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Digital Instrument's BioScope Anyone who has ever taken the time to critically examine a walnut knows that a two-dimensional photograph fails in many respects to truly convey the unique features--the nicks, crannies, valleys, and grooves--of the walnut shell. Researchers use atomic force microscopy (AFM) to literally map the surface of inert and biological samples to obtain three-dimensional images. Whereas technological developments in microscopy1,2 have facilitated the detailed characterization and visualization of large macromolecular assemblies, AFM-generated surface topology maps can portray in explicit detail the surface features of such biological material as protein, DNA, and the membrane surface channels of cells. AFM is just one of a number of novel microscopy technologies collectively known as scanning probe microscopy (SPM). In principle, all SPM technologies are based on the interaction between a submicroscopic probe and the surface of some material. What differentiates SPM technologies is the nature of the interaction and the means by which the interaction is monitored. In electrostatic force microscopy, electrostatic interactions between probe and sample are measured as voltage gradients. By monitoring these gradients, detailed maps of the electrostatic features of a sample's surface can be created. Surfaces with active electrical currents can produce magnetic fields. Magnetic force microscopy measures the subtle forces exerted on a magnetized probe as it moves across a surface with such magnetic fields. The changes in the force are then translated into an electrochemical profile (activity map) of the sample surface. Hippocampus neurons imaged using ThermoMicroscope's Explorer in a liquid environment Lateral force microscopy measures lateral twisting of the probe resulting from changes in friction or slope of the material being analyzed. In scanning tunneling microscopy, sample-probe conductance is used to indirectly image the physical landscape of the sample. The mapping is accomplished by measuring the conductance between the probe and sample, and by monitoring the probe-to-surface distance that is continuously modulated to maintain a constant tunneling current between the probe and the sample surface. AFM produces a topographical map of the sample as the probe moves over the sample surface. Unlike most other SPM technologies, AFM is not dependent on the conductivity of the sample being scanned. Therefore, AFM can be carried out in ambient air or in a liquid environment, a critical feature for biological research. SPM techniques need not be "read-only." Nanolithography, a technique in which the probe exerts sufficient force on the sample to move individual atoms, has applications in circuit miniaturization, data storage, and molecular assembly, among others. In one report, physicists Donald M. Eigler and Erhard K. Schwizer, at IBM's Almaden Research Center in San Jose, Calif., used this technique to spell out "IBM" from 35 xenon atoms on a nickel crystal surface.3 Recently, Eigler and colleagues4 used nanolithography to assemble several dozen cobalt atoms on a copper surface into a "quantum corral." When the authors placed an additional cobalt atom at one focus of the ellipse, they detected a "quantum mirage" at the opposite (empty) focal point. These results have potential implications in nanometer-scale wireless data transmission. The basic atomic force microscope is composed of a stylus-cantilever probe attached to the probe stage, a laser focused on the cantilever, a photodiode sensor (recording light reflected from the cantilever), a digital translator-recorder, and a data processor and monitor. Four features of these microscopes that should be taken into account when making a purchase are the user interface (i.e., Is the design intuitive? Does it allow multitasking?), the quality of the attached optical microscope, the ease of tip replacement and handling, and the degree to which the system software and hardware can be customized. BioForce Laboratory's NanoArrayer Principles and Applications Researchers at Stanford University and the IBM San Jose Research Laboratory invented AFM, also known as scanning force microscopy, in the mid-1980s.5 Initially used to examine the surface features of inert materials, AFM has since gained acceptance in biological research, where it has been used to study a broad range of biological questions, including protein and DNA structure, protein folding/ unfolding, protein-protein and protein-DNA interactions, enzyme catalysis, and protein crystal growth.6-10 AFM has been used to literally dissect specific segments of DNA for the PCR generation of genetic probes11 and to monitor development of new gene therapy