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PubMed1998

Submolecular resolution of single macromolecules with atomic force microscopy.

The intrinsically high signal-to-noise ratio of atomic force microscopy (AFM) permits structural determination of individual macromolecules to, at times, subnanometer resolution directly from unprocessed images, avoiding the conditions and possible consequences of averaging over an ensemble of molecules. In this article, we will review some of the most recent achievements in imaging single macromolecules with AFM.

Membrane Proteins↗
PubMed2004

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy.

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

Benzoquinones↗
PubMed2005

Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization.

Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.

Artifacts↗
PubMed2002

Atomic force microscopy and proteins.

This review briefly introduces the principles of atomic force microscopy (AFM) applied to protein samples. AFM provides three-dimensional surface images of the proteins with high resolution. The advantage of AFM for protein studies is that AFM can visualize directly the molecule under physiological conditions without previous treatment. AFM operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand receptor interactions with the goal of measuring forces at the individual molecule level.

Animals↗
PubMed2000

Combined scanning electrochemical-atomic force microscopy.

A combined scanning electrochemical microscope (SECM)-atomic force microscope (AFM) is described. The instrument permits the first simultaneous topographical and electrochemical measurements at surfaces, under fluid, with high spatial resolution. Simple probe tips suitable for SECM-AFM, have been fabricated by coating flattened and etched Pt microwires with insulating, electrophoretically deposited paint. The flattened portion of the probe provides a flexible cantilever (force sensor), while the coating insulates the probe such that only the tip end (electrode) is exposed to the solution. The SECM-AFM technique is illustrated with simultaneous electrochemical-probe deflection approach curves, simultaneous topographical and electrochemical imaging studies of track-etched polycarbonate ultrafiltration membranes, and etching studies of crystal surfaces.

Electrochemistry↗
PubMed1998

A convenient method of aligning large DNA molecules on bare mica surfaces for atomic force microscopy.

Large DNA molecules remain difficult to be imaged by atomic force microscopy (AFM) because of the tendency of aggregation. A method is described to align long DNA fibers in a single direction on unmodified mica to facilitate AFM studies. The clear background, minimal overstretching, high reproducibility and convenience of this aligning procedure make it useful for physical mapping of genome regions and the studies of DNA-protein complexes.

Aluminum Silicates↗
PubMed2003

The application of atomic force microscopy to the study of living vertebrate cells in culture.

Atomic force microscopy (AFM), a relatively new variant of scanning probe microscopy developed for the material sciences, is becoming an increasingly important tool in other disciplines. In this review I describe in nontechnical terms some of the basic aspects of using AFM to study living vertebrate cells. Although AFM has some unusual attributes such as an ability to be used with living cells, AFM also has attributes that make its use in cell biology a real challenge. This review was written to encourage researchers in the biological and biomedical sciences to consider AFM as a potential (and potent) tool for their cell biological research.

Animals↗
PubMed2004

Atomic force microscopy: mechanical unfolding of proteins.

Mechanical unfolding of proteins using atomic force microscopy is becoming a routine biophysical technique. Mechanistic investigations in this rapidly evolving field are beginning to resolve the factors that contribute to the behaviour of biological macromolecules under force. Here we describe the force-mode apparatus, the experimental set-up, tractable systems of study, and the analysis of the resultant force data. Finally we summarise some of the recent achievements and limitations of this technique.

Cloning, Molecular↗
PubMed2001

Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy.

PURPOSE: We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using energy-filtering transmission electron microscopy (EF-TEM) and atomic force microscopy (AFM). METHODS: Human cornea and sclera were digested with keratanase to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observation, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed. RESULTS: With CB staining, numerous CB-positive short filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic collagen fibrils in keratanase-treated corneal collagens were deeper and more regular than in untreated ones. Moreover, the depth and periodicity of keratanase-untreated corneal collagens were shallow and irregular in comparison with keratanase-untreated scleral collagens. On the other hand, there was no difference in depth or regularity between keratanase-treated and -untreated scleral collagen fibrils. Using AFM imaging, additional thin grooves sub-bands were detected on the surface of keratanase-treated corneal collagen fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestion. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands. CONCLUSION: Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with proteoglycans, we conclude that KSPG is found in ample amounts in the human cornea in comparison with sclera. Moreover, we topologically detected KSPG attached to a and c bands of collagen fibrils.

