142 INSTRUMENTAL METHODS
to study the adsorption of membrane - associated protein cytochrome c to
anionic lipid bilayers of dioleoyl phosphatidyl - glycerol in low ionic strength
physiological buffer. The lipids were supported on polylysinated mica, forming
stable single lipid bilayers. When low concentrations of cytochrome c were
added to the mix, the protein molecules were not topographically visible on
the lipid bilayer – buffer interface. However, the forces required to punch
through the bilayer by indentation using the atomic force microscopy probe
were signifi cantly lower after protein adsorption, suggesting that the protein
inserts into the bilayer. Interestingly, the apparent thickness of the bilayer
remained unchanged after cytochrome c adsorption as well. The researchers
knew that cytochrome c was present in the bilayer during their AFM experi-
ments by confi rming cytochrome c ’ s presence using mass spectrometry and
UV – visible absorption spectroscopy. The reference 46 researchers concluded
that (1) cytochrome c inserts into the bilayer ’ s hydrophobic core, (2) cyto-
chrome c insertion changes the mechanical properties of the bilayer, and
(3) atomic force microscopy is a useful tool for investigating lipid – protein
interactions.
Cell biologists have applied the AFM ’ s unique capabilities to study the
dynamic behavior of living and fi xed cells such as red and white blood cells,
bacteria, platelets, cardiac myocytes, living renal epithelial cells, and glial cells.
AFM imaging of cells usually achieves a resolution of 20 – 50 nm, not suffi cient
for resolving membrane proteins but still suitable for imaging other surface
features, such as rearrangements of plasma membrane or movement of
submembrane fi lament bundles.
It is informative to compare AFM with other techniques. Scanning tunnel-
ing microscopy (STM) is considered the predecessor technique to AFM. The
scanning tunneling microscope has better resolution than the atomic force
microscope, but the technique can only be applied to conducting samples
while AFM can be applied to both conductors and insulators. Compared with
scanning electron microscopy (SEM), AFM provides extraordinary topo-
graphic contrast, direct height measurements, and unobscured views of surface
features (no coating is necessary). Compared with transmission electron
microscopes, three - dimensional AFM images are obtained without expensive
sample preparation and yield far more complete information than the two -
dimensional profi les available from cross - sectioned samples. New approaches
in AFM have provided a solid foundation from which research is expanding
into more and more complex bioorganic and bioinorganic analyses. Conven-
tional atomic force microscopes suffer from one disadvantage: 1 – 100 minutes
is required to obtain a high - quality image in contrast to the millisecond to 1 -
minute image times of scanning electron microscopes. High - speed AFM could
address this problem; however, commercial high - speed instruments are not
currently available. Three innovation areas will address this disadvantage in
the near future according to a perspective inScience magazine in October
200647 : (1) smaller cantilevers with resonant frequencies higher by a factor of
30; (2) increased scanner resonant frequencies with practical ranges of 13 μ m