University of California, Irvine, Department of Molecular Biology and Biochemistry, Room 560, Steinhaus Hall, Irvine, CA 92697-3900, USA1
Author for correspondence: Alex McPherson. Fax +1 949 824 1954. e-mail amcphers{at}uci.edu
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Introduction |
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It is unlikely that any probe technology such as scanning transmission electron microscopy or atomic force microscopy (AFM) can, at this time, compete with cryogenic EM or X-ray crystallography as a means of delineating virus structure, particularly the interior structure. Nonetheless, AFM may have its place in accurately determining the dimensions of virus particles, their mechanical properties and the architecture of their surfaces. In the best of cases, it may be capable of revealing capsomere arrangements and perhaps even the distribution of capsid protein subunits within the capsomeres. With future improvements in AFM technology, images may begin to overlap with those of X-ray crystallography.
AFM was invented by G. Binnig in 1982 (Binnig et al., 1986 ) and has been used in the visualization of biological structure for about the last ten years. Its great advantages over other imaging methods are that it can be carried out in a fluid environment, including physiological medium, and that it does not disturb the specimen from its natural state. Furthermore, it spans a range of dimensions from a nanometre up to a hundred microns, which is only marginally accessible by other techniques.
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AFM technology |
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AFM may be operated in either height mode or deflection mode. In height mode, the sample surface is maintained at a constant distance from the tip of the probe by the piezoelectric positioner below by a feedback mechanism. The cantilever deflection in this case is very small, tending to zero. In deflection mode, the sample is stationary and cantilever deflection data are collected directly.
Microfabricated cantilevers exert a force on the substrate surface and, as one might anticipate, the resolution of the technique depends on the degree of force employed. The greater the force between the probe and the surface, the more sensitive the probe is to surface variations. On the other hand, the greater the force, the more the probe will disturb the surface. Sample damage thus becomes an issue.
Problems arising in imaging in contact mode from unfavourable probesurface interactions, particularly lateral force, have been overcome to some extent by the development of what are known as tapping mode instruments (Hansma et al., 1994 ). In tapping mode, the probe tip is not in continuous contact with the surface (referred to as contact mode), but oscillates rapidly up and down as it is scanned over the surface, essentially tapping its way gently, as with a blind mans cane, but firmly and rapidly, and sensing the height of features it encounters.
In tapping mode, the feedback mechanism adjusts, through the piezoelectric positioner, the vertical height of the sample surface in order to keep the amplitude of the freely oscillating probe constant. Tapping mode minimizes the contact between the cantilever tip and the sample surface and it greatly reduces lateral forces. As with contact mode, cantilevers made of silicon nitride, which have a low spring constant, are used for tapping mode operation in liquids. For some specimens, it is preferable to examine virus samples dried in air. For tapping mode imaging of air-dried samples, a stiffer cantilever made of silicon is used, which has a high spring constant of about 50 N/m.
This tapping mode approach has proven to be a significant blessing to biological researchers, as it has allowed the characterization of samples that would otherwise be too soft or too fragile to withstand contact mode examination. Operating in a liquid environment presents some complications due to fluid dynamics, but these are not severe. A constraint that sometimes presents obstacles is that the specimen under study must be fixed to, or made to adhere firmly to, the substrate surface of the fluid cell, which may be glass, cleaved mica, plastic or any other hard material. To achieve this, it may be necessary to treat the substrate with various reagents in order to induce better adhesion of samples. If this condition is not met, the specimen will move due to interaction with the probe and no useful information will be gathered.
AFM can be applied to scan fields ranging in size from less than 20 nm up to about 150 µm and with a spatial resolution on soft biological materials, in the best of cases, of about 2 to 3 nm, with a height resolution as great as 0·5 nm. Its application extends over the range lying between individual macromolecules, which are accessible by X-ray crystallography, macromolecular assemblies, amenable to EM, and living cells, which can be seen using light microscopy (Allen et al., 1997 ; Bustamante & Keller, 1995
). Because visualization is carried out in a fluid environment, specimens suffer no dehydration, as is generally the case with EM, and they require no fixing or staining. Indeed, specimens can be observed over long periods as long as they stay relatively immobilized. For the most part, specimens seem to be oblivious to the presence of the probe tip.
The measurement of particle size and the dimensions of features of individual particles must be treated with some care. Lateral sizes of individual particles adsorbed onto mica appear considerably larger than might be expected because the image obtained is the convolution of the AFM tip shape with that of the particle. That is, the tip is not infinitely sharp and it has finite width. Thus, the curved surface immediately adjacent to the absolute tip causes vertical displacement of the cantilever and, therefore, gives rise to edges in the image before, as well as after, the absolute tip encounters the object. This does not, however, affect the total vertical displacement of the cantilever. As a consequence, single objects visualized by AFM appear broader than their true dimensions but yield an accurate and precise vertical dimension. For roughly spherical particles, such as icosahedral viruses, although their diameter appears greater than is in fact the case, the vertical height of the particles gives a remarkably accurate value for their true diameter.
