Anal. Chem. 2000, 72, 189R-196R
Scanning Probe Microscopy Peter T. Lillehei and Lawrence A. Bottomley*
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 Review Contents Instrumental Aspects Applications Nanofabrication Chemical Force Microscopy/Spectroscopy Scanning Electrochemical Microscopy Biological Imaging The Future of SPM Literature Cited
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Scanning probe microscopy (SPM) encompasses a family of techniques that measures surface topography and surface properties on the atomic scale. The number of papers devoted to technical advances and applications of SPM continues to rise. The availability of high-quality commercial instruments, and the renewed focus among scientists and engineers to gain insights into molecular interactions and to manipulate matter on the atomic scale, are contributing factors to this increasing interest. The format of this review, covering papers published during the period October 1, 1997 through December 31, 1999, is significantly changed from that published two years ago. Since electronic access to the literature and efficient search engines are readily available, extensive listing of the contributions followed by terse summaries of their content no longer seems appropriate. The intent of this review is to highlight the most important contributions to the field published within the aforementioned period. Our selections illustrate only some of the research avenues currently being explored with SPM and are, without doubt, subjective. INSTRUMENTAL ASPECTS Design specifications for next-generation scanning probe microscopes include faster image acquisition, increased force sensitivity, and reduced noise. To this end, researchers have focused on reducing cantilever dimensions and improving measurement of cantilever deflection. Smaller cantilevers resonate at higher frequency and experience lower viscous dampening. This enables faster imaging and reduces the minimum detectable force. However, small cantilevers require improved optical deflection schemes to avoid optical interference from light spilling over the cantilever and reflecting off the substrate. Scha¨ffer and co-workers (1) have designed an optical beam deflection detection apparatus with a spot size of only 1.6 µm. Viani et al. (2) used this apparatus and small cantilevers to obtain Tapping mode images of DNA (in liquid) on mica at speeds of 1.7 s/image. Paloczi and colleagues (3) and Walters et al. (4) have used smaller cantilevers for highspeed, in situ imaging of crystal growth. Viani and co-workers (2, 5) performed force spectroscopy measurements with small cantilevers. Thermal noise levels were 6 times lower than those 10.1021/a10000108 CCC: $19.00 Published on Web 05/11/2000
© 2000 American Chemical Society
typically encountered with commercially available cantilevers of the same force constant (5). To reduce force constants, cantilevers have been manufactured from materials other than silicon or silicon nitride. For example, plastic cantilevers have been fabricated by Genolet et al. (6) using silicon micromolding techniques. The reduced Young’s modulus of the material afforded lower force constants without reduction in cantilever dimensions. Cantilever spring constants were 40 times smaller than silicon cantilevers of similar dimension. Scha¨ffer and colleagues (1) fabricated highly reflective aluminum cantilevers, thus eliminating much of the thermal bending due to a bimetallic strip effect. Cantilevers with different geometry have also been explored. Paddle-shaped (7), torsional (7), and interdigitated (8) cantilevers have been fabricated. Deflection of interdigitated cantilevers can be determined by the intensity of the diffracted modes since the cantilever is a diffraction grating. Noise levels are significantly reduced with this type of cantilever and detection system. The sources of noise in optical detection of cantilever deflection are as follows: photodector noise, detection circuit noise, fluctuations in laser intensity, mechanical vibration, and thermal motion of the cantilever. Garcia-Valenzuela and Villatoro (9) and Yaralioglu and co-workers (8) have independently shown that thermal motion of the cantilever is the major contributor to noise in SFM. Thus, the use of inexpensive nonmonochromatic light sources will not limit SFM performance. Gittes and Schmidt (10) have theoretically examined strategies for increasing the signal-to-noise ratio in force measurements. They concluded that viscous drag and inertial forces on the cantilever are more important than cantilever compliance in reducing thermal noise. A fabrication scheme for the integration of Schottkey diodes into silicon cantilever tips is outlined by Leinhos et al. (11). These cantilevers will have applications in scanning thermal microscopy, scanning near-field optical microscopy, and scanning electrical force microscopy. For low-temperature imaging, cantilevers made from GaAs/AlGaAs enabled detection of cantilever deflection with an integrated field effect transistor. These cantilevers consume less power and dissipate less heat into the environment compared to conventional piezoresistive cantilevers (12). The development of a feedback-stabilized force sensor by Jarvis et al. (13) allows for the effective stiffness of the cantilever to be tuned up to 30% of its natural stiffness. This is accomplished by attaching a small magnetic particle to the back of the cantilever and positioning an electromagnetic coil underneath the sample. The effective stiffness of the cantilever can be tuned by actively dampening the resonant oscillation of the cantilever with the electromagnetic coil. Schemmel and Gaub (14) designed a force spectrometer with magnetic force control and inductive detection. Magnetic control reduces tip lateral movement and force of contact Analytical Chemistry, Vol. 72, No. 12, June 15, 2000 189R
