Molecular resolution imaging of macromolecular crystals by atomic force microscopy.

Atomic force microscopy (AFM) images at the molecular level have been obtained for a number of different protein and virus crystals. They can be utilized in some special cases to obtain information useful to crystal structure analyses by x-ray diffraction. In particular, questions of space group enantiomer, the packing of molecules within a unit cell, the number of molecules per asymmetric unit, and the dispositions of multiple molecules within the asymmetric unit may be resolved. In addition, because of the increasing sensitivity and resolution of the AFM technique, some molecular features of very large asymmetric units may be within reach. We describe here high-resolution studies, using AFM, to visualize individual molecules and viruses in their crystal lattices. These investigations included fungal lipase, lysozyme, thaumatin, canavalin, and satellite tobacco mosaic virus (STMV).     

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.     

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.     

Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy.

Samples of supported planar lipid-protein membranes and actin filaments on mica were imaged by atomic force microscopy (AFM). The samples were fully submerged in buffer at room temperature during imaging. Individual proteins bound to the reconstituted membrane were distinguishable; some structural details could be resolved. Also, surface-induced, self-assembling of actin filaments on mica could be observed. Monomeric subunits were imaged on individual actin filaments. The filaments could be manipulated on or removed from the surface by the tip of the AFM. The process of the decoupling of the filamentous network from the surface upon changing the ionic conditions was imaged in real time.     

Observation of backbone self-crossings of organismal DNAs through atomic force microscopy

It is demonstrated in our studies that intrinsic curvatures of organismal DNAs could lead to the generation of duplex backbone self-crossings in their relaxed forms. In addition, the backbone self-crossings of the organismal DNAs could be readily confirmed through atomic force microscopic examination.     

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.     

Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy.

Purple membranes adsorbed to mica were imaged in buffer solution using the atomic force microscope. The hexagonal diffraction patterns of topographs from the cytoplasmic and the extracellular surface showed a resolution of 0.7 and 1.2 nm, respectively. On the cytoplasmic surface, individual bacteriorhodopsin molecules consistently exhibited a distinct substructure. Depending on the pH value of the buffer solution, the height of the purple membranes decreased from 5.6 nm (pH 10.5) to 5.1 nm (pH 4). The results are discussed with respect to the structure determined by cryo-electron microscopy.     

Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy

As the application of high-resolution atomic force microscopy (AFM) has led us recently to the discovery of a unique pressure-induced circular amyloid, we used the same approach to examine morphological events accompanying insulin aggregation under ambient conditions. This study presents the multistage, hierarchical character of the spontaneous fibrillation of insulin at low pH and at 60 and 70 degrees C, and-due to the marked enhancement of image resolution achieved-brings new clues as to the fibrils' ultrastructure and mechanisms of its assembly. Specifically, focusing on the prefibrillar amorphous aggregates occurring 30 s after elevating temperature to the nucleation-enhancing 60 degrees C, revealed the tendency of the globule-shaped oligomers to queue and assembly into elongated forms. This suggests that the shape of the nuclei itself predetermines-in part-the fibrillar architecture of the amyloid. Among first fibrillar features, short but relatively thick (8-nm) seedlike forms appeared on a very short timescale within the first minute of incubation. It has been shown that such fibrils are likely to act as lateral scaffolds for the growth of amyloid. By using phase-image AFM as a nanometer-resolved probe of visco-elastic surface properties, we were able to show that bundles of early protofilaments associated into parallel fibrils are capable of a cooperative transformation into twisted, highly ordered superhelices of the mature amyloid. Independently from producing evidence for the step-resolved character of the process, intermediate and morphologically heterogeneous forms were trapped and characterized, which yields direct evidence for the multipathway character of the amyloidogenesis of insulin. Apart from the faster kinetics, the increased temperature of 70 degrees C leads to a higher degree of morphological variability: along straight rods, twisted ribbonlike structures, rod bundles, and ropelike structures become prominent in the corresponding AFM data.     

Imaging biomolecule arrays by atomic force microscopy.

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.     

Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy.

Application of atomic force microscopy (AFM) to biological objects and processes under physiological conditions has been hampered so far by the deformation and destruction of the soft biological materials invoked. Here we describe a new mode of operation in which the standard V-shaped silicon nitride cantilever is oscillated under liquid and damped by the interaction between AFM tip and sample surface. Because of the viscoelastic behavior of the cellular surface, cells effectively "harden" under such a tapping motion at high frequencies and become less susceptible to deformation. Images obtained in this way primarily reveal the surface structure of the cell. It is now possible to study physiological processes, such as cell growth, with a minimal level of perturbation and high spatial resolution (approximately 20 nm).     

Imaging polysaccharides by atomic force microscopy

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides 1-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by κ-carrageenan and gellan gum. © 1996 John Wiley & Sons, Inc.     

Imaging the RecA-DNA complex by atomic force microscopy.

Atomic force microscopy (AFM) was applied to study the RecA protein and its complexes with DNA in air and in aqueous solution. RecA and DNA were reacted under several conditions, and deposited onto a mica substrate pre-treated in various ways. We found that the structure of the RecA and RecA-DNA complexes, especially the height of the molecules, was affected by the sample preparation method such as gel filtration, and environment during imaging.     

Analysis off cereal chromosomes by atomic force microscopy.

Atomic force microscopy has been applied to the study of plant chromosomes from cereal grasses Triticum aestivum (bread wheat), Triticum tauschii, and Hordeum vulgare (barley). Using standard mitotic metaphase squashes, high resolution images have been obtained of untreated chromosomes and also of chromosomes after C-banding, N-banding, and in situ hybridization. The true 3-dimensional nature of the images permits detailed analysis of the surface structure and, on untreated uncoated chromosomes, surface features on a length scale consistent with nucleosome structures have been observed. C+ and N+ regions are manifest as areas of high relief on a slightly collapsed chromosome structure. In situ hybridization leads to a more severe degradation of the native structure, although it is still possible to correlate the optical signal with the topography of the hybridized chromosome. Key words : atomic force microscope, AFM, chromosomes, C-banding, in situ hybridization.     

Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy

Attempts were made to apply atomic force microscopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules. In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing. The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA. Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin–avidin complex. Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites. This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample.     

High resolution thermal denaturation of mammalian DNAs.

High resolution melting profiles of different mammalian DNAs are presented. Melting curves of various mammalian DNAs were compared with respect to the degree of asymmetry, first moment, transition breath and Tmi of individual subtransitions. Quantitative comparison of the shape of all melting curves was made. Correlation between phylogenetical relations among mammals and shape of the melting profiles of their DNAs was demonstrated. The difference between multi-component heterogeneity of mammalian DNAs found by optical melting analysis and sedimentation in CsCl-netropsin density gradient is also discussed.     

Biomolecular interactions measured by atomic force microscopy

Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites. In this review we discuss the important contributions that have led to the development of this field. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible measurements by AFM. This increased reproducibility will pave the way for a better understanding of molecular interactions in cell biology.     

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.