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PubMed2006

Detection of viruses: atomic force microscopy and surface enhanced Raman spectroscopy.

This paper demonstrates the capability of atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS) to function effectively as ultra-sensitive readout tools for chip-scale platforms designed for pathogen detection in complex biological media. AFM allows direct (i.e., label-free) visualization and quantification of nanometer-sized viruses captured on a smooth, selective surface. AFM readout led to optimization of a capture substrate for feline calicivirus (FCV), and yielded a limit of detection of 3 x 10(6) FCV/mL. SERS-based detection of FCV, carried out in a sandwich-type assay, requires labelling of the substrate-bound FCV with a selective extrinsic Raman label (ERL). These studies yielded a limit of detection of 1 x 10(6) FCV/mL. The prospects of these two readout methods as additions to the arsenal of tools in bioterrorism prevention are briefly discussed.

Calicivirus, Feline↗
PubMed2004

Probing small unilamellar EggPC vesicles on mica surface by atomic force microscopy.

Sonicated small unilamellar egg yolk phosphatidylcholine (EggPC) vesicles were investigated using atomic force microscopy (AFM) imaging and force measurements. Three different topographies (convex, planar, and concave shape) of the EggPC vesicles on the mica surface were observed by tapping mode in fluid, respectively. It was found that the topography change of the vesicles could be attributed to the interaction force between the AFM tip and vesicles. Force curves between an AFM tip and an unruptured vesicle were obtained in contact mode. During approach, two breaks corresponding to the abrupt penetration of upper and lower bilayer of vesicle were exhibited in the force curve. Both breaks spanned a distance of around 4 nm close to the EggPC bilayer thickness. Based on Hertz analysis of AFM approach force curves, the Young's modulus (E) and the bending modulus (kc) for pure EggPC vesicles were measured to be (1.97 +/- 0.75) x 10(6)Pa and (0.21 +/- 0.08) x 10(-19)J, respectively. The results show that the AFM can be used to obtain good images of intact and deformed vesicles by tapping mode, as well as to probe the integrity and bilayer structure of the vesicles. AFM force curve compare favorably with other methods to measure mechanical properties of soft samples with higher spatial resolution.

Adsorption↗
PubMed2000

In-situ atomic force microscopy study of beta-amyloid fibrillization.

We report the use of atomic force microscopy to observe the initial stages of beta-amyloid fibrillization in situ. The growth of individual beta-amyloid protofibrils on a mica substrate was followed over several hours. The first in situ visualization of protofibril formation from single aggregate units of beta-amyloid is reported. The growth of these protofibrils through the subsequent addition of these aggregate units is also observed. Growth of the protofibrils is bi-directional and the outgrowth of protofibrils from a common amyloid/heterogeneous core is also observed. Elongation also occurred by the addition of protofibrils from solution. This data provides an exciting insight into the early stages of beta-amyloid fibrillization and can be used to enhance the understanding of the mechanism(s) by which beta-amyloid fibrillizes and may consequently enable inhibition of one or more stages of fibrillization as a potential therapeutic strategy.

Adsorption↗
PubMed1999

High speed atomic force microscopy of biomolecules by image tracking.

An image-tracking procedure for atomic force microscopy is proposed and tested, which allows repeated imaging of the same area without suffering from lateral drift. The drift correction procedure is based on on-line cross-correlation of succeeding images. Using the image-tracking procedure allows zooming in on a small scan area over a long period and thus increases the frame rate inversely proportional to the scan area. Application of the procedure is demonstrated for diffusion of 5.4-kb DNA plasmids. With a scan area of 500 * 500 nm(2), a single plasmid can be imaged for more than 30 min at 4 s per frame, with a drift less than 10 nm. The high temporal resolution allows detailed analysis of the diffusion of DNA molecules. A diffusion coefficient of 30 nm(2)/s is found for most DNA molecules, though many molecules are temporally pinned to the mica surface, restricting diffusion.

Aluminum Silicates↗
PubMed2004

Kinetic study on the elastic change of vascular endothelial cells on collagen matrices by atomic force microscopy.