with the substrate. Force curves obtained with this instrument are free of optical interference artifacts common with optical lever detection. A combined noncontact SFM and STM was designed by Battiston et al. (15) for the imaging of heterogeneous samples with atomic resolution. If the local region of the sample is conductive, topography is determined by tunneling. If the local region is not conductive, topography is determined by noncontact SFM. The instrument switches between the two imaging modes, in real time, using a fuzzy logic controller. Burns and co-workers (16, 17) combined interfacial force microscopy with shear force microscopy to allow simultaneous measurement of friction and attraction forces. With this instrument, they were able to measure friction as a function of load on submicrometer domains. This instrument enables the molecular origins of friction and adhesion to be examined in detail without the jump-to-contact instabilities. Improvements in instrumentation and cantilever design have stimulated renewed interest in understanding cantilever movement in air and fluid. Hazel and Tsukruk (18) performed a finite element analysis of standard commercial V-shaped cantilevers. Vertical and lateral (torsional) spring constants for these cantilevers were computed as a function of Au thickness and resonant frequency. Their results showed that the thin Au coating may account for up to 30% of the mass of the cantilever. Thus, composite cantilevers should not be treated as a single layered material in predicting force constants. Several works describing the fundamentals of dynamic imaging were recently published (19-22). Correlations between amplitude, frequency and phase with tip sharpness, viscoelasticity, and sample mechanical properties have been reported (23-32). Especially noteworthy was Sader’s (33) examination of the frequency response of cantilevers in fluids. He provided a general theoretical model for any cantilever beam immersed in a fluid and driven by any force. Enhanced sensitivity to force gradients can be achieved by driving the cantilever oscillation at higher flexural modes (34). For a detailed description of the cantilever response at these higher modes, see Rabe et al. (35). APPLICATIONS With a scanned probe, one can manipulate matter on the atomic and molecular scale, measure intermolecular forces and mechanical properties, initiate chemical reactions, and determine molecular structure. In the following sections, recent and noteworthy contributions to nanofabrication, chemical force spectroscopy, scanning electrochemical microscopy, and biological applications of SPM are presented. Nanofabrication. Manipulation of atoms with an STM has been practiced for several years. In their review of the manipulation of single molecules with local probes, Gimzewski and Joachim (36) examined the merits of pushing, pulling, and sliding single molecules around on a surface. Insight was provided on the role of scanned probes in molecular-scale electronic device fabrication and single-molecule science. Meyer and co-workers (37) picked up individual CO molecules with a tip and placed them onto specific sites on the copper lattice. The CO-modified tip enhanced contrast between chemically 190R
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different species (CuO, CuCO). Resch et al. (38, 39) built threedimensional structures by pushing individual and connected nanoparticle structures with an SFM tip. Piner and colleagues (40) coined the term “dip-pen” nanolithography for their direct method of writing alkanethiols onto a gold surface. Molecules are transported to the surface via the water meniscus that forms between the tip and gold substrate. This technique has been expanded to a two-ink system (41). In noncontact lithography, the probe tip is kept just slightly above the sample and a field emission current from the tip defines the pattern. This pattern can be either developed in resist (42) or oxidized into silicon (43). The use of a noncontact process eliminates the problem of pattern degradation due to tip wear. Another approach is to use a carbon nanotube modified probe tip to perform local oxidation (44, 45). Nanotube tips are impervious to high compressive forces and are thus less susceptible to wear and pattern degredation. For fabrication of prototype nanoscale devices, SPM techniques are preeminent. The practical drawback to using SPM for nanoscale device fabrication is low throughput. A single tip moving at moderate speeds would take far too long to pattern nanometerscale features over the centimeter-sized areas needed for integrated circuits. Quate’s group is focused on removing this limitation. They have independently controlled two tips to produce nanometer-scale resist patterns (46) and have succeeded in patterning SiO2 lines over 1 cm2 of a silicon wafer in a single pass (47). Pattern formation was accomplished by electric field enhanced oxidation of silicon using 50 cantilevers in parallel. The sample can be simultaneously imaged using this cantilever array. Cooper et al. (48) fabricated a 1.6 Tbit/in.2 data storage device using the SFM. A carbon nanotube, immobilized on a tip, afforded high-resolution imaging and pattern fabrication. Titanium dioxide bits (8 nm in diameter on a 20-nm pitch) were formed on atomically flat titanium surfaces at recording rates of 5 kbits/s. This device, operating at room temperature under ambient conditions, is the first storage device to achieve the terabit per square inch threshold. Also in the area of ultrahigh-density data storage, Binnig et al. (49) developed a technique to write/read/erase/rewrite bits of data into a thin polymer layer, on a silicon surface, at densities