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PubMedDate not supplied

Improvement of DNA-visualization in dynamic mode atomic force microscopy in air.

Near-contact mode atomic force microscopy (AFM) imaging leads to sharper representations of DNA double strands on mica imaged at ambient conditions compared with noncontact mode AFM. Phase shift was used for feedback control yielding height information using a simple model calculation. No contact between tip and sample occurs. Measured DNA widths were up to four times smaller than measured with the same AFM tip in noncontact mode at ambient condition.

Animals↗

PubMed2006

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy.

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).

Biosensing Techniques↗

PubMed2001

Accurate length determination of DNA molecules visualized by atomic force microscopy: evidence for a partial B- to A-form transition on mica.

Achieving the most correct estimate of the contour length of digitized DNA molecules is a key aspect of the microscopic analysis of nucleic acids by either electron microscopy (EM) or atomic force microscopy (AFM). Six different methods, that are mathematically not too complex and suited for common, practical use, have been tested here using simulated polymers in two dimensions and real DNA molecules (564, 1054, 2049 and 4297 bp long) imaged in air by AFM. The main result is that the frequently used Freeman estimator (L(F) = n(e) + square root 2n(o)) overestimates the real contour length of the polymers by about 4%. More accurate estimates are obtained with the Kulpa estimator (L(K) = 0.948n(e) + 1.343n(o)) or with the corner count estimator (L(C) = 0.980)n(e) + 1.406n(o) - 0.091n(c)). In the range of the DNA sizes and magnifications we have considered, however, the best results are obtained with an ad hoc developed routine that smoothes the DNA trace by a polynomial fitting of degree 3 over a moving window of 5 points. Under these conditions, the difference between the measured and the real contour length of the molecules is less than 0.4%. The accuracy of this procedure allowed us to reveal a discrete, size-dependent, shortening of DNA molecules deposited onto mica under low salt conditions and imaged in air by AFM. Awareness of this structural alteration, that can be attributed to a partial transition from B- to A-form DNA, may lead to a more correct interpretation of DNA molecules or protein-DNA complexes imaged by AFM.

Algorithms↗

PubMed2006

Probing molecular recognition sites on biosurfaces using AFM.

Knowledge of the molecular forces that drive receptor-ligand interactions is a key to gain a detailed understanding of cell adhesion events and to develop novel applications in biomaterials science. Until recently, there was no tool available for analyzing and mapping these forces on complex biosurfaces like cell surfaces. During the past decade, however, single-molecule atomic force microscopy (AFM) has opened exciting new opportunities for detecting and localizing molecular recognition forces on artificial biosurfaces and on living cells. In this review, we describe the general principles of the AFM technique, present procedures commonly used to prepare samples and tips, and discuss a number of applications that are relevant to the field of biomaterials.

Animals↗

PubMed2005

Solution AFM studies of human Swi-Snf and its interactions with MMTV DNA and chromatin.

ATP-dependent nucleosome remodeling complexes are crucial for relieving nucleosome repression during transcription, DNA replication, recombination, and repair. Remodeling complexes can carry out a variety of reactions on chromatin substrates but precisely how they do so remains a topic of active inquiry. Here, a novel recognition atomic force microscopy (AFM) approach is used to characterize human Swi-Snf (hSwi-Snf) nucleosome remodeling complexes in solution. This information is then used to locate hSwi-Snf complexes bound to mouse mammary tumor virus promoter nucleosomal arrays, a natural target of hSwi-Snf action, in solution topographic AFM images of surface-tethered arrays. By comparing the same individual chromatin arrays before and after hSwi-Snf activation, remodeling events on these arrays can be monitored in relation to the complexes bound to them. Remodeling is observed to be: inherently heterogeneous; nonprocessive; able to occur near and far from bound complexes; often associated with nucleosome height decreases. These height decreases frequently occur near sites of DNA release from chromatin. hSwi-Snf is usually incorporated into nucleosomal arrays, with multiple DNA strands entering into it from various directions, + or - ATP; these DNA paths can change after hSwi-Snf activation. hSwi-Snf appears to interact with naked mouse mammary tumor virus DNA somewhat differently than with chromatin and ATP activation of surface-bound DNA/hSwi-Snf produces no changes detectable by AFM.

Adenosine Triphosphate↗

PubMed2005

How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations.

We present high-resolution atomic force microscopy (AFM) imaging of the single-ring mutant of the chaperonin GroEL (SR-EL) from Escherichia coli in buffer solution. The native GroEL is generally unsuitable for AFM scanning as it is easily being bisected by forces exerted by the AFM tip. The single-ring mutant of GroEL with its simplified composition, but unaltered capability of binding substrates and the co-chaperone GroES, is a more suited system for AFM studies. We worked out a scheme to systematically investigate both the apical and the equatorial faces of SR-EL, as it binds in a preferred orientation to hydrophilic mica and hydrophobic highly ordered pyrolytic graphite. High-resolution topographical imaging and the interaction of the co-chaperone GroES were used to assign the orientations of SR-EL in comparison with the physically bisected GroEL. The usage of SR-EL facilitates single molecule studies on the folding cycle of the GroE system using AFM.

