Applications of scanning force microscopy (SFM) in polymer studies have flourished in this decade, reflecting (a) the power of SFM to image both structure and propertiesdown to the nanometer scale, and (b) the low cost and ease of getting useful results in ambient environments. One difficulty in SFM of polymers has been damage incurred by soft materials during the imaging process. The problem was alleviated by the development of special dynamic modes of operation, in which the probe spends little or no time in contact with the polymer surface. Such modes were dubbed “tapping”, “intermittent-contact”, “non-contact”, “near-contact”, etc. As studies proliferated, it became apparent that different researchers were using different terms to refer to the same apparent imaging mechanism, or the same term to refer to different imaging mechanisms. This quandary derived from a poor understanding of exactly how the SFM probe interacts with the sample surface.1-3,5

Fourier Transform Infrared (FTIR) Microspectroscopy is a powerful method to examine and characterize domains down to areas of 10х10 μm2. Spatial concentration maps of chemical species were obtained using apertures to sequentially examine areas on a grid and obtain a map by plotting average spectral absorbance data obtained from each of the areas. New Focal Plane Array (FPA) detectors, composed of a large number (in our case, 64х64) of small detectors arranged in a grid pattern, allow simultaneous collection of infrared radiation at many points from a large spatial region (500х500 μm2 in our case). This allows for a rapid acquisition of chemically specific images from a given area and the examination of several real-time processes. This technique has found a wide variety of applications in multi-phase polymers including polymer laminates, phase separated polymer composites, semi-crystalline polymers and blends, and solvent diffusion into polymers have been the main areas of interest in non-biological IR studies using FPA detection. We present some of the recent work in our group on various multi-phase polymer systems using FPA based infrared spectroscopy.

The diffraction of visible light limits the spatial resolution in conventional optical microscopy to about 200-300 nm. In near-field scanning optical microscopy (NSOM), resolution is improved by bringing the light source, such as the end of an optical fiber, very close to the sample surface. Laser light coupled into the opposite end of the fiber propagates down the fiber core and is emitted from the aperture of the tip. When the sample is in the near-field(roughly within one tip diameter of the end of the tip), the spatial resolution is essentially equal to the diameter of the aperture at the end of the tip and is not determined by diffraction effects. Two-dimensional imaging is accomplished by raster-scanning the sample underneath the fiber tip and collecting transmitted or reflected light at a photodetector.

Atomic force microscopy (AFM) allows the surfaces of native biological macromolecules to be imaged in aqueous solution with submolecular resolution. Short range forces govern the interactions between the AFM tip and the sample and produce the submolecular information of high resolution topographs. In contrast, the long range electrostatic double-layer (EDL) force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area [1]. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteineous substructures. Consequently, the deformation of the native protein is minimized and polypeptide loops of individual proteins were imaged in aqueous solution with a lateral resolution < 5 Åand a vertical resolution < 1 Å (Fig. 1).

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.

Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

DNA in the eukaryotic nucleus is not naked. Instead, it is complexed at frequent intervals with an equal molecular mass of histones to form nucleosomes. In the nucleosome, ∼146 bp of DNA are wrapped around a core histone octamer composed of two copies each of histones H4, H3, H2B, and H2A. The family of linker histones (H1, H5 and H1°) bind to the DNA between successive nucleosomes and help maintain the three-dimensional arrangement of nucleosomes within the chromatin fiber. AFM studies of chromatin fibers with the histones either selectively trypsinized or selectively reconstituted demonstrate a specific role for the H3 N-termini in maintaining fiber structure, in conjunction with the 80 amino acid linker histone globular domain. These AFM results structurally agree with the location of the H3 N-termini in the histone octamer or the nucleosome core particle.

The mechanical Q-factor of an AFM cantilever immersed in fluid is reduced to a small value (ca. 3) owing to viscous damping. Thus, a large driving force is needed to excite the cantilever into bending motion in fluid. There are two common methods for exciting cantilevers for dynamic force microscopy in fluids, illustrated in Figure 1. Fig. la illustrates acoustic excitation in which a piezoelectric transducer displaces the base of the cantilever, causing bending motion of the cantilever when the driving frequency is near to a bending resonance of the cantilever. Fig. lb shows magnetic excitation. In magnetic excitation, a magnetic field is used to cause bending of a magnetic cantilever either through magnetostriction or MXB forces.