up to 500 Gbit/in.2. The process uses a thermomechanical probe to both write and read the data. Erasing of individual bits can be done with the probe tip whereas selective heating can erase whole blocks of data. No degradation performance of the film was observed after several erase/rewrite cycles. Magnetic or chargebased storage devices were described by Born and Weissendanger (50). In comparison, these types of systems are capable of data densities as high as 3 Gbit/in.2 and offer data read rates of up to 1 Mbit/s. Chemical Force Microscopy/Spectroscopy. The ease with which tips can be chemically modified has stimulated SPM-related research on intermolecular chemical forces. McKendry et al. (51) showed that interpretation of chemical force microscopy (CFM) images must include consideration of electrostatic, frictional, and adhesive interactions between the tip and the underlying substrate. Wong and co-workers (52) demonstrated that the spatial orientation of the terminal groups on the chemically modified tip/ substrate has a detectable impact on friction. Kerssemakers and
De Hosson (53) assessed slip/stick friction by laterally resonating the tip in close proximity to the surface. McKendry et al. (54) examined chiral interactions using chemical force microscopy. Chiral molecules were attached to the tip and patterned substrates. Enantiomeric interactions were discerned by adhesion measurements (force curves) and friction force imaging. Chemical force microscopy can also be used to evaluate DNA hybridization. Mazzola et al. (55) affixed a latex particle containing a single strand of DNA to a cantilever. Friction force images acquired using this cantilever revealed varying degrees of hybridization on a patterned array of complementary DNA strands. This approach holds promise for “reading” DNA biochips. Force spectroscopy is the currently accepted term for the acquisition and analysis of force-distance curves. Experimentally, cantilever deflection is monitored as the tip and substrate are brought into contact and subsequently separated. Cantilever deflection occurs in response to short- and long-range interaction forces between the tip and the substrate. These include attractive or repulsive electrostatic forces, van der Waals forces, and adhesive interactions. When the tip and/or substrate is chemically modified, force-distance curves can provide direct measurement of specific intermolecular interactions. In comparison with other methods for assessing intermolecular forces, force spectroscopy combines high force sensitivity, wide dynamic range, positional accuracy, and small contact area (less than 10 nm2). When a single molecule is bound to both the tip and substrate, acquisition of the force curve yields mechanical information about the molecule. Use of the term “force spectroscopy” for this experiment implies a dynamic process, i.e., a molecule’s response to an applied strain over a range of rates (frequencies). Thus, force curves should be acquired at several approach/retraction rates even though most investigators use a single piezovelocity. Three superb reviews of force spectroscopy have been published. Cappella and Dietler (56) critically evaluated the use of SFM as a tool for measuring intermolecular forces. Their review leads the reader systematically through theories of tip-substrate interactions in both contact and noncontact regimes. The empirical aspects of making force measurements in air or under liquid are discussed as are calibration and noise assessment techniques. Takano et al. (57) examined, in detail, the chemical and biological applications of force spectroscopy. Heinz and Hoh (58) described how the type of tip-substrate interaction determines force curve shape and image contrast. Artifacts in force-volume imaging are specifically addressed. Most force spectroscopic reports published during the period of this review have focused on single-molecule measurements of titin, polysaccharides, and DNA. Typically, mechanical testing of single polymeric molecules is performed without knowledge of the length of the chain being elongated. Force measurements on polymers such as poly(acrylic acid) (59), poly(methyl methacrylate) (60), polystyrene, poly(ethylene oxide)/poly(methacrylic acid) diblock copolymer (61), poly(vinyl alcohol) (62), and polystyrene-b-poly-2-vinylpyridine (63) usually involve the “smash and grab” procedure. A very thin film of a polymer is coated on a surface, and the cantilever tip is driven into the polymer film. Upon retraction, nonspecific interactions between the polymer and the cantilever tip fix the molecule between the two surfaces. As the polymer is stretched, the force is recorded by the deflection
on the cantilever. This technique often picks up several molecules at once, and the data must then be statistically analyzed to determine the response for one chain. Maaloum and Courvoisier (64) have demonstrated the merits of establishing selective points of attachment. They prepared a poly(ethylene oxide) chain with a hydrophobic end cap and applied this material to a hydrophobic surface. When this substrate was brought in contact with a hydrophobic cantilever, polymer chain elongation resulted from pulling by the hydrophobic ends rather than some random point in the middle. Ludwig et al. (65) showed the utility of AFM force measurements for unfolding of single protein molecules. From force curves, contour length and elasticity of single polymeric biomolecules can be readily determined. Two models are commonly used to evaluate force curve data: the freely joined chain model (or some modification) or a wormlike chain model. Ortiz (60) has provided a comparison of these models and offers selection criteria for their appropriate application. Titin is the modular protein responsible for the