Chemistry Reference
In-Depth Information
phase-contrast imaging will allow us to study dynamic changes in the physicochemi-
cal nature of protein molecules in action.
12.7.2
Recognition Imaging
By attaching to the cantilever tip a molecule that binds speci
cally to complementary
molecules contained in a multi-component sample, phase-contrast imaging can
reveal the arrangement of speci
c molecules in the sample. However, to date the
studies for this type of recognition imaging is minimal. Hinterdorfer and his
colleagues recently developed a recognition imaging technique using a different
approach [36, 37]. A cantilever tip that is functionalized with a probe molecule
through a
flexible short-linker is oscillated at a frequency below resonance.
Its oscillation is split into lower and upper components. The lower-component
oscillation signals re
ect the repulsive tip-sample interaction and provide topography
images. The upper-component signals re
ect the association events between
the probe molecule and its counterpart and provide recognition images. Thus,
topographic and recognition images can be obtained simultaneously, which has
been demonstrated with systems such as antibody
-
antigen and biotin
-
avidin. This
method is very useful to identify locations of speci
c molecules in the topography
image of a multi-component sample.
12.8
Substratum
The
hoice of the substratum on which a sample is placed is very important for AFM
observation of biomolecular processes. Preparations of substrata with very low
roughness have been explored extensively for observing still images by AFM.
However, these preparations have been devised to attach samples
firmly onto them
and are therefore inapplicable for studying biomolecular processes. For our interests,
various properties are required for a substratum including (a) appropriate
binding af
nity for the sample, which ensures the retention of its physiological
function, (b) selective attachment of a speci
c component in a multi-component
sample, (c) attachment ofmolecules in a desired orientation. Mica (natural muscovite
or synthetic
c
fluorophlogopite) has been frequently used as a source substratum
because its surface is
at at the atomic level over a large area . It has net negative
charges and is therefore quite hydrophilic. Bare mica surfaces adsorb various
proteins by electrostatic interaction. Except for some cases (such as GroEL attach-
ment in an end-up orientation), its binding orientation is not unique, meaning that
selective attachment of a speci
c species cannot be expected. Furthermore, its use
sometimes compromises the sample.
If possible, it is best to have a surface to which the sample never attaches. Thus,
the surface can be modi
ed so that only a speci
c species of molecule attaches
