dehydration and coating processes. Several images can then be recorded on the same sample, which is
tilted between recordings, and used to reconstruct the full 3D shape of the sample (with resolution ca.
10Å). Although the ultimate resolution of cryo EM is limited, crucial information about the interaction of
nucleic acids with other macromolecules can be gained by the marriage of this tool with other structural
methods. Cryo EM has also been used recently to investigate more complex nucleic acid incorporated
processes, including those involved in transcription.
11.5.2 Scanning Probe Microscop y
The field of scanning probe microscopy(SPM) refers to a class of techniques, devised originally during
the 1980s, which provide images of samples through use of a tiny probe (tip) to ‘feel’ the outermost prop-
erties of a flat surface. The members include the scanning tunnelling microscope(STM), the atomic
force microscope(AFM), the scanning near-field optical microscope and the scanning capacitance micro-
scope, but it is the first two of these which have contributed most to the structural analysis of nucleic acids.
11.5.2.1 Scanning Tunnelling Microscopy. Scanning tunnelling microscopy was developed in ca.
- This instrument exploits the ‘electron tunnelling’effect: electrons are able to pass through a potential
barrier when the distance of travel is small. The STM uses an atomically sharp conducting probethat scans
closely (typically angstrom) across the sample immobilized on a conducting substrate. The movement of
the probe or sample in x, yand zdirections is realised by a piezoelectric crystal, most often in the form of a
tube scanner. The scanner is capable of sub-angstrom resolution, with the z-axis conventionally perpendicular
to the sample in all scanning probe miscroscopes.
By monitoring the tunnelling current as the probe is scanned over the sample, any spatial variation in the
electronic topography of the surface is recorded. Due to the exponential dependence of the tunnelling current on
the probe-sample separation, the recorded image contrast invariably arises from variations in the surface topog-
raphy of the sample. Any variations in the electronic nature of the substrate under study, however, also con-
tribute to the recorded signal. Although STM is capable of achieving atomic resolution, in analysis of biological
molecules, image resolution is at best several nanometres.^37 Thus, whilst STM has been used for the imaging
of some nucleic acid samples, the interpretation of the images has always been somewhat open to question.
Various sample preparation approaches have been used for STM analysis. The most straightforward
involves deposition from a dilute solution, with the subsequent drying of solvent. The requisite conductive
properties of the sample are normally acquired through deposition on conducting substrates (atomically flat
gold and highly oriented pyrolytic graphite surfaces are frequently used), coating molecules deposited on
mica with thin films of platinum–iridium–carbon or through variation in the relative humidity of the imaging
environment. However, in reality the small number of suitable conducting surfaces, and the restrictions
imposed by the working environment (in air or vacuum) have limited the analysis of nucleic acids by STM.
The limitations of STM subsequently led to the development of the AFMor scanning force microscope
(SFM)(ca.1986). The AFM utilises a similar instrumental setup to the STM, but replaces the conducting
metal probe with a sharp probe (typically silicon or silicon nitride), which is situated at the apex of a can-
tilever spring of known stiffness (spring constant). As the probe is scanned over the surface of the sample,
the cantilever bends or twists in response to forces acting between the probe and the sample. By reflection
of a laser beam off the back surface of the cantilever onto a position sensitive photodiode, variation in the
bending or twisting of the cantilever can be monitored to produce an image (Figure 11.15).
11.5.2.2 Atomic Force Microscopy. The AFM can be used in three different imaging formats,
namely contact, tappingand non-contactmode, describing the way in which the probe interacts with the
surface during imaging. For the imaging of biological samples, tapping mode is more commonly used. In this
mode the probe intermittently comes into and out of contact with (i.e.lightly taps) the sample as is it scanned
over the surface, avoiding the strong lateral forces associated with contact mode, which can denature or sweep
away soft or poorly immobilized samples.
Physical and Structural Techniques Applied to Nucleic Acids 447