passive elasticity of muscle. Particular emphasis has been placed on determining its unfolding mechanism. Elongation occurs sequentially on discrete domains regardless of where chain attachment is made. Reif and co-workers (66) simulated (using Monte Carlo methods) the unfolding or stretching of the protein and found good agreement with their force spectroscopic data. A more detailed, molecular mechanics-based model of titin unfolding has been provided by Lu et al. (67). This model suggests that more than one partially unfolded intermediate state exists during the pulling process. Unfortunately, the unfolding rates used in molecular modeling studies did not coincide with those used in experimental studies. Because of limitations in computational time, the slowest pulling rates examined in molecular dynamic studies were 50 m/s. In contrast, the fastest pulling experiments reported to date were acquired at 100 µm/s (2, 5). Noteworthy additions to our understanding of the dynamics of titin unfolding were contributions by Marszalek, Rief, and their co-workers. Marszalek et al. (68) engineered a new strain of titin by site-directed mutagenesis to eliminate an unfolding intermediate. Single-molecule measurements confirmed that their new strain extended without a partially unfolded intermediate state. Rief and co-workers (69) systematically modified the mechanically active regions of titin by inserting fibronectin III and immunoglobulin into the “I-band” region. The sensitivity of force spectroscopy measurements was sufficient to reveal changes in mechanical properties between molecules differing by just a few amino acids. Polysaccharides have also been a major focus of single-molecule mechanical measurements. Molecules examined include tenasin, nacre, dextran, amylose, cellulose, xanthan, heparin, and pullulan (70-73). Marszalek et al. (73) hypothesized that the intermediate or transition state in polysaccharide elongation is due to the chairboat conformational change by the pyranose ring. To test their hypothesis, they oxidatively opened the pyranose ring. Force extension experiments on this polysaccharide derivative revealed the absence of the transition intermediate. In a similar study, Li and co-workers (74) observed differences in the force extension curves of amylose and cellulose. Force curves of amylose, an R-(1, 4) polysaccharide, contain a plateau region whereas cellulose, a β-(1, 4) polysaccharide with the same Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
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chemical composition, does not. They attributed the plateau region of amylose to a force-induced chair-boat transition. Additional evidence in support of this assignment was provided by force extension experiments on heparin, another R-(1,4) polysaccharide of different composition. Force curves revealed a plateau region indicative of a force-induced transition state. Heymann and Grubmu¨ller (75) performed molecular dynamic simulations on R-(1, 4) and β-(1,4)polysaccharides and determined a molecular basis for the transition state. They postulated that the transition-state elasticity could be tailored by adding saccharide substituents to enhance or hinder the chair-boat transition. Grandbios et al. (76) covalently attached a polysaccharide to different surfaces and empirically determined the forces needed for bond breakage. They found that 2.0 ( 0.3 nN of force was required to break polysaccharide bound to a silicon surface whereas only 1.4 ( 0.3 nN of force was required when the polysaccharide was bound to a gold surface (when loaded at 10 nN/s). Through molecular dynamic calculations, they determined the Si-C bond and Au-S bonds to be the weak linkages and assigned the observed force-induced ruptures to breakage of these bonds. DNA continues to be a focus of single-molecule, force spectroscopy measurements. Strunz et al. (77) immobilized complimentary strands of DNA on a glass slide and the AFM cantilever. Upon allowing the strands to form a duplex, the glass slide is retracted and the forces are measured at the cantilever. Their results are presented as a function of the retraction velocity of the glass slide and as a function of the number of base pairs. Shivashanker and Libchaber (78) coupled an optical tweezer apparatus to an AFM for mechanical testing of DNA. One terminus of DNA was covalently attached to a glass slide, the other to a latex bead. The optical tweezer enabled selection of the latex bead and its attachment to the cantilever. This approach facilitated multiple trials on the same or different molecules, the latter by simply selecting a different bead. MacKerell and Lee (79) used molecular dynamic simulations to gain insight into the tensile loading of DNA, especially under the conditions used in AFM mechanical testing. The agreement between simulation and experiment is quite good; their model predicted transient force events and the length at rupture. They assert that, due to the charged backbone of DNA, the ionic strength of the medium must be considered in all theoretical or experimental analyses of DNA elongation. Since base pairing in nucleic acids involves differing numbers of hydrogen bonds, the mechanical strength of DNA should be dependent upon the sequence of the strand. Rief et al. (80) performed single-molecule force spectroscopic studies on different stands of DNA to test this hypothesis. Random sequences of DNA exhibit a plateau in the force curve at 65 pN, corresponding to the transition from B-DNA to stretched DNA, and a transition at 150 pN, corresponding to strand separation (melting). Force curves acquired on poly(dG-dC) resembled those acquired on random DNA whereas those acquired on poly(dA-dT) had the B-DNA to stretched DNA transition lowered to 35 pN. Also, the strand separation transition shifted to 300 pN for poly(dG-dC) whereas no distinct strand separation transition was observed for poly(dA-dT). Rief and co-workers (80) proposed that the poly192R