delivery particles.12 These biomedical applications have only scratched the surface of what can and will be accomplished with AFM. Recently, Charles M. Lieber and colleagues used a carbon-nanotube-tipped AFM to map single nucleotide polymorphisms (SNPs) to a specific chromosome.13 SNPs are genetic variations that can be markers for disease. To map a disease gene, however, it is usually necessary to track the inheritance pattern of a number of SNPs. The problem these researchers addressed was the fact that it is not easy to determine whether two SNPs are located on the same or opposite chromosome of a pair--that is, whether the pair of SNPs segregates together or separately. The authors literally visualized the answer using two SNP probes that could be distinguished based on the size of the attached labels (one probe was labeled with streptavidin and the other with IRD800). Fibroblasts imaged in a liquid environment AFM is unlike other SPM technologies in that the probe makes physical, albeit gentle, contact with the sample. The cornerstone of this technology is the probe, composed of a surface-contacting stylus attached to an elastic cantilever that is mounted to a probe stage. As the probe is dragged across the sample, the stylus moves up and down in response to surface features. This vertical movement is reflected in the bending of the cantilever, which is measured as changes in the light intensity from a laser beam bouncing off the cantilever and recorded by a photodiode sensor. The data from the photodiode is translated into digital form and processed by specialized software on a computer, then visualized as a three-dimensional topological image. This process is analogous to fingers scrolling across the surface of Braille lettering on paper, only in AFM the fingers (probe stylus) are submicroscopic in size and the Braille (proteins, DNA) even smaller. In AFM, like Braille, samples are scanned in a raster format: the stylus is systematically and slowly advanced across the sample surface from left to right and top to bottom. In the original AFM method, the probe was in constant contact with the specimen throughout the scanning process. This method was and continues to be used primarily with inert, hard specimens. Although this technique has also been used successfully to examine softer biological material, the continuous contact can potentially "tear" the specimen and/or indirectly affect its topology (via friction, adhesion, electrostatic forces, etc.), and also lead to premature breakdown of the probe. To circumvent these problems, a new intermittent-contact ("tapping mode") scanning method was developed.14 In this mode, the probe is made to vibrate and literally taps along the surface of sample, minimizing or eliminating any damage to the specimen. Another improvement in AFM technology is the introduction of the noncontact scanning mode, which provides a mechanism to analyze samples that cannot be immersed in liquid, are especially prone to physical damage by any probe contact, or whose hardness is such that the probe is easily damaged by sample contact. In noncontact scanning, the probe rides slightly above the sample and attractive Van der Waals forces that act between the sample and probe are monitored and then translated into a topological map of the surface. This mode is less sensitive than both contact modes, and generally is not used for biological sample analysis. Most atomic force microscopes available today are capable of operating in each of the above-mentioned modes. The magnification power achieved by atomic force microscopes rivals that of transmission and scanning electron microscopes (TEM and SEM, respectively). Atomic force microscopes also are capable of a wide field of view (similar to SEM) and extreme vertical resolution (like TEM). The ratio of vertical to horizontal magnification can exceed 1,000:1, permitting the discrimination of subtle differences between extremely smooth surfaces; when combined with phase imaging, this can facilitate the discernment of differences in the chemical composition of sample surfaces. In general, AFM illuminates topographic contrasts, permits atomic scale measurements, and provides for the analysis of unmodified surface features (i.e., the sample need not be coated as in electron microscope technology), without the extensive effort, time, and resources needed to prepare samples for other technologies. Thus, atomic force microscopy is a very powerful and cost-efficient tool to employ in biological research. Phase image of Tobacco Mosaic Virus New Horizons SPM technology, especially AFM, continues to expose the molecular detail of biological specimens and probe the mechanics of dynamic biochemical events. Practical applications for this technology for