Abstract
Scanning tunneling microscopy (STM) has the highest resolution of all scanning probe techniques, routinely producing atomic scale images. With such resolution, it is highly desirable to apply STM to investigations of molecular biology and medicine. The difficulty when considering the application of STM to molecular biology is that biological samples are nonconductive. It may be more accurate to describe biological samples as having both local and varying conductivities. These two issues will addressed in this review, and examples of conditions for the successful use of STM for biomedical imaging will be discussed. Successful applications of STM to significant research problems in biology and medicine will be presented. Keywords: scanning tunneling microscopy; STM; DNA; RNA; retinoic acid; pyrimidine; purine; imaging artifacts; data restoration; deconvolution; probe response function
Introduction
Four years after its invention in 1982 (1), the scanning tunneling microscope (STM) was awarded the 1986 Nobel Prize for physics, one of only four such prestigious awards given for a truly significant contribution to scientific instrumentation. Since then, the family of scanning probe microscopy (SPM) techniques, which includes scanning tunneling microscopy, atomic force microscopy (2-4), magnetic force microscopy (5) , near-field optical microscopy (6) , scanning thermal microscopy (7) , and others, has revolutionized studies of semiconductors, polymers, and biological systems. The key capability of SPM is that, through a controlled combination of feedback loops and detectors with the raster motion of piezoelectric actuator, it enables direct investigations of atomic-to-nanometer scale phenomena.
Scanning probe microscopy is based on a piezoelectricactuated relative motion of a tip versus sample surface, while both are held in a near-field relationship with each other. In standard SPM imaging, some type of tip-sample interaction (e.g., tunneling current, Coulombic forces, magnetic field strength) is held constant in z through the use of feedback loops, while the tip relative to the sample undergoes an x-y raster motion, thereby creating a surface map of the interaction. The scan rate of the x-y raster motion per line is on the order of seconds while the tip-sample interaction is on the order of nanoseconds or less. The SPM is inherently cable of producing surface maps with atomic scale resolution, although convolution of tip and sample artifacts must be considered.
Scanning tunneling microscopy is based on a tunneling current from filled to empty electronic states. The selectivity induced by conservation of energy and momentum requirements results in a self-selective interaction that gives STM the highest resolution of all scanning probe techniques. Even with artifacts, STM routinely produces atomic scale (angstrom) resolution.
With such resolution possible, it would be highly desirable to apply STM to investigations of molecular biology and medicine. Key issues in biology and medicine revolve around regulatory signaling cascades that are triggered through the interaction of specific macromolecules with specific surface sites. These are well within the inherent resolution range of STM.
The difficulty when considering the application of STM to molecular biology is that biological samples are nonconductive. It may be more accurate to describe biological samples as having both local and varying conductivities. These two issues will addressed in this article, and examples of conditions for the successful use of STM for biomedical imaging will be discussed. We begin with an overview of successful applications of STM in biology and medicine.
Scanning Tunneling Microscopy In Biology And Medicine: Dna And Rna
The STM imaging for direct analysis of base pair arrangements in DNA was historically the first biological application of the new technique. An amusing piece of scientific history is that the first (and widely publicized) images (8) (9) (10) (11) (12) of (deoxyribonucleic acid) DNA were subsequently shown to correspond to electronic sites on the underlying graphite substrate! However, more careful investigations have resulted in an authentic body of work in which the base pairings and conformations of DNA and RNA are directly investigated by STM. One goal of these investigations is to replace bulk sequencing techniques and crystal diffraction techniques, which both require large amounts of material, with the direct sequencing of single molecules of DNA and RNA. Two examples of DNA and RNA investigation by STM are presented here. One is an investigation of DNA and RNA structures, and the other is an investigation of DNA biomedical function.
Recently reported research from the group at The Institute for Scientific and Industrial Research at Osaka University in Japan (13) has shown detailed STM images of well-defined guanine-cytosine (G-C) and adenine-thymine (A-T) base pairings in double-and single-stranded DNA. Four simple samples involving only G-C and only A-T base pairs in mixed (hetero) and single sided (homo) combinations were chosen for analysis ( Fig. 1 ). These were deposited on a single-crystal copper (111)-orientation [Cu(111)] substrate using a technique developed specially by this group to produce flat, extended strands for imaging. An STM image showing the individual A-T base pairs in the hetero A-T sample is shown in Fig. 2 . Images of the overall structures indicated repeat distances consistent with interpretation as the double helix. Images from mixed samples of hetero G-C and hetero A-T are shown in Fig. 3 . The larger structure is interpreted as hetero G-C and the smaller as hetero A-T, which is consistent with X-ray diffraction data that indicates the A-T combination is more compact.
