140 INSTRUMENTAL METHODS
microscope, but allowing imaging under the “ natural ” conditions usually asso-
ciated with the light microscope. Images can be obtained for samples in air,
water, or vacuum with typical resolutions on the order of 10 nm. AFM offers
the prospect of high - resolution images of biological material, images of mole-
cules and their interactions even under physiological conditions, and the study
of molecular processes in living systems. Applications of AFM in the biosci-
ences include analysis of (1) DNA and RNA, (2) protein – nucleic acid com-
plexes, (3) chromosomes; (4) cellular membranes, (5) proteins and peptides,
(6) molecular crystals, (7) biopolymers and biomaterials, and (8) ligand –
receptor binding.
The atomic force microscope is one of about two - dozen types of scanned -
proximity probe microscopes. All of these microscopes work by measuring a
local property — height, optical absorption, or magnetism — with a probe or
“ tip, ” typically made from silicon nitride, Si 3 N 4 , or elemental silicon, placed
very close to the sample. The small probe – sample separation (on the order of
the instrument ’ s resolution) makes it possible to take measurements over a
small area. To acquire an image, the microscope raster - scans the probe over
the sample while measuring the local property in question. The resulting image
resembles an image on a television screen in that both consist of many rows
or lines of information placed one above the other. Unlike traditional micro-
scopes, scanned - probe systems do not use lenses, so the size of the probe
(rather than diffraction effects) generally limits their resolution.
The concept of resolution in AFM is different from radiation - based micros-
copies because AFM is a three - dimensional imaging technique. There is an
important distinction between images resolved by wave optics and scanning
probe techniques. The former is limited by diffraction, whereas the latter is
limited primarily by apical probe geometry and sample geometry. Usually the
width of a DNA molecule is loosely used as a measure of resolution, because
it has a known diameter of 2.0 nm in its B form.
Intermolecular forces largely govern many biological processes — DNA rep-
lication, protein synthesis, drug interactions, and others. AFM has the ability
to measure these forces, some of which may be in the nanonewton (10 − 9
newton) range. This makes it possible to quantify molecular interactions in
biological systems such as important ligand – receptor interactions. The dynam-
ics of many biological systems depends on the electrical properties of the
sample surface, and AFM is able to image and quantify electrical surface
charges. In addition to measuring binding and electrostatic forces, the atomic
force microscope can also probe the micromechanical properties of biological
samples. Specifi cally, the AFM can observe the elasticity and, in fact, the viscos-
ity of samples ranging from live cells and membranes to bone and cartilage.
The fi rst highly reproducible AFM images of DNA were obtained in 1991.
Four major advances that have enabled clear resolution of nucleic acids are
(1) control of the local imaging environment including sample modifi cation,
(2) TappingMode ™ scanning techniques, (3) improved AFM probes (such as
standard silicon nitride probes modifi ed by electron beam deposition and