Adsorption↗

PubMed2005

UV-vis-infrared optical and AFM study of spin-cast chitosan films.

Optical properties of spin-cast chitosan films have been determined in the infrared, visible, and ultraviolet region of the spectrum using spectroscopic ellipsometry. Optical constants for the UV-vis-near IR spectra from 130 to 1700 nm were determined using Cauchy dispersion forms combined with Lorentzian oscillator models in the absorptive shorter wavelength regions. Infrared index of refraction and extinction coefficients from 750 to 4000 cm(-1) were determined using ellipsometric data fits to dispersion models based on harmonic oscillators. This modeling determined that optical anisotropy was present and measurable over all wavelength regions of ellipsometric data. To obtain information on the micro- and nano-scale surface structure, tapping mode atomic force microscopy (AFM) imaging was employed to determine morphology and roughness information of dry spin-cast chitosan films.

Algorithms↗

PubMed2002

Characterization of the adhesive mucilages secreted by live diatom cells using atomic force microscopy.

Atomic Force Microscopy (AFM) resolved the topography and mechanical properties of two distinct adhesive mucilages secreted by the marine, fouling diatom Craspedostauros australis. Tapping mode images of live cells revealed a soft and cohesive outer mucilage layer that encased most of the diatom's siliceous wall, and force curves revealed an adhesive force of 3.58 nN. High loading force, contact mode imaging resulted in cantilever 'cleaned' cell walls, which enabled the first direct observation of the active secretion of soft mucilage via pore openings. A second adhesive mucilage consisted of strands secreted at the raphe, a distinct slit in the silica wall involved in cell-substratum attachment and motility. Force measurements revealed a raphe adhesive strand(s) resistant to breaking forces up to 60 nN, and these strands could only be detached from the AFM cantilever probe using the manual stepper motor.

Adhesiveness↗

PubMed2005

Sample preparation procedures for biological atomic force microscopy.

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.

Animals↗

PubMed2001

Novel methods for studying lipids and lipases and their mutual interaction at interfaces. Part I. Atomic force microscopy.

Mono-layers of lipids and their interaction with surface active enzymes (lipases) have been studied for more than a century. During the past decade new insight into this area has been obtained due to the development of scanning probe microscopy. This novel method provides direct microscopic information about the system in question and allows in situ investigations under near physiological conditions. In the present review the theory, experimental set-up and sample requirements of atomic force microscopy (AFM) are described. An overview of recent results is also presented with special emphasis on lipase hydrolysis and kinetics investigated in situ using AFM.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

Mass transfer limitations at crystallizing interfaces in an atomic force microscopy fluid cell: a finite element analysis.

Although atomic force microscopy (AFM) has emerged as the preeminent experimental tool for real-time in situ measurements of crystal growth processes in solution, relatively little is known about the mass transfer limitations that may impact these measurements. We present a continuum analysis of flow and mass transfer in an atomic force microscope fluid cell during crystal growth, using data acquired from calcium oxalate monohydrate (COM) crystal growth measurements as a comparison. Steady-state flows and solute concentration fields are computed using a three-dimensional, finite element method implemented on a parallel supercomputer. Steady-state flow results are compared with flow visualization experiments to validate the model. Computations of the flow field demonstrate how nonlinear momentum transport alters the spatial structure of the flow with increasing flow volume, altering mass transport conditions near the AFM cantilever and tip. The simulations demonstrate that the combination of solute depletion from crystal growth and mass transfer resistance lowers the solute concentration in the region between the tip and the crystal compared with the solute concentration at the inlet of the AFM cell. For example, using experimentally measured growth rates for COM, the solute concentration in this region is 3.1% lower than the inlet value because the solute consumed by crystal growth beneath the AFM tip cannot be replenished fully due to mass transport limitations. The simulations also reveal that increasing the flow rate through the cell does not affect this difference significantly because of the inherent shielding by the AFM tip in proximity with the crystal surface. Models such as the one presented here, used in conjunction with AFM measurements, promise more precise interpretations of measurement data.

Journal Article↗

PubMed2000

Nuclear pore function viewed with atomic force microscopy.

In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.

Adenosine Triphosphate↗

PubMed2003

Use of AFM to probe the adsorption strength and time-dependent changes of albumin on self-assembled monolayers.

