Fine structure of cell wall surfaces in the giant-cellular xanthophycean alga <Emphasis Type="Italic">Vaucheria terrestris</Emphasis>

The mechanical strength of cell walls in the tip-growing cells of Vaucheria terrestris is weakened by treatment with proteolytic enzymes. To clarify the morphological characteristics of the components maintaining cell wall strength, the fine structures of the cell walls, with and without protease treatment, were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations indicated that cellulose microfibrils were arranged in random directions and overlapped each other. Most of the microfibrils observed in the inner surface of the cell wall were embedded in amorphous materials, whereas in the outer surface of the cell wall, microfibrils were partially covered by amorphous materials. The matrix components embedding and covering microfibrils were almost completely removed by protease treatment, revealing layers of naked microfibrils deposited deeply in the cell wall. Topographic data taken from AFM observations provided some additional information that could not be obtained by TEM, including more detailed images of the granular surface textures of the matrix components and the detection of microfibrils in the interior of the cell wall. In addition, quantitative AFM data of local surface heights enabled us to draw three-dimensional renderings and to quantitatively estimate the extent of the exposure of microfibrils by the enzymatic treatment.     

Atomic force microscopy of cell growth and division in Staphylococcus aureus

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.     

Atomic force microscopy of pollen grains,cellulose microfibrils,and protoplasts

Summary Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.Abbreviations AFM atomic force microscope - FESEM field emission scanning electron microscope - PyMS pyrolysis mass spectrometry - TEM transmission electron microscope     

Photoinactivation effects of hematoporphyrin monomethyl ether on Gram-positive and -negative bacteria detected by atomic force microscopy

The photodynamic antimicrobial chemotherapy as a promising approach for efficiently killing pathogenic microbes is attracting increasing interest. In this study, the cytotoxic and phototoxic effects of hematoporphyrin monomethyl ether (HMME) on the Gram-positive and Gram-negative bacteria were investigated. The cell viability was assessed by colony-forming unit method, and the results indicated that there was no significant cytotoxicity but high phototoxicity in the examined concentrations. Notably, the Gram-positive bacteria were more sensitive to HMME in phototoxicity. Simultaneously, an atomic force microscope (AFM) was used to detect the changes in morphological and nanomechanical properties of bacteria before and after HMME treatment. AFM images indicate that upon photoinactivation, the bacterial surface changed from a smooth, homogeneous architecture to a heterogenous, crackled morphology. The force spectroscopy measurements reveal that the cell wall became less rigid and the Young’s modulus decreased about 50%, whereas the tip-cell-surface adhesion forces increased significantly compared to those of native cells. It was speculated that the photodynamic effects of HMME induced the changes in the chemical composition of the outer membrane and exposure of some proteins inside the envelope. AFM can be utilized as a powerful and sensitive method for studying the interaction between bacteria and drugs.     

Dynamics of the Antimicrobial Peptide PGLa Action on Escherichia coli Monitored by Atomic Force Microscopy

Summary Atomic force microscopy (AFM) images of living cells in physiological solution were used to monitor the different stages involved in the interaction between Escherichia coli and the antimicrobial peptide PGLa. Damage on bacterial membranes was observed in the past using standard electron microscopy; stiffness measurements and images scanned in physiological solution demonstrate the advantage of AFM for such studies. From force versus separation curve measurements it is possible to determine the variation of the cellular stiffness. PGLa action on components of the cell structure like the outer membrane, the bacterial pili, the peptidoglycan wall and the inner membrane was determined by the comparison of AFM images of bacteria before and after PGLa addition. The interaction of Escherichia coli with PGLa in the culture medium has two stages. The first is characterized by the loss of surface stiffness and the formation of micelles probably originating from the disruption of the outer membrane and the loss of the bacteria’s ability to adhere to the substrates. In the second stage there is further damage, which resulted in total cell rupture. AFM images of bacteria in air and surface roughness measurements were also used to estimate peptide damage.     