The elasticity of vascular endothelial cells (HUVEC) was measured with an atomic force microscopy (AFM, Olympus), and analyzed by applying the Hertz model, and those data were compared with ones reported by us previously. The latter elasticity data were measured with AFM Instruments (Seiko) on the basis of the Young's modulus of gelatin gel, which was obtained from the measurement with a tensile tester. The elasticity of HUVEC was concluded to depend on the culture period. The elasticity of the cells cultured on type IV collagen for longer than 4 days led to average elasticity values higher than ca. 10(4)Pa. Moreover, the scattered values of elasticity decreased eminently in the AFM measurement of the cells at room temperature. A few cells, however, appeared to adhere long and tensely on matrix, which seems to one of reasons for relatively high elasticity in our previous works. The possibility of such high elasticity was considered to change in cell adhesion, anchoring on matrix during a long culture period.

Cell Adhesion↗
PubMed2004

Cytochrome c adsorption to supported, anionic lipid bilayers studied via atomic force microscopy.

The adsorption of membrane-associated protein cytochrome c to anionic lipid bilayers of dioleoyl phosphatidylglycerol was studied in low ionic strength physiological buffer using atomic force microscopy. The bilayers were supported on polylysinated mica. The formation of stable, single lipid bilayers was confirmed by imaging and force spectroscopy. Upon addition of low concentrations of cytochrome c, protein molecules were not topographically visible on the lipid bilayer-buffer interface. However, the forces required to punch through the bilayer by indentation using the atomic force microscopy probe were significantly lower after protein adsorption, which suggest that the protein inserts into the bilayer. Moreover, the apparent thickness of the bilayer remained unchanged after cytochrome c adsorption. Yet, mass spectroscopy and visible light absorption spectroscopy confirmed the presence of cytochrome c in the lipid bilayers. These results suggest that 1), cytochrome c inserts into the bilayer and resides in its hydrophobic core; 2), cytochrome c insertion changes the mechanical properties of the bilayer significantly; and 3), bilayer force spectroscopy may be a useful tool in investigating lipid-protein interactions.

Adsorption↗
PubMed2005

Atomic force microscopy study of the structural effects induced by echinomycin binding to DNA.

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.

2-Aminopurine↗
PubMed2001

Single molecule study of xanthan conformation using atomic force microscopy.

Conformations of individual macromolecules of the biopolymer xanthan were investigated using atomic force microscopy (AFM). Xanthan from very dilute solutions (1 ppm) was allowed to adsorb onto freshly cleaved mica and examined using tapping mode AFM under ambient conditions. The secondary structure of xanthan was probed by heating the polymer and gradually cooling, which denatured and renatured the polymer. When salt was present, renatured xanthan formed a double helical structure, consistent with the structure of native xanthan. In pure water, renaturation was not complete as what appeared to be single helical structures were observed. The number-average contour length (L(n)) of the polymer in its single helical state was 1651 nm. In the double helical state, induced by the addition of salt, L(n) decreased to 450 nm (in 0.5 M KCl). The chains also became less rigid as salt was added. The persistence length decreased from 417 nm in pure water to approximately 150 nm in 0.1 or 0.5 M KCl. This indicated a trend toward more flexible molecules when salt was present. Calculations of end-to-end distances based on equilibrium and projected conformations confirmed that the xanthan chain conformation on the mica surface was at equilibrium and was therefore representative of the conformation of xanthan in solution. The single-molecule AFM technique eliminates one common bias of solution techniques, which is the determination of an average signal between aggregates and dissolved molecules. It is thus a useful complement to solution-based methods for determining physical-chemical properties of biopolymers.

Carbohydrate Conformation↗
PubMed1997

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles.

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm. Lumbricus terrestris, were imaged in NH4Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h1 and h2, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h1 and 1.23 and 13.6 nm for h2. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2 +/- 10.5 nm, n = 75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h1 and h2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 +/- 3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 +/- 0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.

Animals↗
PubMed1999

Hyaluronic acid by atomic force microscopy.

Hyaluronic acid (HA) of different molecular weights has been examined by atomic force microscopy (AFM) in air. This technique allows 3-D surface images of soft samples without any pretreatment, such as shadowing or staining. In the present study we examined the supermolecular organization of HA chains when deposited on mica and graphite, to better understand the interchain and intrachain interactions of HA molecules in solution. The concentration of the solution deposited varied from 0.001 to 1 mg/ml. On both substrates, and independent of the concentration, high-molecular-mass HA formed networks in which molecules ran parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and rejoin into similar neighboring aggregates. Accurate measurements of the thickness of the thinnest sheets were consistent with a monolayer of HA molecules, 0.3 nm thick, strongly indicating lateral aggregation forces between chains as well as rather strong hydrophilic interactions between mica and HA. The results agree with an existing model of HA tertiary structure in solution in which the network is stabilized by both hydrophilic and hydrophobic interactions. Our images support this model and indicate that hydrophobic interactions between chains may exert a pivotal role in aqueous solution.

