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Viewing molecules with scanning tunneling microscopy and atomic force microscopy.

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.

Brain

Imaging and nanodissection of individual supercoiled plasmids by atomic force microscopy.

The atomic force microscope (AFM) was used to image supercoiled plasmid DNA deposited on a mica surface in either a hydrated or desiccated state. Hydrated plasmid was precisely cut by the scanning tip at a location determined by the instrument operator. Small pieces of DNA (100-150 nm in length) were excised and deposited adjacent to the dissected plasmid, demonstrating that it is possible to remove and manipulate genomic DNA fragments, unresolvable by light microscopy, from defined chromosomal locations by AFM.

DNA, Superhelical

High-resolution imaging of chromosome-related structures by atomic force microscopy.

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein.

Animals

Molecular-resolution images of aspirin crystals with atomic force microscopy.

The atomic force microscope was developed in order to image arrays of molecules on the (001), (100) and (011) faces of aspirin crystals in water. Lattice spacings and symmetries of methyl groups and the part of the phenyl groups on the (001) face in water are consistent with X-ray diffraction data. Surfaces of the (100) face that show the best-developed cleavage, in general, are difficult to image. Although the lattice constant derived from (100) image is consistent with the X-ray diffraction data, the c-glide plane symmetry is lost and diffused spots are observed that are 1.8-3.1 times more elongated than (001) spots. It is not possible to image the (011) surface. This initial success in imaging the molecular crystal surfaces of drug compounds has clarified the different submolecular structures of the three different crystal faces, and the close relationship between the imaging difficulties and the differences in the dissolution process in water.

Aspirin

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.

Animals

Imaging isolated strands of DNA molecules by atomic force microscopy.

We have employed an atomic force microscope (AFM) to image in air isolated strands of pBS+ plasmid DNA adsorbed onto freshly cleaved mica. At a DNA concentration below 0.3 micrograms/ml isolated strands of the plasmid DNA are usually seen, while for concentrations higher than 3 micrograms/ml a uniform coverage of interconnected DNA strands was observed. We found that the contrast and the width of DNA were dependent upon humidity. When the relative humidity exceeds 60%, negative contrast images with strand widths 20 times the width of DNA are found, while positive contrast images with 7 to 10 times the width of DNA are found when the humidity is below 30%. By placing the AFM in an environment where the humidity could be controlled, we were able to switch between positive and negative contrasts.

Adsorption

Atomic force microscopy of DNA on mica and chemically modified mica.

Atomic force microscopy (AFM) was used to image circular DNA adsorbed on freshly cleaved mica and mica chemically modified with Mg(II), Co(II), La(III), and Zr(IV). Images obtained on unmodified mica show coiling of DNA due to forces involved during the drying process. The coiling or super twisting appeared to be right handed and the extent of super twisting could be controlled by the drying conditions. Images of DNA observed on chemically modified surfaces show isolated open circular DNA that is free from super twisting, presumably due to strong binding of DNA on chemically modified surfaces.

Adsorption

Atomic force microscopy of reovirus dsRNA: a routine technique for length measurements.

Atomic force microscopy (AFM) was used to image reovirus double stranded RNA (dsRNA) deposited from diluted buffer solution onto a chemically treated mica surface. This procedure allows AFM images of dsRNA molecules to be obtained with a quality close to that obtained with conventional electron microscopy. The length of the molecules were measured directly on a computer display using the digitally acquired images. The lengths of the molecules varied between 0.2 and 1.8 microns. Statistical analysis showed a multimodal distribution with clear maxima at 0.4, 0.65 and 1.05 microns. These data are in a good agreement with those obtained by electron microscopy and gel electrophoresis.

Microscopy, Scanning Tunneling

Cisplatin induced alterations in oriented fibers of DNA studied by atomic force microscopy.

Oriented fibers of DNA, prepared by the wet spin method, were imaged by atomic force microscopy. It was found that an oriented fiber's substructure is an array of assemblages of DNA. For native DNA, the assemblages exhibit a characteristic width of 76 nm, a thickness of 20 nm, and appear to carry a right handed twist. Treatment with the anticancer drug cisplatin prior to wet spinning induces geometric irregularities, in the form of kinks and width distortions, into the assemblages of DNA.

Cisplatin

Atomic force microscopy and dissection of gap junctions.

An atomic force microscope (AFM) was used to study the structure of isolated hepatic gap junctions in phosphate-buffered saline (PBS). The thickness of these gap junctions appears to be 14.4 nanometers, close to the dimensions reported by electron microscopy (EM). When an increasing force is applied to the microscope tip, the top membrane of the gap junction can be "dissected" away, leaving the extracellular domains of the bottom membrane exposed. When such "force dissection" is performed on samples both trypsinized and fixed with glutaraldehyde, the hexagonal array of gap junction hemichannels is revealed, with a center-to-center spacing of 9.1 nanometers.

Animals

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.

Actins

Atomic force microscopy of DNA molecules.

DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.

Cytochrome c Group

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.

Aluminum Silicates

Atomic force microscopy: seeing molecules of lipid and immunoglobulin.

The atomic force microscope (AFM) can image individual molecules by raster-scanning a sharp tip over a surface. In this paper we present molecular-resolution images of immunoglobulin M (IgM) and of ultraviolet light-polymerized films of the lipid dimethyl-bis(pentacosadiynoyloxyethyl) ammonium bromide ("BRONCO"). The polar head groups of individual lipid molecules can be resolved on the surface of this and other lipid films. These lipid films also provide a good substrate for AFM imaging of DNA and of other molecules such as antibodies. Because the AFM scans surfaces, it is most often successful at imaging either molecules that can form an array on a surface or molecules that are quite firmly attached to a surface. The ability of the AFM to operate under water, buffers, and other liquids makes it possible to study biological molecules under conditions in which they are physiologically active. Imaging of the actual molecular process of fibrin polymerization shows the potential of the AFM for studying biological processes. In the six years since its invention, the AFM has excited much interest and has imaged molecules in a wide range of systems.

Antibodies

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.

1,2-Dipalmitoylphosphatidylcholine

Atomic force microscopy of conventional and unconventional nucleic acid structures.

Images of conventional (Watson-Crick base paired) and unconventional (G4 RNA) nucleic acid structures have been obtained by atomic force microscopy. The images are reproducibly generated from samples deposited on freshly cleaved mica. Periodic substructural features are evident in fibres observed in both cases. In the case of G4 RNA, tip-induced formation of large fibres is observed.

DNA

Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM.

Animals