Predicting the chemical composition and structure of Aspergillus nidulans hyphal wall surface by atomic force microscopy

In fungi, cell wall plays an important role in growth and development. Major macromolecular constituents of the aspergilli cell wall are glucan, chitin, and protein. We examined the chemical composition and structure of the Aspergillus nidulans hyphal wall surface by an atomic force microscope (AFM). To determine the composition of the cell wall surface, the adhesion forces of commercially available β-glucan, chitin, and various proteins were compared to those of corresponding fractions prepared from the hyphal wall. In both setups, the adhesion forces of β-glucan, chitin, and protein were 25–50, 1000–3000, and 125–300 nN, respectively. Adhesion force analysis demonstrated that the cell surface of the apical tip region might contain primarily chitin and β-glucan and relatively a little protein. This analysis also showed the chemical composition of the hyphal surface of the mid-region would be different from that of the apical region. Morphological images obtained by the tapping mode of AFM revealed that the hyphal tip surface has moderate roughness.     

Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy

Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.     

Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy

We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.     

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.     

SEM and AFM images of pyrite surfaces after bioleaching by the indigenous<Emphasis Type="Italic"> Thiobacillus thiooxidans</Emphasis>

The bioleaching mechanism of pyrite by the indigenous Thiobacillus thiooxidans was examined with the aid of scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of the pyrite surface. The presence of pyrite eliminated the lag phase during growth of this microorganism. This was due to the stimulatory effect on cell growth of the slight amount of Cu2+ that had leached from the pyrite. Zn2+ was found to be much more readily solubilized than Cu2+. The efficiency of bioleaching was four times higher than that of chemical leaching. SEM images provided evidence of direct cell attachment onto the pyrite surface, thereby enhancing the bioleaching rate. Furthermore, extracellular polymeric substances (EPSs) were found on the pyrite surface after 4 days of oxidation. AFM images showed that the pyrite surface area positively correlated with the oxidation rate. A combination of direct and indirect mechanism is probably responsible for the oxidation of pyrite by T. thiooxidans.     

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.     

Atomic Force Microscopy: A Promising Tool for Deciphering the Pathogenic Mechanisms of Fungi in Cystic Fibrosis

During the past decades, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Although most of the works concerned bacteria, AFM also permitted major breakthroughs in the understanding of physiology and pathogenic mechanisms of some fungal species associated with cystic fibrosis. Complementary to electron microscopies, AFM offers unprecedented insights to visualize the cell wall architecture and components through three-dimensional imaging with nanometer resolution and to follow their dynamic changes during cell growth and division or following the exposure to drugs and chemicals. Besides imaging, force spectroscopy with piconewton sensitivity provides a direct means to decipher the forces governing cell–cell and cell–substrate interactions, but also to quantify specific and non-specific interactions between cell surface components at the single-molecule level. This nanotool explores new ways for a better understanding of the structures and functions of the cell surface components and therefore may be useful to elucidate the role of these components in the host–pathogen interactions as well as in the complex interplay between bacteria and fungi in the lung microbiome.

     

Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.     

Heterogeneous cell mechanical properties: an atomic force microscopy study.

Atomic force microscopy (AFM) is a non-invasive microscopy to explore living biological systems like cells in liquid environment. Thus AFM is an appropriate tool to investigate surface chemical modification and its influence on biological systems. In particular, control over biomaterial surface chemistry can result in a regulated cell response. This report investigates the influence of adhesive and non-adhesive surfaces on the cell morphology and the influence of the cytoskeleton structure on the local mechanical properties. In this study, the main work concerns a thorough investigation of the height images obtained with an AFM as therecorded images provide the evolution of the mechanical properties of the cell as function of its local structure. Information on the cell elasticity due to the cytoskeleton organization is deduced when comparing the AFM tip indentation depth versus the distance between the cytoskeleton bundles for the different samples.     

