Biomedical Engineering Reference
In-Depth Information
Increasing the concentration of protein or their aggregates on a surface can lead to
increased stability and imaging resolution, as at high surface concentrations the proteins
can stabilize each other. The ultimate extension of this is to form two-dimensional crystals
at a surface, which can lead to greatly improved imaging quality, and has been shown to
allow sub-molecular resolution in some systems [102, 600]. However, despite the impres-
sive resolution this technique is applicable only to some proteins, and removes one of the
great advantages of AFM, i.e. single-molecule imaging and relevance to biological
conditions [100].
Nucleic acids are highly suited to AFM imaging, and can be imaged in air and in
liquid, and by contact, non-contact and intermittent-contact modes [300, 319, 601].
Samples have included single stranded DNA (ssDNA), double stranded DNA
(dsDNA), and even the unusual triple stranded DNA [602]. RNA in both the common
single stranded form (with tertiary structure) [603, 604], double stranded RNA [605,
606], and ssRNA in an extended configuration have been imaged [479]. Complexes of
proteins (usually enzymes that have the nucleic acid as a substrate) with both RNA (anti-
RNA antibodies-RNA complexes [605]) and DNA (e.g. DNAse-DNA complexes) have
been imaged in air [607].
High-quality images of DNA can be obtained by deposition of a solution onto freshly-
cleaved mica, followed by imaging by NC- or IC-AFM. Reproducible and high-resolution
images require some way of binding DNA to the mica, because both mica and DNA are
negatively charged under common conditions. This is usually done by either treating
the mica with a divalent cation solution before deposition, or including such a cation
(e.g. Ni
2
þ
or Mg
2
þ
) in the deposition buffer. The divalent cations are thought to act as a
salt bridge [319], and this treatment allows imaging in air (after washing away most of the
salts, followed by drying), or in liquid (the imaging liquid must then contain the divalent
cation). Alternative methods include treating mica with an amino-terminated silane [300,
302], although caution must be taken not to increase greatly the roughness of the mica by
this method, as imaging the DNA well requires a very clean surface. See Chapter 4 for
more sample-preparation details.
One great advantage of the electrostatic absorption via divalent cations, is that if
imaging in liquid, careful control of the ionic conditions in the imaging buffer can ensure
the DNA stays on the surface, while allowing it freedom to move in two dimensions, and
even to carry out physiological functions [2, 4, 478, 608]. This has led to some very
elegant experiments in which both DNA and proteins are 'bound' to a surface well-enough
to be imaged by AFM, while being free enough to carry out their interactions in real-time.
Of course, such molecules bound to a mica surface are not under true physiological
conditions, but no other technique allows molecular biologists real-time single-molecule
imaging of these sorts of reactions at all [609]. Some stills from a time-lapse 'movie' that
can be generated by this technique are shown in Figure 7.19. More applications of AFM to
studies of DNA are discussed in the review [610].
The IC-AFM images shown in Figure 7.19 were acquired at a rate of 1 image
per second. This imaging rate is remarkably fast for AFM, especially for IC-AFM
images of such delicate structures. However a long-term goal for fast-AFM imaging
researchers is to improve the speed of IC-AFM data acquisition even further, allowing
AFM to probe protein-nucleic acid reactions with high time and spatial resolution
[476].