Acoustic excitation has the highest amplitude at mechanical resonances of the cantilever housing, with the result that the response is dominated by these sharp features,Fig. 2a. In contrast, the response to magnetic excitation is intrinsic to the cantilever, Fig. 2b. Thus, magnetic excitation permits the cantilever to be driven over a wide range of frequencies. This is important for calibration of the amplitude and for experiments involving time and concentration dependence in tip-sample interactions, e.g., anti-body recognition imaging.

Repair of damaged or incorrectly matched DNA is essential to the survival of all organisms. Consequently cells have devised a plentitude of pathways for repair. We have been investigating the mechanisms of mismatch repair and base excision repair. Both of these repair processes involve a large number of proteins that interact with one another as well as with DNA. Our long-term goal is to assemble complexes that are fully functional for DNA repair and to image the process of DNA repair. In addition, we wish to i) determine the stoicheometry of binding of the protein complexes to each other and to DNA, ii) monitor conformational changes due to substrate binding, iii) measure physical properties of DNA and the complexes. To accomplish this end, we have endeavored to improve techniques for solution imaging as well as those for data analysis. In this presentation I will discuss data on the stoicheometry of binding in several protein complexes and data on the physical properties of DNA.

To measure the physical properties of DNA, we utilize a nanoManipulator, a modified Scanning Force Microscope with a novel, user-friendly interface that allows easy and controlled manipulation of nanometer-sized samples.

Microtubules are long, hollow, stiff polymers that extend throughout the cytoplasm. They are involved in such diverse functions as governing the location of membrane-bounded organelles and chromosome separation during mitosis. The basic structural unit is tubulin, which is a heterodimer consisting of two closely related and tightly linked globular polypeptides called α and β-tubulin. Alternating α and β tubulin subunits form protofilaments, 13 of which bundle around a central core to form microtubule. The detailed structures of cytoplasmic microtubules have been studied extensively using various electron microscopic techniques. As microtubules are dynamic structures in constant transitions between growing and shrinking phases, it would be extremely interesting to investigate the structural organization of the subunit tubulin molecules in a buffer close to physiological conditions. With its high resolution and ability to image in fluid, atomic force microscope (AFM) makes it possible to study the biological structures in a native environment.

A major advantage of atomic force microscopy (AFM) is its intrinsic high contrast, owing to its extremely high sensitivity to height variations. For clean, flat specimens, high-resolution images of a variety of bio-molecules have been obtained, demonstrating the usefulness of AFM in structural biology. However, for large molecular complexes, the effectiveness of AFM is seriously limited, partly owing to the excessive deformation induced by the probe force exerted on the specimen. To overcome this difficulty, we have developed a cryo-AFM, operated under liquid nitrogen vapor at a temperature only a few degrees above 77K. In this system, specimen or tip contamination is significantly reduced, and the resolution achieved with isolated macromolecules is generally higher than that obtainable at room temperature with AFM. Therefore, cryo-AFM has been proven to be a worthy alternative for conventional electron microscopy (EM) with the potential of higher resolution without image averaging.

A particularly fruitful application of cryo-AFM is the study of the spatial structure of several immuno-proteins, where the 3D surface topology of the molecule has been obtained.

Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supported by a soft but tough dentin structure, the tooth is an advanced nanocomposite that can endure mastication stresses throughout a lifetime. A detail understanding the structure of the tooth, and sepecifically detin-enamel junction (DEJ), not only provides a sound basis for a model for synthetic dental restoration, but also provides lessons from nature on biomimetic regeneration with mechanical integrity. Enamel is a non-growing mineralized tissue and is subjected to most mechanical abuse. Dentin-enamel junction plays a critical role of distributing load between two very dissimilar materials - enamel and dentin. Bulk scale mechanical tests have shown that induced cracks on enamel tend to be arrested DEJ. Furthermore, nanoindentation measurements have also shown that there is a gradual decrease in hardness from enamel to dentin in the DEJ zone suggests a strong mechanical coupling in both deciduous and adult incisors. The objective of this investigation, through microscopical study, is to understand how these two dental hard tissues structurally couple through their junction zone.