Aluminum Silicates↗
PubMed1999

Imaging and force-distance analysis of human fibroblasts in vitro by atomic force microscopy.

The structure of human fibroblasts have been characterised in vitro by atomic force microscopy (AFM) operated in the imaging or in the force versus distance (F-d) modes. The choice of cell substrate is important to ensure good adhesion. Of greater significance in the context of AFM analysis, is the observation that the substrate affects the imaging conditions for in vitro analysis of live cells. For instance, very rarely will glass coverslips lead to acceptable outcomes (i.e., resolved cytoskeletal structure). Activated tissue culture dishes, on the other hand, promote conditions that routinely result in good quality images. Those conditions are then unaffected by adoption of relatively high force loadings (more than 10 nN), large fields of view (100 x 100 microm2) and high scan speeds (up to ca. 200 microm/sec), all of which exceed values recommended in the literature. Plasma membranes are fragile in the context of AFM analysis (F-d analysis gives an equivalent Young's Modulus of ca. 5 kPa). However, the present work suggests that fragility per se need not be a problem, rather it is the adhesive interactions with the tip, which under some circumstances may exceed 20 nN, that are the source of poor imaging conditions. The present results, being supported by a qualitative model, suggest that the activated substrate acts as a preferential scavenger of cellular debris thus preventing the tip from biofouling, and will therefore promote low adhesion between tip and membrane. Good imaging conditions provide non-destructive in vitro information about cytoskeletal structure and dynamics, as shown in two examples concerned with cytochalasin treatment and with the MTT assay.

Cell Division↗
PubMed2006

Enhancement of topographic images obtained in liquid media by atomic force microscopy.

The open liquid-cell atomic force microscope (AFM) has potential for studies of biomaterials and surface morphology in liquid media, and a variety of fluids can be used as buffer solutions. The dependence of image distortion on fluid properties (kinematic viscosity) has been studied with edge friction force obtained in lateral images and will shortly appear elsewhere [Appl. Phys. Lett. 2006, 88, 173121]. Previous studies indicate that the scan rate should be slower for obtaining a nondistorted image. However, the time required for the scan is greatly increased. Therefore, we introduced the vector concept to evaluate the net force for scanning in the y-direction and found two solutions to achieve a zero force difference introduced by the cantilever-fluid and the tip-surface. When the scan rate approaches zero or a specific velocity (30 microm/s in this study), the force of the interaction induced by the cantilever-fluid and tip-surface is reduced to a considerable extent. Among the two solutions, a scan with a specific velocity is an easy, rapid method for obtaining a nondistorted image, compared to the previously proposed method (scan rate approaches zero). This proposed model was confirmed in a proof-of-concept test using 2-propanol.

2-Propanol↗
PubMed2005

Gold-coated conducting-atomic force microscopy probes.

Some aspects of the performance of gold-coated conductive probes used in conducting atomic force microscopy (C-AFM) technique are discussed. The resistance of the nanocontact between the gold-coated AFM tip and the graphite substrate has been monitored at various applied forces. For small forces (<50 nN), resistance on the order of a few kiloohms was observed. Minimal contact resistance was observed for forces in the range 100-150 nN, beyond which the tip seems to undergo plastic deformation. The resistance of the nanocontact increased when current on the order of 100 microA was allowed to pass through, finally resulting in melting of the gold coating.

Chemical Phenomena↗
PubMed1994

Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy.

