with a spatial resolution on biological, soft materials of ~1 nm, and a height resolution as fine as 0.1 nm. Thus it provides precise visual detail over a size range that is inaccessible for most other techniques. Its application extends over the size range lying between that of individual macromolecules (which are accessible by X-ray crystallography) to macromolecular assemblies (which

FIGURE 13.3 Schematic of an atomic force microscope.

are amenable to electron microscopy) and living cells (which can be seen using light microscopy).

Figure 13.3 is a schematic diagram of an atomic force microscope. In AFM an extremely sharp probe is brought into atomic contact with a surface and is scanned systematically over the surface. Topographic features on the surface and the tip of the probe interact with aggregate atomic forces as the probe is scanned over the surface. The sharp probe is attached to a flexible cantilever extending from a rigid substrate. A laser is reflected from the back surface of the cantilever; deflection of the tip, as it moves over surface topography, is detected as the reflection changes position on four elements of a positionsensitive diode. The differential signal between the sum signal of the top two elements and the sum signal of the bottom two elements provide a measure of the deflection of the cantilever. Similarly, the differential signal between the sum signal of the left two elements and the sum signal of the right two elements provides a measure of the torsion of the cantilever, which is a measure of the friction between the probe and sample.

When operating in "contact mode," the tip touches the sample continuously during the measurement, exerting constant normal and lateral forces as the tip is scanned over the sample. AFM applications investigating soft materials such as biological samples typically use "tapping mode" operation,7 which minimizes contact between the probe tip and the sample surface and greatly

FIGURE 13.4 AFM images of DNA. (a) linear DNA, (b) linear DNA with ABFp2 packing protein, (c) highly supercoiled DNA.

FIGURE 13.4 AFM images of DNA. (a) linear DNA, (b) linear DNA with ABFp2 packing protein, (c) highly supercoiled DNA.

reduces lateral forces. In tapping mode, the vertical position of the sample is continually adjusted by a feedback mechanism, in order to maintain constant amplitude for the freely oscillating probe. Changes in the oscillation amplitude, in response to attractive or repulsive forces between the sample and the tip, are monitored. These changes are transformed into topographic information.

The past decade has seen continuous progress in the quality of AFM imaging of biological samples. In particular, membrane proteins,8,9 DNA,10,11 and DNA-protein complexes12 have been intensively studied. Examples of DNA imaged using an atomic force microscope with a standard commercial silicon tip and operating in tapping mode are shown in Figure 13.4. The image in Figure 13.4a shows linear DNA; in Figure 13.4b, ABF2p, a packing protein, was added to the DNA sample, resulting in compaction. Figure 13.4c shows highly supercoiled DNA resulting from adding TOTO-1, a dye that intercalates between base pairs.

Despite decades of study of viruses and their pressing importance in human medicine, many of their structural properties are poorly understood. Because of their heterogeneity and lack of symmetry, large viruses are often not amenable to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). AFM can be effectively used to image the intact structures of large viruses,13 and their internal structures can be revealed by AFM in combination with chemical and enzymatic dissection.14,15 Development of AFM as a diagnostic tool to probe the structures and function of human viruses has the capacity to provide important information on their structure, function, and assembly.

Figure 13.5 shows turnip yellow mosaic virus (TYMV), a 28 nm diameter, T = 3 icosahedral plant virus in which the capsomeric structure of a small virus was visualized by AFM for the first time. In these images, pentameric and hexa-

FIGURE 13.5 (a,b) In situ AFM images of turnip yellow mosaic virus particles immobilized in the crystalline lattice clearly display capsomers on the surface of the T = 3 icosahedral virions. (c) The structure of the capsid of turnip yellow mosaic virus based on X-ray diffraction analysis.

meric clusters which are roughly 60 A across can be discriminated from one another, and the difference between the highest and lowest points on the capsid surface, about 45 A,16 was accurately reflected by AFM. Recently, the cap-someric structures and orientations of individual brome grass mosaic and cucumber mosaic virions were also visualized by AFM.17 These studies demonstrated that AFM could provide a means for obtaining structural information directly from individual virus particles immobilized on a substrate.17

The clarity with which structural detail can be recorded on the surfaces of small plant viruses, such as TYMV, suggested that AFM may be even more broadly useful as an analytical tool for macromolecular structural investigations and provide important topographical information on large macromolec-ular ensembles such as human and other animal viruses that would otherwise be lacking. Fragility and structural heterogeneity can render such viruses troublesome targets for X-ray crystallography. The only alternative approach to date, cryo-EM, yields resolutions from 8 A upwards.18 A limitation of this method, however, is that it benefits greatly from high particle symmetry, such as icosahedral symmetry, and is far less powerful for irregular, polymorphic virus particles. In addition, because of a low contrast in biological EM, the high resolution requires computer averaging and processing of thousands of images.

Much of the technique used for imaging other biological structures with AFM can be applied to the imaging of bacterial spores. Figure 13.6 is an AFM image of a Bacillus subtilis niger spore. Figure 13.6a shows the entire spore with some structure of the protein coat visible. When a smaller area of the surface is imaged, as shown in Figure 13.6b, much more detail of the protein coat is visible. We have found that, similar to many other biological molecules, spores can be somewhat soft and sometimes exhibit regions of increased

FIGURE 13.6 (a) AFM image of Bacillus globigii spore. (b) High-resolution image of the protein coat of the spore.

adhesion to the AFM tip. The force placed on the sample by the tip needs to be kept to a minimum so as not to deform the surface during the measurement, yet the cantilever needs to be stiff enough to overcome adhesion between the tip and sample. Imaging spores in fluid should increase resolution, since the tip does not have to penetrate the water layer that is typically on samples measured in air under ambient conditions.

Currently, the principal limitation on application of the promising technique of AFM to structural biology is the resolution limit imposed by the finite tip radius—which is typically larger than 10 nm. Overall tip sharpness becomes more important in imaging the relatively rough structures of pathogens having deep crevasses, and in delineating details of protein complexes. Recently, carbon nanotubes (CNTs) have emerged as the next generation of force microscopy probes.19-22 These probes typically have an aspect ratio of more than 100, and the end radius of curvature of a single walled nanotube is ~10 A. This small tip radius, combined with the high aspect ratio and mechanical robustness, presents an obvious advantage over conventional AFM probes and permits resolution about one order of magnitude better than that achievable with commercial AFM tips. High-resolution imaging capabilities of CNTs were demonstrated recently on various biomolecules, such as DNA, antibodies, proteins, and nucleosomes.21

0 0

Post a comment