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(dA-dT) strands melt as they undergo the B-DNA to stretched DNA transition. After force-induced strand separation, both poly(dG-dC) and poly(dA-dT) can form hairpins upon relaxation. This phenomenon was utilized to advantage in directly measuring G-C and A-T base-pairing forces. Ligand-Receptor Interactions. For the most part, force spectroscopy measurements have relied on nonspecific interactions to make opposing points of contact. The use of ligand-receptor coupling allows for known, reproducible points of contact to a single molecule. Mapping of several force measurements is also possible to provide simultaneous force spectroscopy data and topography. Biotin-avidin and biotin-streptavidin coupling has been widely used. Lo et al. (81) presented a new statistical analysis method that uses a Poisson distribution to determine the strength of a single biotin-avidin interaction. The method presents a straightforward strategy for deducing the strength of a single interaction from force curve data acquired on an unknown number of ligand-receptor pairs. Stevens and co-workers (82) compared the histogram, Poisson and the JKR continuum methods for analysis of bond rupture from force curve data. Often, force spectroscopy experiments are cut short because immobilized biomolecules lose activity during sample preparation or use (in response to imposed mechanical stresses). Green and co-workers (83) assessed the mechanical thresholds for streptavidin inactivation. They found that the activity of streptavidin films diminishes under mechanical loads greater than 2 nN. When mechanical or chemical degradation of tip functionality occurs, the tip must be exchanged. This procedure often exposes the substrate to contamination. The same group demonstrated the utility of using tipless cantilevers and tip arrays to circumvent this problem (84). If a tip on the array loses its functionality, a simple translation of the cantilever to a new tip will allow experiments to continue. Since the contact area of the tip is small, the number of possible fresh interaction sites with a tipless cantilever is very large. The tip array enables probing large arrays of different biomolecules immobilized on one surface. Gad et al. (85) mapped the distribution of polysaccharides on the surface of living yeast cells. Gold-coated tips were functionalized with concanavalin A. Force-volume imaging with these tips provided not only estimates of the binding force between concanavalin A and mannaN but also a measure of this polymer’s distribution on the surface. Lehenkari and co-workers (86) examined integrin-binding forces between several Arg-Gly-Aspcontaining peptides and extracellular receptors on intact cells. Similarly, Holland and colleagues (87) mapped the surface of platelets with tips covalently modified with hexapeptides containing the Arg-Gly-Asp sequence. These studies reaffirm the advantages of force spectroscopy for investigating receptor-ligand interactions in cell membranes. Scanning Electrochemical Microscopy. Scanning electrochemical microscopy (SECM) is a scanned probe technique focusing on nanoscale electrochemistry. Through the use of highresolution positioning, spatially resolved electrochemical reactions can be performed. Recent contributions applied SECM to studies of liquid-liquid and liquid-solid interfaces (88-91). Wilhelm and co-workers (92) used SECM to simultaneously map the topography and enzyme activity of glucose oxidase covalently immobilized on glass electrodes. Yasukawa et al. (93) imaged living cells with
SECM to determine their respiration rate. This rate depended upon the oxygen reduction current measured by the ultramicroelectrode tip. Living cells deplete oxygen in the surrounding medium. Addition of cyanide to the culture reduced the degree of oxygen consumption. Readers interested in learning more about SECM are referred to an excellent review written by Mirkin (94). In this review, the fundamentals of SECM operation, theory, and application are presented. Biological Imaging. New and interesting applications of SFM in biology resulted from ingenuity in sample preparation and exploitation of the enhanced capabilities provided by Tapping mode imaging in fluid. For example, Scheuring et al. (95) grew streptavidin crystals on biotinylated lipid monolayers at an airwater interface and transferred them onto HOPG. SFM images acquired on these substrates provided unit-cell parameters and resolved details of the two-dimensional crystals on the submolecular level. Mu¨ller and Engel (96) imaged two-dimensional crystals of Escherichia coli porin OmpF. Porins are trimeric channels that facilitate passage of small solutes through the outer membrane of Gram-negative bacteria. Conformational changes resulting in the closure of the channel entrance were observed as a function of applied voltage and pH. The spectacular images presented suggest an evolutionary adaptation that enables cells to protect themselves from drastic changes in their environment. Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. To examine the dynamics of fibril assembly, Goldsbury and co-workers (97) monitored the spontaneous formation of synthetic human amylin fibrils with SFM. They proved the efficacy of this technique for monitoring the growth, directionality, and changes in morphology for individual fibrils. SFM has merit over spectroscopy-based methods that average the growth over many fibrils and require up to 1000 times more protein. Their approach for characterizing human amylin fibrils should prove applicable to fibril formation from other amyloid proteins and peptides. In situ imaging of growing crystals continues to be a productive application for SFM. Li and colleagues (98, 99) presented a new approach for precise determination of the molecular packing arrangements on lysozyme crystal faces. Images were obtained during active growth and dissolution. After correcting for tip shapeinduced artifacts, high-resolution images revealed the occurrence of surface reconstruction of the (110) crystal face and, more importantly, that the growth unit is tetrameric. The latter finding contradicts the long-held belief that protein crystals grow by singlemolecule addition to the lattice. Kuznetsov and co-workers (100-102) visualized metastable phases at the surfaces of several macromolecular crystals growing from solution. Small, linear and highly branched aggregates were incorporated into growing crystals as impurities and produced defects in the crystal whereas ordered aggregates formed distinct microcrystals. Their SFM results suggested that small proteinrich clusters play an important role in macromolecular crystallization. Other workers focused on overcoming the challenges imposed while imaging the surface of intact cells. For example, Braet et al. (103) imaged living and glutaraldehyde-fixed liver endothelial cells. Only after fixation were arrays of pores in the cell membrane visible in the images. The increase in resolution was attributed
to fixation-induced changes in cell modulus. The elastic modulus increased from 2 kPa for the living cell to more than 100 kPa for the fixed cell. Rotsch et al. (104) investigated the morphology and elastic properties of living cultured rat liver microphages (Kupffer cells). Individual cells in physiological buffer were continuously imaged for several hours without damage. Elasticity mapping in the presence of cytochalasin B revealed a 7-fold decrease in the cell’s average elastic modulus, consistent with the chemical disassembly of the actin network by this agent. In an effort to gain insight into the structure and function of nuclear pore complexes, Danker and co-workers (105) used nuclear patch clamp techniques to mount the nuclear envelope of Xenopus laevis oocytes onto mica. Although they were unable to obtain high-resolution SFM images of nuclear pore complexes, this paper is notable because of the extensive experimental details regarding sample preparation by patch clamp methods. Tanaka and colleagues (106) measured adhesive forces between antigens on the surface of E. coli cell walls and anti-E. coli antibody-conjugated probe at the single-cell level. When the same probe was used to image Pseudomonas aeruginosa cells, no adhesive interactions were observed. These results illustrate the promise of force-volume SFM imaging in identifying individual bacterial cells. Force-volume imaging of anti-ferritin antibody-coated surfaces with ferritin-coated tips enabled Allen et al. (107, 108) to map the distribution of the antibody on a silicon surface. Similarly, Willemsen and co-workers (109, 110) monitored reversible binding between intercellular adhesion molecule (ICAM-1) and leukocyte function-associated antigen (LFA-1). These contributions merit mention because of the careful consideration by the authors of nonspecific interactions. Enhancement in image resolution will be possible when methods for controlling the contact area between the tip and the substrate as well as the orientation of the antigen and antibody on each surface become available. The structure of DNA and its complexes continues to be a major focus area for SFM. Shlyakhtenko and colleagues (111) developed a new procedure for covalent binding of DNA to a functionalized mica substrate. Their approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. The shapes of molecules immobilized in this fashion are comparable to those immobilized electrostatically onto mica. Fang et al. (112) reexamined the effect of ethanol on the structure of duplex DNA. Contour length measurements showed that DNA undergoes a conformational transition from the B-DNA to A-DNA form with increasing ethanol concentration. This transition is commensurate with a decrease in the apparent persistence length of DNA. At higher concentrations, DNA formed several higher-order condensed structures. Zuccheri and co-workers (113) used SFM to determine the dynamics of supercoiled circular DNA molecules adsorbed on mica under both water and buffer. Changes in the ionic strength of the medium produced variations in the rate conformational conversions in immobilized molecules. These motions were interpreted in terms of locally fluctuating supercoiling tensions. Kelley and colleagues (114) prepared ordered monolayer films of thiol-derivatized DNA duplexes by self-assembly onto gold surfaces. They found that, under potential control, monolayer Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
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thickness changed dramatically with applied potential. This observation confirmed that helical orientation is very sensitive to surface charges on the metal substrate. Depending upon the applied potential relative to the potential of zero charge, duplex DNA can be oriented either normal or parallel to the electrode surface. Klinov and co-workers (115) performed high-resolution mapping of individual plasmids and cosmids using RNA probes specific for long terminal repeats within these DNA. The RNA probes formed so-called R-loops which, when stabilized by glyoxal, were readily imaged following chemisorption of the conjugate onto Mg2+-modified mica. R-Loop positions were accurate to 0.5% of the cosmid length. Fang and Hoh (116) and Lin and co-workers (117) independently acquired SFM images of DNA as a function of spermidine concentration. Their images revealed that