Many viruses with dimensions ranging from 17 to 150 nm whose capsid sizes were accurately established by EM, light scattering or X-ray crystallography have now been examined. Images of these particles consistently showed them to have lateral dimensions of about 2·5 times their actual diameter, which serves as a reasonable rule of thumb. In all cases, however, heights measured by AFM were accurate to within a few per cent. Therefore, precise free particle diameters must be based on vertical measurements.
In the case of virus particle crystals, the situation is different. With virus crystals, each particle is embedded in a lattice composed of similar members. As the AFM tip passes over the surface, the tip never approaches the bottom of a single virion (i.e. a surface equivalent to the flat mica substrate) before it encounters a neighbouring virion. Thus, the height of the particle in crystalline form does not give a valid measure of its true diameter. However, it is a simple matter to measure the centre-to-centre distances of the virus particles in the lattice and, because the virions are, in general, closely packed, these distances do yield precise diameters of better than a few per cent. Fourier transformation of the lattice arrays (Kuznetsov et al., 1997 ) can improve these values.
More distinct images of virus particles can generally be obtained from virus crystals, rather than from single, free particles adsorbed onto mica or glass. This is not due to any averaging process or Fourier filtering, but seems to be an inherent property of the technique. Presumably, the immobilization of particles and their physical stability is responsible for the quality of the crystals images. When crystals cannot be obtained, slowly drying the virus particles onto the mica substrate may induce them to form aggregates and clusters, often ordered into closely packed, two-dimensional paracrystals. These sometimes serve nearly as well as crystals. In these semi-ordered arrays, the centre-to-centre distance of particles also provides a reasonably accurate estimate of diameters.
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Examples of AFM images of viruses |
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Fig. 4 shows a small area on the surface of a turnip yellow mosaic virus (TYMV) crystal (Malkin et al., 1999
; Hirth & Givord, 1988
). Groups of six crystallographically related T=3 particles of 28 nm diameter gather around large, open channels with widths that are roughly equal to a virion diameter, presumably filled with solvent. Even at this resolution, each of the virions appears as a roughly spherical cluster of grapes. In many cases, individual grapes or substructures in the clusters can be seen to exhibit hexagonal or pentagonal outlines characteristic of icosahedral capsomeres. The capsomeres are well-resolved from one another on the top surfaces of the virions and have dimensions consistent with those obtained from X-ray crystallography, about 5 to 6 nm (Canady et al., 1996
).
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Fig. 5(a) shows a larger, spherical plant virus, cauliflower mosaic virus (CaMV), adsorbed onto mica. Height measurements suggest its diameter to be 48 nm, though its width in the AFM images is about 100 nm. X-ray crystallography indicates it to be 52 nm (Gong et al., 1990
). With AFM, even at a relatively low magnification, quilting on the virion surface suggests the emergence of substructure. At higher magnifications, the underlying architecture begins to develop and individual, large capsomeric units become visible on the virion surface (Fig. 5bd)
. These have centre-to-centre distances of 10 to 12 nm and are organized into hexameric patterns.
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Some preliminary conclusions |
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Heights of single particles on mica and centre-to-centre distances of particles in ordered arrays are the most reliable measures of a virions dimensions. If these are used, shapes and dimensions obtained by AFM agree well, to within a few per cent, with results based on X-ray structure determination. The size of the virus particle does not appear to be a limitation in terms of resolving capsid detail. That is, 17 nm virus capsids, as seen here for BMV T=1 particles, and 28 nm viruses, such as TYMV, yield as much detail as 50 or 100 nm viruses. The favourable increase in virion curvature with increase in diameter is probably compensated for negatively by increasing virion deformity. Even membrane-encapsidated virions yield some information regarding the core or protein capsid substructure.
Unrelated T=3 plant viruses, such as TYMV and BMV, can be discriminated from one another on the basis of shape, size and capsomere structure, even with current technology. Resolution of very closely related viruses, such as cowpea chlorotic mottle virus and BMV, would probably not be possible at this time. Nonetheless, AFM can give some useful information regarding the presence or distribution of different viruses in a sample. As AFM technology progresses and better, more acute tips are developed (Woolley et al., 2000 ), the technique will become increasingly powerful and may begin to challenge and supplement low-resolution X-ray crystallography.
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Acknowledgments |
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References |
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