visualized by AFM. In the current paper we discuss facilities and challenges of using this technique for further embryo researching.

[Atomic force microscopy of living cells: advances and future outlooks].

The advances of the method of atomic force microscopy for investigating the animal cells and an analysis of its development have been reviewed, with much attention being given to studies of living cells. The features and problems of the method have been considered, and a number of special methods based on the use of atomic force microscopy have been analyzed. The problems of choosing the geometry of probes for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches have been discussed. The problems of combining the atomic force microscopy with optical and laser scanning confocal microscopy have been considered. Possible applications of the method in biotechnology and medicine are discussed.

[A study of lamellas of the web recombinant protein by atomic force microscopy].

Lamellas formed on the mica by protein 1F9, a recombinant analogue of the web protein, have been studied by atomic force microscopy. It has been shown that the molecules of 1F9 dissolved in strong solvents are capable of aggregating on the mica surface to form lamellas less than 1 nm in height and more than 1 microm in length. A model of a plane zigzag has been constructed to describe the conformation of 1F9 molecules on the mica surface.

Atomic force microscopy study of the arrangement and mechanical properties of astrocytic cytoskeleton in growth medium.

Astrocytes are quite interesting to study because of their role in the development of various neurodegenerative disorders. The present work describes an examination of the arrangement and mechanical properties of cytoskeleton of living astrocytes using atomic force microscopy (AFM). The experiments were performed with an organotypic culture of dorsal root ganglia (DRG) obtained from a chicken embryo. The cells were cultivated on a gelatinous substrate and showed strong adhesion. AFM allows one to observe cytoskeleton fibers, which are interpreted as actin filaments and microtubules. This assumption is supported by confocal microscopy fluorescence imaging of α-tubulin and fibrillar actin. Mapping of the local Young's modulus of a living astrocyte showed that the stiff areas correspond to the sites where the cytoskeleton fibers are located. Thus, the data obtained indicate that AFM is a promising method to study neural cells cytoskeleton integrity and arrangement inin vitromodels of neurodegeneration.

[A comparative study of biodegradation kinetics of biopolymer systems based on poly(3-hydroxybutyrate)].

The aim of this study was to evaluate and to compare of long-term kinetics curves of biodegradation of poly(3-hydroxybutyrate) (PHB), its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and PHB/polylactic acid blend. The total weight loss and the change of average viscosity molecular weight were used as an index of biodegradation degree. The rate of biodegradation was analyzed in vitro in presence oflipase and in vivo when the films were implanted in animal tissues. The morphology of PHB films surface was studied by atomic force microscopy technique. It was shown that biodegradation of PHB is occurred by means of as polymer hydrolysis, and as its enzymatic biodegradation. The obtained data can be used for development of medical devices on the base of PHB.