spermidine-induced DNA condensation involves multiple, well-defined structural intermediates. Ono and Spain (118) observed bacterial plasmid DNA condensation dynamics with a SFM. Their images revealed a condensate structure that is a possible intermediate for a multicondensate toroid. The Seeman group has pioneered the use of DNA in constructing multiply connected objects, networks, and devices. SFM has played an important role in the characterization of their DNA constructs. For example, an antiparallel DNA double-crossover molecule was incorporated into one edge of a DNA triangle. Ligation of this construct resulted in long linear molecules. Predictable changes in image features were obtained when triangles were interspersed with double-crossover molecules of specific lengths (119). In a second report, this group synthesized double-crossover DNA molecules specifically designed to promote their self-assembly into two-dimensional crystals. SFM images provided unequivocal proof of the existence of highly ordered crystals (120). The sticky ends that held the two-dimensional crystals together were varied to include diverse periodic arrangements of molecules in the crystal. The inclusion of hairpins designed to protrude from the plane in the crystal produced readily observable features in SFM images. When the hairpin contained a restriction site, image features attributed to protruding hairpins were absent following digestion of the two-dimensional crystal with the appropriate restriction enzyme (121). Perhaps the most exciting application of SFM lies in the realtime visualization of protein-DNA complexation. Schulz and coworkers (122) imaged complexes of E. coli RNA polymerase and a linear DNA fragment containing the glnA promoter. An apparent bend angle of 26((34)° was determined for the specific complexes bound at the promoter. Yokota et al. (123, 124) mapped the binding of GAL4 protein onto straightened plasmid DNA. They cautioned that the observed bend angles in the complex may reflect either a preferred conformation of protein-bound DNA or a higher affinity of the protein compared to DNA for the substrate. Thomson and co-workers (125) prepared recombinant RNA polymerase containing histidine tags (hisRNAP) on the C-terminus and immobilized them onto ultraflat gold via a mixed monolayer of alkanethiols. Specific binding of this molecule to a 42-base circular single-stranded DNA template was confirmed by in situ SFM images showing the production of huge RNA transcripts. 194R
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Margeat and colleagues (126) visualized the protein-protein and the protein-DNA complexes involved in transcriptional regulation by the trp repressor (TR). Plasmid fragments bearing the natural operators trp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged. Specific and nonspecific complexes of TR with DNA are found, as well as free TR assemblies directly deposited onto the mica surface. Their findings suggest protein-protein interactions serve a role in transcriptional regulation by the trp repressor. Bustamante and co-workers (127) obtained tapping mode SFM images that demonstrated the diffusion of E. coli RNA polymerase along DNA. Direct evidence of facilitated targeting of RNAP by intersegment transfer and possibly hopping (intradomain association and dissociation) was obtained for the first time. Ternary intermediates, in which RNAP appears to be simultaneously bound to two DNA segments, were directly observed during intersegment transfer events. In some image sets, transfer events were preceded and followed by sliding processes. Their results provide additional insight into the mechanism by which RNA polmerase searches for the promoter, a key step in transcription. Their highresolution images and analysis provided the first direct evidence of individual complexes involved in transcription. Current developments in SFM technology will allow faster scanning and will improve the temporal resolution of so-called SFM movies. These improved capabilities will enable SFM visualization of these and other complex biological processes as they occur, one molecule at a time. THE FUTURE OF SPM Carbon nanotube tips will revolutionize scanning probe microscopy. Their high aspect ratio and small effective radius significantly improve image resolution and enable images of surfaces with deep crevices and trench structures. Nanotubes buckle elastically, reducing the force applied while imaging soft samples. Compared with other chemically modified tips, nanotube tips are exceedingly robust. Chemical modification of the terminus of the nanotube affords improved resolution in chemical sensitivity (128, 129). Binnig and Rohrer, the inventors of STM, have published a retrospective on the development of proximal probe techniques (130). Their invention forever changed the way we perceive atomic structures. With proximal probes, we now have the capability to manipulate matter on the atomic and molecular scale, measure intermolecular forces and mechanical properties of single molecules, initiate chemical reactions, and determine molecular structure. Binnig and Rohrer have challenged the practitioners of SPM to exploit existing capabilities, and invent new ones, while fabricating technologically relevant nanometer-scale devices and gaining insights into the structure of complex biological assemblies. The future of SPM appears limited only by the diligence, ingenuity, and creativity of its practitioners. ACKNOWLEDGMENT
The papers selected for this review were a subset of literature found in the STN International and Current Contents databases. The authors are grateful to Mr. Don Stickel from STN for his assistance. Financial support from the ONR-sponsored Georgia Tech Molecular Design Institute is gratefully acknowledged.