Aged↗
PubMed2001

An in situ study of metal complexation by an immobilized synthetic biopolymer using tapping mode liquid cell atomic force microscopy.

Near-field scanning optical microscopy and tapping mode, liquid cell atomic force microscopy were used to study the conformational changes in simple short-chain silica-immobilized biopolymer, poly(L-cysteine) (PLCys), as the polymer was exposed to reducing, metal-rich, and acidic environments, respectively, to simulate on-line metal preconcentration. In a reducing environment (0.01 M dithiothreitol in pH 7.0 ammonium acetate buffer), the PLCys features resembled islands on the surface of the glass, 36 +/- 7 nm in height and 251 +/- 60 nm in diameter. Upon exposure to metal (Cd2+ buffered at pH 7.0), the PLCys islands broke up into smaller metal binding clusters whose features were lower in height, 22 +/- 5 nm, and diameter, 213 +/- 53 nm. Exposure to 0.01 M HCl used for metal stripping resulted in protonation of the polymer chains and further reduction in the polymer height to 12 +/- 5 nm. These changes in molecular structure have given new insight into the mechanisms involved to achieve strong binding as well as rapid, quantitative release of bound metals to flexible short-chain synthetic biopolymers.

Biopolymers↗
PubMed2004

Atomic force microscopy imaging and pulling of nucleic acids.

Recent advances in atomic force microscopy (AFM) imaging of nucleic acids include the visualization of DNA and RNA incorporated into devices and patterns, and into structures based on their sequences or sequence recognition. AFM imaging of nuclear structures has contributed to advances in telomere research and to our understanding of nucleosome formation. Highlights of force spectroscopy or pulling of nucleic acids include the use of DNA as a programmable force sensor, and the analysis of RNA flexibility and drug binding to DNA.

DNA↗
PubMed1995

Biological atomic force microscopy: from microns to nanometers and beyond.

In recent years, atomic force microscopy (AFM) has become the most rapidly developing imaging method. We summarize its recent applications in structural biology, with emphasis on high-resolution imaging, and illustrate these applications with images of cell membranes, DNA, and soluble and membrane proteins. With present technology, and at room temperature, nanometer resolution of DNA and soluble proteins is achievable, although the resolution attained on cell surfaces is more limited (10-50 nm). We suggest that high-resolution imaging of cell surfaces and very high (sub-nanometer) resolution molecular imaging requires an approach that increases specimen rigidity. Operation at cryogenic temperatures, the most promising approach, is discussed, and a cryo-AFM designed for operating at ambient pressure and liquid nitrogen temperature is described. The results include high-resolution images and evidence of increased molecular rigidity at low temperatures.

Animals↗
PubMed1997

Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.

Crystallization↗
PubMed2004

Moisture-induced surface crystallization of spray-dried amorphous lactose particles studied by atomic force microscopy.

The aim of this study was to show that atomic force microscopy (AFM) can be used to obtain mechanistic and kinetic information about the process of moisture-induced surface crystallization of single particles of amorphous lactose. Completely amorphous lactose particles were prepared by spray-drying a solution of alpha-lactose monohydrate, and moisture-induced crystallization was monitored for a bed of particles by microcalorimetry and for single particles by AFM. From the AFM images it was found that crystallization of the surface of single particles can be described in terms of a sequence of three events: an initial smoothening of the surface, formation of crystalline nanostructures dispersed in amorphous material, and growth of these structures to a complete crystalline surface. The surface roughness parameter rugosity was used to estimate the fraction crystalline surface, and the growth kinetics were found to obey the JMAK equation. The fraction crystalline surface at different times could also be estimated by determining the growth rate of individual crystals. It was concluded that AFM offers a unique means of visualizing the process of moisture-induced surface crystallization of amorphous particles and enables mechanistic and kinetic information about the process to be extracted.

Calorimetry↗
PubMed2006

Atomic force microscopy characterization of Xenopus laevis oocyte plasma membrane.