The adsorption kinetics of human serum albumin (HSA) on CH3- and COOH-terminated self-assembled monolayers (SAMs) has been investigated using radioassays and atomic force microscopy (AFM). On both surfaces, the amount of HSA adsorbed reached a plateau after 30 min. The plateau level was higher on the CH3 compared to the COOH surface. The adhesion force (Fadh), measured using Si3N4 AFM tips in water, decreased with time of contact with the HSA solution on the CH3 surface. This time-dependent change in the adhesiveness of the adsorbed protein is best explained by a change in the conformation or orientation. In contrast, Fadh was independent of the time of contact with the HSA solution on the COOH surface, indicating that once adsorbed, the HSA molecules do not undergo further conformation or orientation changes. The perturbation induced by scanning with the AFM in water on the adsorbed HSA layers was greater on CH3 surfaces than on COOH surfaces, suggesting a weaker protein-substratum interaction on the CH3-terminated SAMs. This was further confirmed by a stronger desorption of HSA following sodium dodecyl sulfate (SDS) treatment on the CH3 surface compared to the COOH surface. Taken together, these data suggest that for COOH SAMs, (1) there is a strong interaction between HSA and the substratum; (2) there is an absence of reorientation with time; and (3) there is a smaller amount of adsorbed protein at 24 h, possibly due to increased but rapid spreading/denaturation of the protein. On the CH3 surface, less deformation of HSA occurs and the molecules maintain a higher mobility at short adsorption times. AFM measurements performed after aging of an adsorbed HSA layer in buffer suggests the role played by HSA in solution in determining the time-dependent conformation and/or orientation changes.

Biofilms↗

PubMed2006

Two-chamber AFM: probing membrane proteins separating two aqueous compartments.

Biological membranes compartmentalize and define physical borders of cells. They are crowded with membrane proteins that fulfill diverse crucial functions. About one-third of all genes in organisms code for, and the majority of drugs target, membrane proteins. To combine structure and function analysis of membrane proteins, we designed a two-chamber atomic force microscopy (AFM) setup that allows investigation of membranes spanned over nanowells, therefore separating two aqueous chambers. We imaged nonsupported surface layers (S layers) of Corynebacterium glutamicum at sufficient resolution to delineate a 15 A-wide protein pore. We probed the elastic and yield moduli of nonsupported membranes, giving access to the lateral interaction energy between proteins. We combined AFM and fluorescence microscopy to demonstrate the functionality of proteins in the setup by documenting proton pumping by Halobacterium salinarium purple membranes.

Binding Sites↗

PubMed2005

The interaction between OPH and paraoxon at the air-water interface studied by AFM and epifluorescence microscopies.

The paraoxon hydrolysis reaction catalyzed by organophosphorus hydrolase (OPH) monolayer at the air-water interface was studied. OPH-paraoxon interactions, occurring at the two-dimensional interface, by close-packed, highly orientated OPH monolayer, were investigated by several different surface chemistry techniques; e.g. surface pressure area isotherms, atomic force microscopy (AFM), and in situ epifluorescence microscopy. The characterization of OPH Langmuir and Langmuir-Blodgett films prepared in both the presence and absence of paraoxon, demonstrated significantly distinctive feature when compared with one another. Continuous growth of the OPH aggregates is a distinct phenomenon associated with hydrolysis, in addition to the pH changes in the local environment of the enzyme macromolecules.

Adsorption↗

PubMed2005

Experimentally derived sticking efficiencies of microparticles using atomic force microscopy.

The sticking efficiencies (alpha) of colloidal particles have been derived from the intersurface potential energy between 2 microm carboxylated polystyrene microspheres and a silica glass plate using the interaction force boundary layer model. The intersurface potential energies were calculated from force-distance data measured using atomic force microscopy (AFM) and from calculations based on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. AFM forces were measured in aqueous solutions over a range of pH and ionic strength conditions, and DLVO calculations were performed on identical systems. In most conditions, sticking efficiencies that were calculated from AFM data are considerably largerthan values calculated from DLVO predictions. Sticking efficiencies vary between 0 and 1 and are strongly dependent upon solution chemistry. AFM-derived sticking efficiencies are consistent with measured microsphere and collector zeta-potentials; sticking efficiencies are lower for more negatively charged surfaces. These results provide the first alpha estimates of a microparticle-collector system that are calculated directly from physically measured interfacial nanoforces. This study clearly demonstrates that significant differences exist between DLVO- and AFM-derived sticking efficiencies.

Colloids↗

PubMed2003

AFM imaging of lipid domains in model membranes.

Characterization of the two-dimensional organization of biological membranes is one of the most important issues that remains to be achieved in order to understand their structure-function relationships. According to the current view, biological membranes would be organized in in-plane functional microdomains. At least for one category of them, called rafts, the lateral segregation would be driven by lipid-lipid interactions. Basic questions like the size, the kinetics of formation, or the transbilayer organization of lipid microdomains are still a matter of debate, even in model membranes. Because of its capacity to image structures with a resolution that extends from the molecular to the microscopic level, atomic force microscopy (AFM) is a useful tool for probing the mesoscopic lateral organization of lipid mixtures. This paper reviews AFM studies on lateral lipid domains induced by lipid-lipid interactions in model membranes.

Animals↗

PubMed1998

Measuring elasticity of biological materials by atomic force microscopy.

Atomic force microscopy (AFM) has proved its value not only for resolving the topographical structure of biological samples, but also for probing inherent properties of biological structures, like local interaction forces, mechanical properties or dynamics in a natural (physiological) environment. This minireview focuses on the acquisition of elasticity data of biomaterials by AFM. A possible theoretical model is presented, followed by a practical 'how to do it with AFM', and, finally, a brief overview of publications in this field is given.

Animals↗