Peter T. Lillehei is a graduate student completing his doctoral degree in analytical chemistry at the Georgia Institute of Technology. He obtained his baccalaureate degree in chemistry and chemical engineering at the University of Minnesota. His current research interests include singlemolecule mechanical testing of biopolymers, especially nucleic acids. Lawrence A. Bottomley is Professor of Chemistry at the Georgia Institute of Technology. He obtained his baccalaureate degree in chemistry at California State University, Fullerton, and his doctoral degree in analytical chemistry at the University of Houston. His current research interests include the biological and nanotechnological applications of scanning probe microscopy and electrochemistry. LITERATURE CITED (1) Schaffer, T. E.; Viani, M.; A., W. D.; Drake, B.; Runge, E. K.; Cleveland, J. P.; Hansma, P. K. Proc. SPIE-Int. Soc. Opt. Eng. 1997, 3009, 48. (2) Viani, M. B.; Schaffer, T. E.; Paloczi, G. T.; Pietrasanta, L. I.; Smith, B. L.; Thompson, J. B.; Reif, M.; Gaub, H. E.; Cleland, A. N.; Hansma, H. G.; Hansma, P. K. Rev. Sci. Instrum. 1999, 70, 4300. (3) Paloczi, G. T.; Smith, B. L.; Hansma, P. K.; Walters, D. A.; Wendman, M. A. Appl. Phys. Lett. 1998, 73, 1658-1660. (4) Walters, D. A.; Viani, M.; Paloczi, G. T.; Schaffer, T. E.; Cleveland, J. P.; Wendman, M. A.; Gurley, G.; Elings, V.; Hansma, P. K. Proc. SPIE-Int. Soc. Opt. Eng. 1997, 3009, 43-47. (5) Viani, M. B.; Schaffer, T. E.; Chand, A.; Reif, M.; Gaub, H. E.; Hansma, P. K. J. Appl. Phys. 1999, 86, 2258. (6) Genolet, G.; Brugger, J.; Despont, M.; Drechsler, U.; Vettiger, P.; de Rooij, N. F.; Anselmetti, D. Rev. Sci. Instrum. 1999, 70, 2398-2401. (7) Streckeisen, P.; Rast, S.; Wattinger, C.; Meyer, E.; Vettiger, P.; Gerber, C.; Guentherodt, H. J. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S341-S344. (8) Yaralioglu, G. G.; Atalar, A.; Manalis, S. R.; Quate, C. F. J. Appl. Phys. 1998, 83, 7405. (9) Garcia-Valenzuela, A.; Villatoro, J. J. Appl. Phys. 1998, 84, 58. (10) Gittes, F.; Schmidt, C. F. Eur. Biophys. J. 1998, 27, 75. (11) Leinhos, T.; Stopka, M.; Oesterschulze, E. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S65-S69. (12) Beck, R. G.; Eriksson, M. A.; Topinka, M. A.; Westervelt, R. M.; Maranowski, K. D.; Gossard, A. C. Appl. Phys. Lett. 1998, 73, 1149-1151. (13) Jarvis, S. P.; Duerig, U.; Lantz, M. A.; Yamada, H.; Tokumoto, H. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S211-S213. (14) Schemmel, A.; Gaub, H. E. Rev. Sci. Instrum. 1999, 70, 13131317. (15) Battiston, F. M.; Bammerlin, M.; Loppacher, C.; Guggisberg, M.; Luethi, R.; Meyer, E.; Eggimann, F.; Guentherodt, H. J. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S49-S53. (16) Burns, A. R.; Houston, J. E.; Carpick, R. W.; Michalske, T. A. Langmuir 1999, 15, 2922-2930. (17) Burns, A. R.; Houston, J. E.; Carpick, R. W.; Michalske, T. A. Phys. Rev. Lett. 1999, 82, 1181-1184. (18) Hazel, J. L.; Tsukruk, V. V. Thin Solid Films 1999, 339, 249257. (19) Behrend, O. P.; Oulevey, F.; Gourdon, D.; Dupas, E.; Kulik, A. J.; Gremaud, G.; Burnham, N. A. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S219-S221. (20) Anczykowski, B.; Cleveland, J. P.; Krueger, D.; Elings, V.; Fuchs, H. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S885-S889. (21) Attard, P.; Schulz, J. C.; Rutland, M. W. Rev. Sci. Instrum. 1998, 69, 3852-3866. (22) Attard, P.; Carambassis, A.; Rutland, M. W. Langmuir 1999, 15, 553-563. (23) Bar, G.; Brandsch, R.; Whangbo, M.-H. Surf. Sci. 1998, 411, L802-L809. (24) Whangbo, M.-H.; Bar, G.; Brandsch, R. Surf. Sci. 1998, 411, L794-L801. (25) Bar, G.; Brandsch, R.; Whangbo, M. H. Surf. Sci. 1999, 422, L192-L199. (26) Bar, G.; Brandsch, R.; Whangbo, M.-H. Langmuir 1998, 14, 7343-7347. (27) Whangbo, M. H.; Bar, G.; Brandsch, R. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S1267-S1270. (28) Garcia, R.; Tamayo, J.; Calleja, M.; Garcia, F. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S309-S312. (29) Garcia, R.; Tamayo, J.; Paulo, A. S. Surf. Interface Anal. 1999, 27, 312-316. (30) Hoummady, M.; Rochat, E.; Farnault, E. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S935-S938. (31) Anczykowski, B.; Gotsmann, B.; Fuchs, H.; Cleveland, J. P.; Elings, V. B. Appl. Surf. Sci. 1999, 140, 376-382. (32) Gotsmann, B.; Anczykowski, B.; Seidel, C.; Fuchs, H. Appl. Surf. Sci. 1999, 140, 314-319. (33) Sader, J. E. J Appl. Phys. 1998, 84, 64. (34) Hoummady, M.; Farnault, E. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S361-S364. (35) Rabe, U.; Turner, J.; Arnold, W. Appl. Phys. A: Mater. Sci. Processes 1998, A66, S277-S282. (36) Gimzewski, J. K.; Joachim, C. Science 1999, 283, 1683-1688. (37) Meyer, G.; Bartels, L.; Rieder, K. H. Superlattices Microstruct. 1999, 25, 463-471.
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