Recent AFM research in our laboratory has covered such diverse biomaterials as laminin and other macromolecules from basement membranes (Fig. 1) (1), DNA condensed for gene therapy (Fig. 2) (2), DNA-protein complexes in the yeast kinetochore (Fig. 3) (3), and biofilms of the bacterium Pseudomonas putida (Fig. 4) (4).

Laminin is a major protein of basement membranes. When analyzed by AFM in air, it shows a variety of conformations of its cruciform structure (Fig. 1A). Time-lapse images of a single laminin molecule in aqueous solution show the flexibility of the laminin arms as they move and bend (Fig. 1B)(1).

AFM analysis of DNA condensed for receptor-mediated gene therapy poses a puzzle (Fig. 2)(2). Polylysine condenses DNA poorly (Fig. 2A), while AsOR-polylysine condenses DNA into compact toroids and short rods (Fig. 2B). AsOR (asialoorosomucoid) is a 38-kDa glycoprotein with a net negative charge of 5 carboxylic acid groups per molecule. The puzzle is why this negative glycoprotein, covalently attached to polylysine, can enhance the condensation of DNA so much over the condensation caused by the polycation, polylysine, alone.

The Atomic Force Microscope is a simple instrument capable of imaging single macromolecules with submolecular resolution and measure the forces required to unfold a single protein domain (Fig. 1).

The AFM method of unfolding a tandem modular protein generates a characteristic sawtooth pattern in the force-extension curves (Fig. 2). The measured spaces between the peaks are thought to correspond to the increase in contour length (ΔΔLC) of the protein caused by the unfolding of one of its modules. In order to demonstrate the validity of this model and measure the sensitivity of the AFM we have designed an experiment where we have inserted a cluster of 5 Gly into the loop joining the F and G b-strands of the I27 module of the I band from human cardiac titin. This loop is clearly within the folded structure of the module and hence, if our predictions are correct, unfolding of the I27-5Gly module should reveal an expanded ΔLC. We expect to measure an increase in the contour length increment of ΔΔLC = 5 ✗ 0.38 nm =1.9 nm.

The high spatial resolution and the force sensitivity of the Atomic Force Microscope have made it possible to perform mechanical experiments with single molecules. These experiments give direct access to the forces that stabilize biomolecule structure. In a variety of examples we show how different molecular interactions determine the mechanical stability of polysaccharides, proteins, DNA and other biomolecules.

Under the influence of a stretching force of around 750 pN the polysaccharide dextran undergoes a transition into a stretched conformation during which bond angles flip into a new conformation. Molecular dynamics simulations corroborated this conformational change.

Proteins fold into compact domains. Especially for structural proteins the mechanical stability of these domains is of importance. We show that the immunoglobulin and fibronectin III domains of the muscle protein titin exhibit exceptionally high unfolding forces (150-250 pN) when stretched at speeds of normal muscle operation (Fig. 1).

Recently, a quantum leap has been achieved in the analysis of DNA and proteins through the advent of the biochip technology. This technology is a product of a broad interdisciplinary approach combining biochemical analysis, semiconductor manufacturing and computer software. Biochips can be defined as miniaturized ordered arrays of macro molecules or pieces thereof that are immobilized in a precise spatial manner on support media and can be used in highly automated, large-scale and high-throughput fashion to analyze biological material. The biochip can be used in a wide variety of areas related to basic research and can find versatile applications in almost all areas of human activities connected to biotechnology, medicine, agriculture, and environment monitoring and bioremediation.

The power of the technology has already been demonstrated in areas like gene sequencing and proofreading, detection of single-nucleotide mutation and polymorphism, identification of genes, identification of viruses and microorganisms, gene expression analysis, analysis of sequencespecific ligands and proteins, and others.

Connexins oligomerize into hemichannels, traffic to the cell surface and dock with hemichannels from an adjacent cell to form intercellular gap junction channels. Pulsechase studies have revealed that connexins are subject to a short half-life ranging from 1- 5 hrs. To examine the mechanisms involved in connexin trafficking, gap junction assembly and internalization in living cells we fused green fluorescent protein (GFP) to the amino or carboxyl terminus of full length connexins (Cx). When cDNA encoding Cx43-GFP was transfected into communication-competent NRK cells, Cx43-negative MDCK cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into functional gap junctions (Figure 1). Most notably, the fusion of GFP to the amino terminal of Cx43 (GFP-Cx43) or Cx32 (GFP-Cx32) did not inhibit the co-translational insertion of these polytopic type IV connexins into the membrane and as a result gap junction plaques assembled at cellcell interfaces.