investigating biological phenomena have grown considerably over the past few years. Advances in SPM technology and instrumentation will continue to broaden and enhance basic science research. One interesting application is the detection of protein-protein interactions on a nanometer-scale array. Because AFM has a Z-height resolution of 0.5 nm, whereas the typical globular protein diameter is approximately 3 nm or greater, AFM has the ability to rapidly detect protein-protein interactions without labeling. BioForce Laboratory Inc. of Ames, Iowa, is currently developing such a system in the NanoReader(tm), an atomic force microscope designed to analyze the company's nanometer-scale array assays for biological molecules. According to Michael Clark, the company's chief operating officer, the NanoReader is expected to be available about the middle of this year. The box below provides a list of companies offering a broad selection of integrated, turnkey, and custom AFM systems, probes, peripherals, and software. The Web sites for Digital Instruments (www.di.com), Pacific Scanning Corp. (www.pacificscanning.com), and Thermo Microscopes (www.thermomicro.com), are rich sources of information on SPM technology, instrumentation, and applications. A publication entitled "Practical Guide to Scanning Probe Microscopy," is available online at www.thermomicro.com/spmguide/contents.htm, or as a CD available free from ThermoMicroscopes. It is also an excellent source of information on SPM technology and techniques. Carol Wright-Smith and Christopher M. Smith (csmith@sdsc.edu) are freelance writers in San Diego. References 1. C. Wright-Smith, C. Smith, "Science in a new light," The Scientist, 14[18]:24, Sept. 18, 2000. 2. J. McAdara, "The resolution solution," The Scientist, 14[3]:24, Feb. 7, 2000. 3. D.M. Eigler, E.K. Schweizer. "Positioning single atoms with a scanning tunneling microscope," Nature, 344:524-6, 1990. 4. H.C. Manoharan et al., "Quantum mirages formed by coherent projection of electronic structure," Nature, 403:512-5, 2000. 5. G. Binnig et al., " Atomic force microscope," Physics Review Letters, 56:930-3, 1986. 6. C.H. Chen, H.G. Hansma, "Basement membrane macromolecules: Insights from atomic force microscopy," Journal of Structural Biology, 131:44-55, 2000. 7. F. Oesterhelt et al., "Unfolding pathways of individual bacteriorhodopsins," Science, 288:143-6, 2000. 8. M. Stolz et al., "Monitoring biomolecular interactions by time-lapse atomic force microscopy," Journal of Structural Biology, 131:171-80, 2000. 9. M. Grandbois et al., "Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2," Biophysics Journal, 74:2398-404, 1998. 10. A. McPherson et al., "Atomic force microscopy in the study of macromolecular crystal growth," Annual Reviews of Biophysics and Biomolecular Structure, 29:361-410, 2000. 11. S. Thalhammer et al., "The atomic force microscope as a new microdissecting tool for the generation of genetic probes," Journal of Structural Biology, 119:232-7, 1997. 12. H.G. Hansma et al., "DNA condensation for gene therapy as monitored by atomic force microscopy," Nucleic Acids Research, 26:2481-7, 1998. 13. A.T. Woolley et al., "Direct haplotyping of kilobase-size DNA using carbon nanotube probes," Nature Biotechnology, 18:760-3, 2000. 14. P.K. Hansma et al., "Tapping mode atomic force microscopy in liquids," Applied Physics Letters, 64:1738-40, 1994. Atomic Force Microscopy Hardware and Service Providers Advanced Surface Microscopy Inc. (800) 374-8557 www.asmicro.com BioForce Laboratory Inc. (562) 930-1324 www.bioforcelab.com Burleigh Instruments Inc. (716) 924-9355 www.burleigh.com Digital Instruments Veeco Metrology Group (800) 873-9750 www.di.com JEOL USA Inc. (978) 535-5900 www.jeol.com Molecular Imaging Corp. (800) 819-2519 www.molec.com Nanofactory Instruments AB +46 31-772-81-75 (Sweden) www.nanofactory.com Nanonics Ltd. +972 2-678-9573 (Israel) www.nanonics.co.il Nanosurf AG +41 61-903-06-11 (Switzerland) www.nanosurf.com NT-MDT Co. +7 095-535-0305 (Russia) www.ntmdt.ru Omicron Vakuumphysik GmbH +49 6128-987-0 (Germany) www.omicron-instruments.com Pacific Scanning Corp. (626) 796-2626 www.pacificscanning.com Photometrics Inc. (714) 895-4465 www.photometrics.com Quesant Instrument Corp. (818) 597-0311 www.quesant.com RHK Technology, Inc. (248) 577-5426 www.rhk-tech.com Surface Imaging Systems +49 2407-96147 (Germany) www.sis-gmbh.com U.S. Distributor: Accurion LLC. (510) 445-1533 www.accurion.com ThermoMicroscopes (408) 747-1600 www.thermomicro.com Triple-O Microscopy GmbH +49 331-23129-0 (Germany) www.triple-o.de WITec (Wissenschaftliche Instrumente und Technologie) GmbH +49 700 94832 366 (Germany) www.witec.de

Atomic Force Microscopy

Jan 21, 2001

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