We used atomic force microscopy (AFM) to characterize the plasma membrane of Xenopus laevis oocytes. The samples were prepared according to novel protocols, which allowed the investigation of the extra- and intracellular sides of the membrane, both of which showed sparsely distributed spherical-like protrusions. Regions with comparably sized and densely packed structures arranged in an orderly manner were visualized and dimensionally characterized. In particular, two different arrangements, hexagonal and square packing, were recognizable in ordered regions. The lateral dimension of structures visualized on the external side had a normal distribution centered on 25.5 +/- 0.3 nm (mean value +/- SE), whereas that on the intracellular side showed a normal distribution centered on 30.2 +/- 0.8 nm. The height of the protrusions was 2-5 nm on the external side and 1-3 nm on the intracellular side. The mean number of structures on the external and intracellular sides of the plasma membrane was about 1000 microm(-2) and 850 microm(-2) respectively. Trypsin treatment greatly decreased the size of the membrane protrusions, thus confirming the proteic nature of the structures. These results show that AFM is a useful tool for structural characterization of proteins in a native eukaryotic membrane.

Animals↗
PubMed2000

An atomic force microscopy investigation of bioadhesive polymer adsorption onto human buccal cells.

Atomic force microscopy (AFM) was used to examine the buccal cell surface in order to image the presence of adsorbed bioadhesive polymers identified from previous work. Isotonic saline solution (5 ml) containing either polycarbophil (pH 7.6), chitosan (pH 4.5) or hydroxypropyl methylcellulose (pH 7.6) (0.5% w/v) was exposed to freshly collected buccal cells (ca. 48x10(4) cells/test) for 15 min at 30 degrees C. The cells were then rinsed with a small volume of double distilled water, allowed to air-dry on a freshy cleaved mica surface and imaged using contact mode AFM. Untreated cells showed relatively smooth surface characteristics, with many small 'crater-like' pits and indentations spread over cell surfaces. Cells that had been treated with all the investigated polymers appeared to have lost the crater and indentation characteristic and gained a higher surface roughness. These results suggest that polymer chains had adsorbed onto the cell surfaces. Quantitative image analysis of cell topography showed significant increases (P<0.05) in arithmetic roughness average (R(a)) for all the investigated polymer treated cells surfaces with respect to untreated control specimens. The changes in surface topography indicate the presence of adsorbed polymer, confirming previous work. This study demonstrates the suitability of AFM as a powerful and sensitive technique for detecting and imaging bioadhesive polymers present on mucosal cell surfaces.

Acrylic Resins↗
PubMed2003

Application of atomic force microscopy and grating coupler for the characterization of biosensor surfaces.

Atomic force microscopy (AFM) and an optical grating coupler system were used to improve the understanding of the biosensing layer on a Ta(2)O(5)-light-guiding surface. Exemplary, we investigated the immobilization of the protein avidin, the subsequent binding of biotinylated oligonucleotides and hybridization of a complementary 12-mer. The AFM measurements revealed the height of approximately 1.6 nm for a single avidin molecule, while the thickness of the avidin layer on the biosensor surface seemed to be 2.8-3.0 nm. This result lead to the conclusion that the protein was not forming a simple monolayer. However, the thickness of the avidin layer could not be determined directly, but only after shifting of protein by the tip of the AFM leading to grooves of 1 micro m(2) and approximately 3 nm depth. As the height of oxide particles forming the waveguide surface was also in the range of 1.5 nm, the depth of these grooves could also be a result of the deposition of proteins on top of the oxide particles. This was consistent with the increased roughness of the surface after protein binding. Thus, investigations with the grating coupler were used to determine quantitatively the amount of immobilized avidin. On a biotinylated surface the amount of immobilized avidin lead to the assumption of a complete monolayer, whereas simple adsorption proved to be less efficient. A binding ratio of 1:1.3 for avidin and a biotinylated oligonucleotide was achieved. Up to 83% of the bound single strand were accessible for a subsequent hybridization reaction with a 12-mer. These results supported the model of avidin being deposited mainly on top of the oxide particles leading to the picture of a 'rough' complete protein monolayer, which was postulated from the AFM investigations.

Avidin↗