Solid tumors frequently contain cells that are heterogeneous in the copy number of DNA loci. This fact implies the existence of genetic instability, which may be associated with disease aggressiveness. Accurate measurement of this phenomenon requires analysis of intact nuclei within their natural tissue context. We perform these measurements by preparing >30μm thick tissue sections, labeling them with fluorescent labels for total DNA and for specific DNA loci using fluorescence in situ hybridization (FISH) which retain the transparency of the tissue and acquiring 3D images of the tissue using confocal microscopy (figure 1). In this study, we combined automated 3D image analysis (IA) algorithms for segmenting individual nuclei based on the total DNA stain1 and for segmenting the punctuate FISH signals of DNA loci. This enables us to efficiently enumerate the copy number of specific DNA loci in individual cells and as a function of the cell's location in the tissue.

Dynamin is a 100 kD GTPase that plays an essential role in clathrin-coated vesicle formation during receptor mediated endocytosis, and in caveolae internalization and may play a role in intracellular membrane trafficking (1). It shares an extensive sequence homology (70% identity) to shibiregene product in Drosophila(2,3). The shibiretsmutants exhibit a rapid and reversible paralysis at non-permissive temperature due to a depletion of synaptic vesicles in their nerve termini which is believed to be caused by a block in endocytosis since there is an accumulation of “collared” clathrin-coated pits at the plasma membrane (4). Synaptosomes treated with GTPγs produces elongated necks surrounded by dynamin (6). Purified recombinant dynamin itself can assemble to form spirals and bind to lipid vesicles to form tubes, which resemble the “collar” at the necks of coated pits (5). These dynamin tubes vesiculate upon GTP treatment (7), suggesting a unique role of dynamin acting as a mechanoenzyme which causes clathrin-coated vesicles to be pinched off plasma membrane.

The chemical synapse is a key structure used by neurons to communicate with neighboring cells. This is an asymmetric structure composed of two elements. The presynaptic part contains small synaptic vesicles filled with neurotransmitter. In an active synapse synaptic vesicles fuse with the presynaptic membrane and release neurotransmitter into the synaptic cleft. This results in activation of receptor molecules and respective ion channels on the postsynaptic cell. After a fusion event, the vesicle membrane is retrieved to form a new vesicle. Clathrin-mediated endocytosis is one of the main pathways for synaptic vesicle recycling. It involves a series of steps that starts with a flat plasma membrane and ends with an internalized coated vesicle. Four distinct stages have been defined: (1) the assembly of the clathrin coat, (2) the invagination of the coated membrane into a pit, (3) the constriction of the coated pit, and (4) the fission reaction that liberates the coated vesicle (e.g. De Camilli and Takei, 1996; Brodin et al., 1997). Analysis of mutant yeast and animal cells have linked a number of proteins to each of these stages. Taken together with data from in vitro binding studies, these observations indicate the existence of a complex molecular network that regulate the endocytic reactions.

Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane proteins that function as cation selective, ligand-gated ion channels and are widely distributed throughout the vertebrate nervous system. One of the most abundant nAChRs is a species that contains the α7 gene product (α7-AChRs), binds the snake venom oc-bungarotoxin, and rapidly desensitizes. These receptors have been shown to function at presynaptic sites to modulate transmitter release, as well as on postsynaptic cells where they mediate transmission. Interestingly, these receptors have an exceptionally high relative permeability to calcium.

In the chick ciliary ganglion, the α 7-AChRs play a prominent role, by generating large synaptic currents, but the receptors appear to be excluded from postysnaptic densities on the cell. Immunohistochemical studies have shown that the receptors form large clusters on the surface of the ciliary ganglion neurons. We have recently shown that the α 7-containing receptors are concentrated on mats of somatic spines in close proximity to putative sites of presynaptic transmitter release.