We describe the morphology and mechanical stability of the apical surface of MDCK monolayers by atomic force microscopy (AFM). Living cells could be imaged in physiological solution for several hours without noticeable deterioration. Cell boundaries appear as ridges that clearly demarcate neighboring cells. In some cases the nucleus of individual cells could be seen, though apparently only in very thin areas of the monolayer. Two types of protrusions on the surface could be visualized. Smooth bulges that varied in width from a few hundred nanometers to several micrometers, which appear to represent relatively rigid subapical structures. Another type of protrusion extended well above the membrane and was swept back and forth during the imaging. However, the microvilli that are typically present on the apical surface could not be resolved. For comparison, a transformed MDCK cell line expressing the K-ras oncogene was also examined. When cultured on solid substrata at low density, the R5 cells spread out and are less than 100 nm thick over large areas with both extensive processes and rounded edges. Many intracellular structures such as the nucleus, cytoskeletal elements and vesicles could be visualized. None of the intracellular structures seen in the AFM images could be seen by scanning electron microscopy. Both R5 cells and MDCK monolayers required imaging forces of > 2 nN for good image contrast. Force measurements on the MDCK monolayers show that they are very soft, with an effective spring constant of approximately 0.002 N/m for the apical plasma membrane, over the first micrometer of deformation, resulting in a height deformation of approximately 500 nm per nanoNewton of applied force. The mechanical properties of the cells could be manipulated by addition of glutaraldehyde. These changes were monitored in real time by collecting force curves during the fixation reaction. The curves show a stiffening of the apical plasma membrane that was completed in approximately 1 minute. On the basis of these measurements and the imaging forces required, we conclude that deformation of the plasma membrane is an important component of the contrast mechanism, in effect 'staining' structures based on their relative rigidity.

Animals↗
PubMed2000

High-resolution imaging of antibodies by tapping-mode atomic force microscopy: attractive and repulsive tip-sample interaction regimes.

A force microscope operated with an amplitude modulation feedback (usually known as tapping-mode atomic force microscope) has two tip-sample interaction regimes, attractive and repulsive. We have studied the performance of those regimes to imaging single antibody molecules. The attractive interaction regime allows determination of the basic morphologies of the antibodies on the support. More importantly, this regime is able to resolve the characteristic Y-shaped domain structure of antibodies and the hinge region between domains. Imaging in the repulsive interaction regime is associated with the irreversible deformation of the molecules. This causes a significant loss in resolution and contrast. Two major physical differences distinguish the repulsive interaction regime from the attractive interaction regime: the existence of tip-sample contact and the strength of the forces involved.

Aluminum Silicates↗
PubMed2006

Combined atomic force microscopy and optical microscopy measurements as a method to investigate particle uptake by cells.

We propose a combination of atomic force microscopy (AFM) and optical microscopy for the investigation of particle uptake by cells. Positively and negatively charged polymer microcapsules were chosen as model particles, because their interaction with cells had already been investigated in detail. AFM measurements allowed the recording of adhesion forces on a single-molecule level. Due to the micrometer size of the capsules, the number of ingested capsules could be counted by optical microscopy. The combination of both methods allowed combined measurement of the adhesion forces and the uptake rate for the same model particle. As a demonstration of this system, the correlation between the adhesion of positively or negatively charged polymer microcapsules onto cell surfaces and the uptake of these microcapsules by cells has been investigated for several cell lines. As is to be expected, we find a correlation between both processes, which is in agreement with adsorption-dependent uptake of the polymer microcapsules by cells.

Adhesiveness↗
PubMed2005

Numerical simulation of scanning electrochemical microscopy experiments with frame-shaped integrated atomic force microscopy--SECM probes using the boundary element method.

Integrated submicroelectrodes for combined AFM-SECM measurements are characterized with numerical simulations using the boundary element method. SECM approach curves and SECM images are calculated and analyzed for a model substrate containing pronounced topographical and electrochemical features. The theoretically calculated image has been compared to the experimental data and shows excellent quantitative agreement. Hence, the applicability of integrated AFM-SECM electrodes for combined electrochemical and topographical imaging and a profound theoretical description including quantification of the obtained results are demonstrated.

Electrochemistry↗
PubMed2003

Direct observations of freeze-etching processes of ice-embedded biomembranes by atomic force microscopy.

We have fabricated a cryogenic atomic force microscope that is designed for structural investigation of freeze-fractured biological specimens. The apparatus is operated in liquid nitrogen gas at atmospheric pressure. Freeze-fracturing, freeze-etching and subsequent imaging are carried out in the same chamber, so that the surface topography of a fractured plane is easily visualized without ice contamination. A controlled superficial sublimation of volatile molecules allows us to obtain three-dimensional views of ultrastructures of biological membranes.

Animals↗