Atomic Force Microscopy as a Tool in Nanobiology Part I : Imaging and Manipulation in Cytogenetics

Shortly after the physics Nobel prize award for the invention of the scanning tunneling microscope (STM), Gerd Binnig and Calvin Quate built the atomic force microscope (AFM) in order to avoid the limitations of the STM to image only conductive matter or thin layers of organics (1). Using the miniaturized record player principle similar to a stylus profilometer, it was then possible to image the surface of biological (non conducting) objects such as DNA and chromosomes down to the molecular scale (2-4). Most important in the development of the AFM to a universal instrument in bio-nanotechnology applications was the fact, that the tip of the cantilever used for imaging could also be used for measuring forces at the nanoscale and moreover as a nanoscale tool, down to the single atom level (5). Here we show how techniques like cutting, gripping and extracting biomaterial at the submicron scale under high-resolution image control have been developed, especially in cytogenetic studies. The combination of the nanomechanics tool box and modern biochemical techniques like PCR has immense potential for the future development of single molecule techniques, ranging from applications in DNA mechanics to cytogenetic studies and biochip development. AFM as an imaging tool Structural analysis with high resolution provides detailed information not only on high molecular complexes but can also be used for in vivo experiments of biological systems. Data can be recorded in real time. Besides structural information of biological systems, 3-dimensional topological data, micro mechanical behavior, dynamic processes and molecular interactions can be recorded. Table I shows a short comparison of AFM to other microscopic techniques, including the required sample preparation. Cytogenetics is basically a visual science. Established microscopic techniques, such as light and electron microscopy, have been widely been used for the study of chromosomes. After the invention of the atomic force microscope, it has been applied in different fields of genetics. Double-stranded DNA fixed on freshly cleaved mica was imaged in air by several groups (7,8). Hansma and coworkers successfully imaged plasmid-DNA fixed on mica in propanol (9). By adding new spreading chemicals, e.g. quaternary ammonium salts, it was possible to reduce surface impurities so that the surface density of the molecules could be reproducibly measured (10). After the introduction of the tapping mode, it became possible to image DNA with less potentially destructive shear forces during scanning, which resulted in a more detailed image of the macromolecule (11). In Figure 2a we have imaged a double-stranded plasmid, pUC19, in tapping mode in air. DNA in a higher condensation status in sperm cells was imaged by Allen and coworkers in air and in liquids (12). 3 Correspondence to: Prof. Dr. Wolfgang M. Heckl, Department for Geoand Environmental Sciences, GeobioCenter and Center for Nanoscience, Ludwig-Maximilians-Universität, Theresienstr. 41, 80333 München, Germany. Tel: 0049-89-2180-4331, Fax: 0049-892180-4331, e-mail: [email protected] / www.nano-science.de