激光作用质粒DNA和小牛胸腺DNA的AFM研究

激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ产生损伤效应,导致ＤＮＡ结构变化,利用一种改进的试样制备过程和纳米显微镜--原子力显微镜（ＡＦＭ）能够获得可重现的激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ的ＡＦＭ图象,显示它们的特殊的表面结构。     

Ultrastructural and physico-chemical heterogeneities of yeast surfaces revealed by mapping lateral-friction and normal-adhesion forces using an atomic force microscope

Scanning force microscopy has been used to probe the surface of the emerging pathogenic yeast Candida parapsilosis, in order to get insight into its surface structure and properties at submicrometer scales. AFM friction images eventually show patches with a very strong contrast, showing high lateral interaction with the tip. Adhesion force measurement also reveals a high normal interaction with the tip, and patches show extraordinarily high pull off values. The tip eventually sticks completely at the center of the patches. While an extraordinarily high interaction is measured by the tip at those zones, topographic images show extraordinarily flat topography over those zones, both of which characteristics are consistent with a liquid-like area. High resolution friction images show those zones to be surrounded by microfibrillar structures, concentrically oriented, of a mean width of about 25 nm, structures that become progressively less defined as we move away from the center of the patches. No structure can be appreciated inside the zones of maximum contrast. Also some helical or ribbon-like structure can be resolved from friction images. There is not only an ordered disposition of the microfibrillar structures, but also the adhesion force increases radially in the direction towards the center of the patches. These structures responsible for the high adhesion are thought to be incipient-emerging budding zones. Microfibrillar structures are thought to represent the first steps of chitin biosynthesis and cell wall digestion, with chitin polymers being biosynthesized, associated with other macromolecules of the yeast cell wall. They can be also beta glucan helical structures, made visible in the zone of yeast division due to the action of autolysins. The observed gradient in surface adhesion and elastic properties correlates well with that expected from a biochemical point of view. The higher adhesion force measured could be either due to the different macromolecular nature of the patches, or to a mechanical adhesion effect due to the different plasticity of that zone. This work reveals the importance of taking into account the dynamic nature of the cell wall physico-chemical properties. Processes related to the normal cell-cycle, as division, can strongly alter the surface morphology and physico-chemical properties and cause important heterogeneities that might have a profound impact on the adhesion behavior of a single cell, which could not be detected by more macroscopic methods.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

PROBING THE SURFACE OF LIVING DIATOMS WITH ATOMIC FORCE MICROSCOPY: THE NANOSTRUCTURE AND NANOMECHANICAL PROPERTIES OF THE MUCILAGE LAYER1

Atomic force microscopy (AFM) is used to investigate the topography and material properties of the mucilage layer of live cells of three benthic diatoms, the marine species Crasepdostauros australis E. J. Cox and Nitzschia navis‐varingica Lundholm et Moestrup and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. Contrary to previous studies, we show that this surface mucilage layer displays unique nanostructural features. In C. australis, tapping mode images revealed a soft mucilage layer encasing the silica cell wall, consisting of a smooth flat surface that was interrupted by regions with groove‐like indentations, whereas force measurements revealed the adhesive binding of polymer chains. The elastic responses of these polymer chains, as they were stretched during force measurements, were successfully fitted to the worm‐like chain model, indicating the stretching of mostly single macromolecules from which quantitative information was extracted. In P. viridis, tapping mode images of cells revealed a mucilage layer that had the appearance of densely packed spheres, whereas force measurements exhibited no adhesion. In N. navis‐varingica, tapping mode images of the outer surface of this cell in the girdle region revealed the absence of a mucilage layer, in contrast to the other two species. In addition to these topographic and adhesion studies, the first quantitative measurement of the elastic properties of microalgal extracellular polymeric substance is presented and reveals significant spatial variation in the C. australis and P. viridis mucilage layers. This study highlights the capacity of AFM in elucidating the topography and mechanical properties of hydrated microalgal extracellular polymeric substance on a nanoscale.     

Atomic force microscopic study of the effects of ethanol on yeast cell surface morphology

The detrimental effects of ethanol toxicity on the cell surface morphology of Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354) were investigated using an atomic force microscope (AFM). In combination with culture viability and mean cell volume measurements AFM studies allowed us to relate the cell surface morphological changes, observed on nanometer lateral resolution, with the cellular stress physiology. Exposing yeasts to increasing stressful concentrations of ethanol led to decreased cell viabilities and mean cell volumes. Together with the roughness and bearing volume analyses of the AFM images, the results provided novel insight into the relative ethanol tolerance of S. cerevisiae and Sc. pombe.